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Sample records for receptor bcr signaling

  1. Modulation of BCR Signaling by the Induced Dimerization of Receptor-Associated SYK

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    Mark L. Westbroek

    2017-12-01

    Full Text Available Clustering of the B cell antigen receptor (BCR by polyvalent antigens is transmitted through the SYK tyrosine kinase to the activation of multiple intracellular pathways that determine the physiological consequences of receptor engagement. To explore factors that modulate the quantity and quality of signals sent by the crosslinked BCR, we developed a novel chemical mediator of dimerization to induce clustering of receptor-associated SYK. To accomplish this, we fused SYK with E. coli dihydrofolate reductase (eDHFR, which binds the small molecule trimethoprim (TMP with high affinity and selectivity and synthesized a dimer of TMP with a flexible linker. The TMP dimer is able to induce the aggregation of eDHFR-linked SYK in live cells. The induced dimerization of SYK bound to the BCR differentially regulates the activation of downstream transcription factors, promoting the activation of Nuclear Factor of Activated T cells (NFAT without affecting the activation of NFκB. The dimerization of SYK enhances the duration but not the amplitude of calcium mobilization by enhancing the extent and duration of its interaction with the crosslinked BCR at the plasma membrane.

  2. Systems-wide analysis of BCR signalosomes and downstream phosphorylation and ubiquitylation

    DEFF Research Database (Denmark)

    Satpathy, Shankha; Wagner, Sebastian A; Beli, Petra

    2015-01-01

    B-cell receptor (BCR) signaling is essential for the development and function of B cells; however, the spectrum of proteins involved in BCR signaling is not fully known. Here we used quantitative mass spectrometry-based proteomics to monitor the dynamics of BCR signaling complexes (signalosomes......) and to investigate the dynamics of downstream phosphorylation and ubiquitylation signaling. We identify most of the previously known components of BCR signaling, as well as many proteins that have not yet been implicated in this system. BCR activation leads to rapid tyrosine phosphorylation and ubiquitylation...... of the receptor-proximal signaling components, many of which are co-regulated by both the modifications. We illustrate the power of multilayered proteomic analyses for discovering novel BCR signaling components by demonstrating that BCR-induced phosphorylation of RAB7A at S72 prevents its association...

  3. MicroRNAs regulate B-cell receptor signaling-induced apoptosis

    NARCIS (Netherlands)

    Kluiver, J. L.; Chen, C-Z

    Apoptosis induced by B-cell receptor (BCR) signaling is critical for antigen-driven selection, a process critical to tolerance and immunity. Here, we examined the roles of microRNAs (miRNAs) in BCR signaling-induced apoptosis using the widely applied WEHI-231 model. Comparison of miRNA levels in

  4. The BTK Inhibitor Ibrutinib (PCI-32765) Blocks Hairy Cell Leukaemia Survival, Proliferation and BCR Signalling: A New Therapeutic Approach

    Science.gov (United States)

    Sivina, Mariela; Kreitman, Robert J.; Arons, Evgeny; Ravandi, Farhad; Burger, Jan A.

    2014-01-01

    B cell receptor (BCR) signalling plays a critical role in the progression of several B-cell malignancies, but its role in hairy cell leukaemia (HCL) is ambiguous. Bruton tyrosine kinase (BTK), a key player in BCR signalling, migration and adhesion, can be targeted with ibrutinib, a selective, irreversible BTK inhibitor. We analysed BTK expression and function in HCL and analysed the effects of ibrutinib on HCL cells. We demonstrated uniform BTK protein expression in HCL cells. Ibrutinib significantly inhibited HCL proliferation and cell cycle progression. Accordingly, ibrutinib also reduced HCL cell survival after BCR triggering with anti-immunoglobulins (A, G, and M) and abrogated the activation of kinases downstream of the BCR (PI3K and MAPK). Ibrutinib also inhibited BCR-dependent secretion of the chemokines CCL3 and CCL4 by HCL cells. Interestingly, ibrutinib inhibited CXCL12-induced signalling, a key pathway for bone marrow homing. Collectively, our data support the clinical development of ibrutinib in patients with HCL. PMID:24697238

  5. TGFβ activated kinase 1 (TAK1 at the crossroad of B cell receptor and Toll-like receptor 9 signaling pathways in human B cells.

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    Dániel Szili

    Full Text Available B cell development and activation are regulated by combined signals mediated by the B cell receptor (BCR, receptors for the B-cell activating factor of the tumor necrosis factor family (BAFF-R and the innate receptor, Toll-like receptor 9 (TLR9. However, the underlying mechanisms by which these signals cooperate in human B cells remain unclear. Our aim was to elucidate the key signaling molecules at the crossroads of BCR, BAFF-R and TLR9 mediated pathways and to follow the functional consequences of costimulation.Therefore we stimulated purified human B cells by combinations of anti-Ig, B-cell activating factor of the tumor necrosis factor family (BAFF and the TLR9 agonist, CpG oligodeoxynucleotide. Phosphorylation status of various signaling molecules, B cell proliferation, cytokine secretion, plasma blast generation and the frequency of IgG producing cells were investigated. We have found that BCR induced signals cooperate with BAFF-R- and TLR9-mediated signals at different levels of cell activation. BCR and BAFF- as well as TLR9 and BAFF-mediated signals cooperate at NFκB activation, while BCR and TLR9 synergistically costimulate mitogen activated protein kinases (MAPKs, ERK, JNK and p38. We show here for the first time that the MAP3K7 (TGF beta activated kinase, TAK1 is responsible for the synergistic costimulation of B cells by BCR and TLR9, resulting in an enhanced cell proliferation, plasma blast generation, cytokine and antibody production. Specific inhibitor of TAK1 as well as knocking down TAK1 by siRNA abrogates the synergistic signals. We conclude that TAK1 is a key regulator of receptor crosstalk between BCR and TLR9, thus plays a critical role in B cell development and activation.

  6. N-wasp is essential for the negative regulation of B cell receptor signaling.

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    Chaohong Liu

    2013-11-01

    Full Text Available Negative regulation of receptor signaling is essential for controlling cell activation and differentiation. In B-lymphocytes, the down-regulation of B-cell antigen receptor (BCR signaling is critical for suppressing the activation of self-reactive B cells; however, the mechanism underlying the negative regulation of signaling remains elusive. Using genetically manipulated mouse models and total internal reflection fluorescence microscopy, we demonstrate that neuronal Wiskott-Aldrich syndrome protein (N-WASP, which is coexpressed with WASP in all immune cells, is a critical negative regulator of B-cell signaling. B-cell-specific N-WASP gene deletion causes enhanced and prolonged BCR signaling and elevated levels of autoantibodies in the mouse serum. The increased signaling in N-WASP knockout B cells is concurrent with increased accumulation of F-actin at the B-cell surface, enhanced B-cell spreading on the antigen-presenting membrane, delayed B-cell contraction, inhibition in the merger of signaling active BCR microclusters into signaling inactive central clusters, and a blockage of BCR internalization. Upon BCR activation, WASP is activated first, followed by N-WASP in mouse and human primary B cells. The activation of N-WASP is suppressed by Bruton's tyrosine kinase-induced WASP activation, and is restored by the activation of SH2 domain-containing inositol 5-phosphatase that inhibits WASP activation. Our results reveal a new mechanism for the negative regulation of BCR signaling and broadly suggest an actin-mediated mechanism for signaling down-regulation.

  7. Rictor positively regulates B cell receptor signaling by modulating actin reorganization via ezrin.

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    Lu Huang

    2017-08-01

    Full Text Available As the central hub of the metabolism machinery, the mammalian target of rapamycin complex 2 (mTORC2 has been well studied in lymphocytes. As an obligatory component of mTORC2, the role of Rictor in T cells is well established. However, the role of Rictor in B cells still remains elusive. Rictor is involved in B cell development, especially the peripheral development. However, the role of Rictor on B cell receptor (BCR signaling as well as the underlying cellular and molecular mechanism is still unknown. This study used B cell-specfic Rictor knockout (KO mice to investigate how Rictor regulates BCR signaling. We found that the key positive and negative BCR signaling molecules, phosphorylated Brutons tyrosine kinase (pBtk and phosphorylated SH2-containing inositol phosphatase (pSHIP, are reduced and enhanced, respectively, in Rictor KO B cells. This suggests that Rictor positively regulates the early events of BCR signaling. We found that the cellular filamentous actin (F-actin is drastically increased in Rictor KO B cells after BCR stimulation through dysregulating the dephosphorylation of ezrin. The high actin-ezrin intensity area restricts the lateral movement of BCRs upon stimulation, consequently reducing BCR clustering and BCR signaling. The reduction in the initiation of BCR signaling caused by actin alteration is associated with a decreased humoral immune response in Rictor KO mice. The inhibition of actin polymerization with latrunculin in Rictor KO B cells rescues the defects of BCR signaling and B cell differentiation. Overall, our study provides a new pathway linking cell metablism to BCR activation, in which Rictor regulates BCR signaling via actin reorganization.

  8. Pan-SRC kinase inhibition blocks B-cell receptor oncogenic signaling in non-Hodgkin lymphoma.

    Science.gov (United States)

    Battistello, Elena; Katanayeva, Natalya; Dheilly, Elie; Tavernari, Daniele; Donaldson, Maria C; Bonsignore, Luca; Thome, Margot; Christie, Amanda L; Murakami, Mark A; Michielin, Olivier; Ciriello, Giovanni; Zoete, Vincent; Oricchio, Elisa

    2018-05-24

    In diffuse large B-cell lymphoma (DLBCL), activation of the B-cell receptor (BCR) promotes multiple oncogenic signals, which are essential for tumor proliferation. Inhibition of the Bruton's tyrosine kinase (BTK), a BCR downstream target, is therapeutically effective only in a subgroup of patients with DLBCL. Here, we used lymphoma cells isolated from patients with DLBCL to measure the effects of targeted therapies on BCR signaling and to anticipate response. In lymphomas resistant to BTK inhibition, we show that blocking BTK activity enhanced tumor dependencies from alternative oncogenic signals downstream of the BCR, converging on MYC upregulation. To completely ablate the activity of the BCR, we genetically and pharmacologically repressed the activity of the SRC kinases LYN, FYN, and BLK, which are responsible for the propagation of the BCR signal. Inhibition of these kinases strongly reduced tumor growth in xenografts and cell lines derived from patients with DLBCL independent of their molecular subtype, advancing the possibility to be relevant therapeutic targets in broad and diverse groups of DLBCL patients. © 2018 by The American Society of Hematology.

  9. B-cell receptor signaling as a driver of lymphoma development and evolution.

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    Niemann, Carsten U; Wiestner, Adrian

    2013-12-01

    The B-cell receptor (BCR) is essential for normal B-cell development and maturation. In an increasing number of B-cell malignancies, BCR signaling is implicated as a pivotal pathway in tumorigenesis. Mechanisms of BCR activation are quite diverse and range from chronic antigenic drive by microbial or viral antigens to autostimulation of B-cells by self-antigens to activating mutations in intracellular components of the BCR pathway. Hepatitis C virus infection can lead to the development of splenic marginal zone lymphoma, while Helicobacter pylori infection is associated with the development of mucosa-associated lymphoid tissue lymphomas. In some of these cases, successful treatment of the infection removes the inciting antigen and results in resolution of the lymphoma. Chronic lymphocytic leukemia has been recognized for decades as a malignancy of auto-reactive B-cells and its clinical course is in part determined by the differential response of the malignant cells to BCR activation. In a number of B-cell malignancies, activating mutations in signal transduction components of the BCR pathway have been identified; prominent examples are activated B-cell-like (ABC) diffuse large B-cell lymphomas (DLBCL) that carry mutations in CD79B and CARD11 and display chronic active BCR signaling resulting in constitutive activation of the NF-κB pathway. Despite considerable heterogeneity in biology and clinical course, many mature B-cell malignancies are highly sensitive to kinase inhibitors that disrupt BCR signaling. Thus, targeted therapy through inhibition of BCR signaling is emerging as a new treatment paradigm for many B-cell malignancies. Here, we review the role of the BCR in the pathogenesis of B-cell malignancies and summarize clinical results of the emerging class of kinase inhibitors that target this pathway. Copyright © 2013. Published by Elsevier Ltd.

  10. Ibrutinib inhibits pre-BCR+ B-cell acute lymphoblastic leukemia progression by targeting BTK and BLK.

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    Kim, Ekaterina; Hurtz, Christian; Koehrer, Stefan; Wang, Zhiqiang; Balasubramanian, Sriram; Chang, Betty Y; Müschen, Markus; Davis, R Eric; Burger, Jan A

    2017-03-02

    Targeting B-cell receptor (BCR) signaling is a successful therapeutic strategy in mature B-cell malignancies. Precursor BCR (pre-BCR) signaling, which is critical during normal B lymphopoiesis, also plays an important role in pre-BCR + B cell acute lymphoblastic leukemia (B-ALL). Here, we investigated the activity and mechanism of action of the BTK inhibitor ibrutinib in preclinical models of B-ALL. Pre-BCR + ALL cells were exquisitely sensitive to ibrutinib at therapeutically relevant drug concentrations. In pre-BCR + ALL, ibrutinib thwarted autonomous and induced pre-BCR signaling, resulting in deactivation of PI3K/Akt signaling. Ibrutinib modulated the expression of pre-BCR regulators (PTPN6, CD22, CD72, and PKCβ) and substantially reduced BCL6 levels. Ibrutinib inhibited ALL cell migration toward CXCL12 and beneath marrow stromal cells and reduced CD44 expression. CRISPR-Cas9 gene editing revealed that both BTK and B lymphocyte kinase (BLK) are relevant targets of ibrutinib in pre-BCR + ALL. Consequently, in mouse xenograft models of pre-BCR + ALL, ibrutinib treatment significantly prolonged survival. Combination treatment of ibrutinib with dexamethasone or vincristine demonstrated synergistic activity against pre-BCR + ALL. These data corroborate ibrutinib as a promising targeted agent for pre-BCR + ALL and highlight the importance of ibrutinib effects on alternative kinase targets. © 2017 by The American Society of Hematology.

  11. BCR Signaling Inhibitors: an Overview of Toxicities Associated with Ibrutinib and Idelalisib in Patients with Chronic Lymphocytic Leukemia

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    Falchi, Lorenzo; Baron, Jessica M.; Orlikowski, Carrie Anne; Ferrajoli, Alessandra

    2016-01-01

    The B-cell receptor (BCR) signaling inhibitors ibrutinib and idelalisib are revolutionizing the treatment of chronic lymphocytic leukemia (CLL) and other B-cell malignancies. These oral agents, both alone and in combination with other drugs, have shown remarkable clinical activity in relapsed or refractory CLL across all risk groups, and have been approved by the Food and Drug Administration for this indication. Preliminary data suggest that an even greater benefit can be expected in treatment-naïve CLL patients. Both ibrutinib and idelalisib are well tolerated by most patients, including older, frailer individuals. Toxicities are usually mild and self-resolving. Clinicians must, however, be aware of a number of peculiar adverse events, the effects of which can be severe enough to limit the clinical use of these agents. In this review, we survey the salient aspects of the pharmacology and clinical experience with the use of BCR signaling inhibitors for the treatment of patients with CLL. We next focus on both the most common and the most clinically significant toxicities associated with these drugs. PMID:26977270

  12. Bruton's tyrosine kinase mediates the synergistic signalling between TLR9 and the B cell receptor by regulating calcium and calmodulin.

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    Elaine F Kenny

    Full Text Available B cells signal through both the B cell receptor (BCR which binds antigens and Toll-like receptors (TLRs including TLR9 which recognises CpG DNA. Activation of TLR9 synergises with BCR signalling when the BCR and TLR9 co-localise within an auto-phagosome-like compartment. Here we report that Bruton's tyrosine kinase (BTK is required for synergistic IL6 production and up-regulation of surface expression of MHC-class-II, CD69 and CD86 in primary murine and human B cells. We show that BTK is essential for co-localisation of the BCR and TLR9 within a potential auto-phagosome-like compartment in the Namalwa human B cell line. Downstream of BTK we find that calcium acting via calmodulin is required for this process. These data provide new insights into the role of BTK, an important target for autoimmune diseases, in B cell activation.

  13. PD-1 immunoreceptor inhibits B cell receptor-mediated signaling by recruiting src homology 2-domain-containing tyrosine phosphatase 2 to phosphotyrosine

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    Okazaki, Taku; Maeda, Akito; Nishimura, Hiroyuki; Kurosaki, Tomohiro; Honjo, Tasuku

    2001-01-01

    PD-1 is an immunoreceptor that belongs to the immunoglobulin (Ig) superfamily and contains two tyrosine residues in the cytoplasmic region. Studies on PD-1-deficient mice have shown that PD-1 plays critical roles in establishment and/or maintenance of peripheral tolerance, but the mode of action is totally unknown. To study the molecular mechanism for negative regulation of lymphocytes through the PD-1 receptor, we generated chimeric molecules composed of the IgG Fc receptor type IIB (FcγRIIB) extracellular region and the PD-1 cytoplasmic region and expressed them in a B lymphoma cell line, IIA1.6. Coligation of the cytoplasmic region of PD-1 with the B cell receptor (BCR) in IIA1.6 transformants inhibited BCR-mediated growth retardation, Ca2+ mobilization, and tyrosine phosphorylation of effector molecules, including Igβ, Syk, phospholipase C-γ2 (PLCγ2), and ERK1/2, whereas phosphorylation of Lyn and Dok was not affected. Mutagenesis studies indicated that these inhibitory effects do not require the N-terminal tyrosine in the immunoreceptor tyrosine-based inhibitory motif-like sequence, but do require the other tyrosine residue in the C-terminal tail. This tyrosine was phosphorylated and recruited src homology 2-domain-containing tyrosine phosphatase 2 (SHP-2) on coligation of PD-1 with BCR. These results show that PD-1 can inhibit BCR signaling by recruiting SHP-2 to its phosphotyrosine and dephosphorylating key signal transducers of BCR signaling. PMID:11698646

  14. Modulation of B-cell receptor and microenvironment signaling by a guanine exchange factor in B-cell malignancies

    International Nuclear Information System (INIS)

    Liao, Wei; Sharma, Sanjai

    2016-01-01

    Objective: Chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) cells over-express a guanine exchange factor (GEF), Rasgrf-1. This GEF increases active Ras as it catalyzes the removal of GDP from Ras so that GTP can bind and activate Ras. This study aims to study the mechanism of action of Rasgrf-1 in B-cell malignancies. Methods: N-terminus truncated Rasgrf-1 variants have a higher GEF activity as compared to the full-length transcript therefore a MCL cell line with stable over-expression of truncated Rasgrf-1 was established. The B-cell receptor (BCR) and chemokine signaling pathways were compared in the Rasgrf-1 over-expressing and a control transfected cell line. Results: Cells over-expressing truncated form of Rasgrf-1 have a higher proliferative rate as compared to control transfected cells. BCR was activated by lower concentrations of anti-IgM antibody in Rasgrf-1 over-expressing cells as compared to control cells indicating that these cells are more sensitive to BCR signaling. BCR signaling also phosphorylates Rasgrf-1 that further increases its GEF function and amplifies BCR signaling. This activation of Rasgrf-1 in over-expressing cells resulted in a higher expression of phospho-ERK, AKT, BTK and PKC-alpha as compared to control cells. Besides BCR, Rasgrf-1 over-expressing cells were also more sensitive to microenvironment stimuli as determined by resistance to apoptosis, chemotaxis and ERK pathway activation. Conclusions: This GEF protein sensitizes B-cells to BCR and chemokine mediated signaling and also upregulates a number of other signaling pathways which promotes growth and survival of these cells

  15. Rac-mediated Stimulation of Phospholipase Cγ2 Amplifies B Cell Receptor-induced Calcium Signaling*♦

    Science.gov (United States)

    Walliser, Claudia; Tron, Kyrylo; Clauss, Karen; Gutman, Orit; Kobitski, Andrei Yu.; Retlich, Michael; Schade, Anja; Röcker, Carlheinz; Henis, Yoav I.; Nienhaus, G. Ulrich; Gierschik, Peter

    2015-01-01

    The Rho GTPase Rac is crucially involved in controlling multiple B cell functions, including those regulated by the B cell receptor (BCR) through increased cytosolic Ca2+. The underlying molecular mechanisms and their relevance to the functions of intact B cells have thus far remained unknown. We have previously shown that the activity of phospholipase Cγ2 (PLCγ2), a key constituent of the BCR signalosome, is stimulated by activated Rac through direct protein-protein interaction. Here, we use a Rac-resistant mutant of PLCγ2 to functionally reconstitute cultured PLCγ2-deficient DT40 B cells and to examine the effects of the Rac-PLCγ2 interaction on BCR-mediated changes of intracellular Ca2+ and regulation of Ca2+-regulated and nuclear-factor-of-activated-T-cell-regulated gene transcription at the level of single, intact B cells. The results show that the functional Rac-PLCγ2 interaction causes marked increases in the following: (i) sensitivity of B cells to BCR ligation; (ii) BCR-mediated Ca2+ release from intracellular stores; (iii) Ca2+ entry from the extracellular compartment; and (iv) nuclear translocation of the Ca2+-regulated nuclear factor of activated T cells. Hence, Rac-mediated stimulation of PLCγ2 activity serves to amplify B cell receptor-induced Ca2+ signaling. PMID:25903139

  16. The bruton tyrosine kinase inhibitor ibrutinib (PCI-32765) blocks hairy cell leukaemia survival, proliferation and B cell receptor signalling: a new therapeutic approach.

    Science.gov (United States)

    Sivina, Mariela; Kreitman, Robert J; Arons, Evgeny; Ravandi, Farhad; Burger, Jan A

    2014-07-01

    B cell receptor (BCR) signalling plays a critical role in the progression of several B-cell malignancies, but its role in hairy cell leukaemia (HCL) is ambiguous. Bruton tyrosine kinase (BTK), a key player in BCR signalling, as well as B cell migration and adhesion, can be targeted with ibrutinib, a selective, irreversible BTK inhibitor. We analysed BTK expression and function in HCL and analysed the effects of ibrutinib on HCL cells. We demonstrated uniform BTK protein expression in HCL cells. Ibrutinib significantly inhibited HCL proliferation and cell cycle progression. Accordingly, ibrutinib also reduced HCL cell survival after BCR triggering with anti-immunoglobulins and abrogated the activation of kinases downstream of the BCR (PI3K and MAPK). Ibrutinib also inhibited BCR-dependent secretion of the chemokines CCL3 and CCL4 by HCL cells. Interestingly, ibrutinib inhibited also CXCL12-induced signalling, a key pathway for bone marrow homing. Collectively, our data support the clinical development of ibrutinib in patients with HCL. © 2014 John Wiley & Sons Ltd.

  17. Functional Independence and Interdependence of the Src Homology Domains of Phospholipase C-γ1 in B-Cell Receptor Signal Transduction

    Science.gov (United States)

    DeBell, Karen E.; Stoica, Bogdan A.; Verí, Maria-Concetta; Di Baldassarre, Angela; Miscia, Sebastiano; Graham, Laurie J.; Rellahan, Barbara L.; Ishiai, Masamichi; Kurosaki, Tomohiro; Bonvini, Ezio

    1999-01-01

    B-cell receptor (BCR)-induced activation of phospholipase C-γ1 (PLCγ1) and PLCγ2 is crucial for B-cell function. While several signaling molecules have been implicated in PLCγ activation, the mechanism coupling PLCγ to the BCR remains undefined. The role of PLCγ1 SH2 and SH3 domains at different steps of BCR-induced PLCγ1 activation was examined by reconstitution in a PLCγ-negative B-cell line. PLCγ1 membrane translocation required a functional SH2 N-terminal [SH2(N)] domain, was decreased by mutation of the SH3 domain, but was unaffected by mutation of the SH2(C) domain. Tyrosine phosphorylation did not require the SH2(C) or SH3 domains but depended exclusively on a functional SH2(N) domain, which mediated the association of PLCγ1 with the adapter protein, BLNK. Forcing PLCγ1 to the membrane via a myristoylation signal did not bypass the SH2(N) domain requirement for phosphorylation, indicating that the phosphorylation mediated by this domain is not due to membrane anchoring alone. Mutation of the SH2(N) or the SH2(C) domain abrogated BCR-stimulated phosphoinositide hydrolysis and signaling events, while mutation of the SH3 domain partially decreased signaling. PLCγ1 SH domains, therefore, have interrelated but distinct roles in BCR-induced PLCγ1 activation. PMID:10523627

  18. Protein sorting by lipid phase-like domains supports emergent signaling function in B lymphocyte plasma membranes.

    Science.gov (United States)

    Stone, Matthew B; Shelby, Sarah A; Núñez, Marcos F; Wisser, Kathleen; Veatch, Sarah L

    2017-02-01

    Diverse cellular signaling events, including B cell receptor (BCR) activation, are hypothesized to be facilitated by domains enriched in specific plasma membrane lipids and proteins that resemble liquid-ordered phase-separated domains in model membranes. This concept remains controversial and lacks direct experimental support in intact cells. Here, we visualize ordered and disordered domains in mouse B lymphoma cell membranes using super-resolution fluorescence localization microscopy, demonstrate that clustered BCR resides within ordered phase-like domains capable of sorting key regulators of BCR activation, and present a minimal, predictive model where clustering receptors leads to their collective activation by stabilizing an extended ordered domain. These results provide evidence for the role of membrane domains in BCR signaling and a plausible mechanism of BCR activation via receptor clustering that could be generalized to other signaling pathways. Overall, these studies demonstrate that lipid mediated forces can bias biochemical networks in ways that broadly impact signal transduction.

  19. Enhancement of B-cell receptor signaling by a point mutation of adaptor protein 3BP2 identified in human inherited disease cherubism.

    Science.gov (United States)

    Ogi, Kazuhiro; Nakashima, Kenji; Chihara, Kazuyasu; Takeuchi, Kenji; Horiguchi, Tomoko; Fujieda, Shigeharu; Sada, Kiyonao

    2011-09-01

    Tyrosine phosphorylation of adaptor protein c-Abl-Src homology 3 (SH3) domain-binding protein-2 (3BP2, also referred to SH3BP2) positively regulates the B-cell antigen receptor (BCR)-mediated signal transduction, leading to the activation of nuclear factor of activated T cells (NFAT). Here we showed the effect of the proline to arginine substitution of 3BP2 in which is the most common mutation in patients with cherubism (P418R) on B-cell receptor signaling. Comparing to the wild type, overexpression of the mutant form of 3BP2 (3BP2-P416R, corresponding to P418R in human protein) enhanced BCR-mediated activation of NFAT. 3BP2-P416R increased the signaling complex formation with Syk, phospholipase C-γ2 (PLC-γ2), and Vav1. In contrast, 3BP2-P416R could not change the association with the negative regulator 14-3-3. Loss of the association mutant that was incapable to associate with 14-3-3 could not mimic BCR-mediated NFAT activation in Syk-deficient cells. Moreover, BCR-mediated phosphorylation of extracellular signal regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) was not affected by P416R mutation. These results showed that P416R mutation of 3BP2 causes the gain of function in B cells by increasing the interaction with specific signaling molecules. © 2011 The Authors. Journal compilation © 2011 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.

  20. Coupling between p210bcr-abl and Shc and Grb2 adaptor proteins in hematopoietic cells permits growth factor receptor-independent link to ras activation pathway.

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    Tauchi, T; Boswell, H S; Leibowitz, D; Broxmeyer, H E

    1994-01-01

    Enforced expression of p210bcr-abl transforms interleukin 3 (IL-3)-dependent hematopoietic cell lines to growth factor-independent proliferation. It has been demonstrated that nonreceptor tyrosine kinase oncogenes may couple to the p21ras pathway to exert their transforming effect. In particular, p210bcr-abl was recently found to effect p21ras activation in hematopoietic cells. In this context, experiments were performed to evaluate a protein signaling pathway by which p210bcr-abl might regulate p21ras. It was asked whether Shc p46/p52, a protein containing a src-homology region 2 (SH2) domain, and known to function upstream from p21ras, might form specific complexes with p210bcr-abl and thus, possibly alter p21ras activity by coupling to the guanine nucleotide exchange factor (Sos/CDC25) through the Grb2 protein-Sos complex. This latter complex has been previously demonstrated to occur ubiquitously. We found that p210bcr-abl formed a specific complex with Shc and with Grb2 in three different murine cell lines transfected with a p210bcr-abl expression vector. There appeared to be a higher order complex containing Shc, Grb2, and bcr-abl proteins. In contrast to p210bcr-abl transformed cells, in which there was constitutive tight association between Grb2 and Shc, binding between Grb2 and Shc was Steel factor (SLF)-dependent in a SLF-responsive, nontransformed parental cell line. The SLF-dependent association between Grb2 and Shc in nontransformed cells involved formation of a complex of Grb2 with c-kit receptor after SLF treatment. Thus, p210bcr-abl appears to function in a hematopoietic p21ras activation pathway to allow growth factor-independent coupling between Grb2, which exists in a complex with the guanine nucleotide exchange factor (Sos), and p21ras. Shc may not be required for Grb2-c-kit interaction, because it fails to bind strongly to c-kit.

  1. Induction of autophagy by Imatinib sequesters Bcr-Abl in autophagosomes and down-regulates Bcr-Abl protein.

    LENUS (Irish Health Repository)

    Elzinga, Baukje M

    2013-06-01

    Chronic Myeloid Leukemia (CML) is a disease of hematopoietic stem cells which harbor the chimeric gene Bcr-Abl. Expression levels of this constitutively active tyrosine kinase are critical for response to tyrosine kinase inhibitor treatment and also disease progression, yet the regulation of protein stability is poorly understood. We have previously demonstrated that imatinib can induce autophagy in Bcr-Abl expressing cells. Autophagy has been associated with the clearance of large macromolecular signaling complexes and abnormal proteins, however, the contribution of autophagy to the turnover of Bcr-Abl protein in imatinib treated cells is unknown. In this study, we show that following imatinib treatment, Bcr-Abl is sequestered into vesicular structures that co-localize with the autophagy marker LC3 or GABARAP. This association is inhibited by siRNA mediated knockdown of autophagy regulators (Beclin 1\\/ATG7). Pharmacological inhibition of autophagy also reduced Bcr-Abl\\/LC3 co-localization in both K562 and CML patient cells. Bcr-Abl protein expression was reduced with imatinib treatment. Inhibition of both autophagy and proteasome activity in imatinib treated cells was required to restore Bcr-Abl protein levels to those of untreated cells. This ability to down-regulate Bcr-Abl protein levels through the induction of autophagy may be an additional and important feature of the activity of imatinib.

  2. Bcr-Abl oncoproteins bind directly to activators of the Ras signalling pathway.

    OpenAIRE

    Puil, L; Liu, J; Gish, G; Mbamalu, G; Bowtell, D; Pelicci, P G; Arlinghaus, R; Pawson, T

    1994-01-01

    The cytosolic 185 and 210 kDa Bcr-Abl protein tyrosine kinases play important roles in the development of Philadelphia chromosome positive (Ph+) chronic myelogenous leukemia (CML) and acute lymphoblastic leukemia (Ph+ ALL). p185 and p210 Bcr-Abl contain identical abl-encoded sequences juxtaposed to a variable number of bcr-derived amino acids. As the mitogenic and transforming activities of tyrosine kinases involve stimulation of the Ras pathway, we analyzed Bcr-Abl oncoproteins for interacti...

  3. Induction of IgG3 to LPS via Toll-like receptor 4 co-stimulation.

    Directory of Open Access Journals (Sweden)

    Francisco J Quintana

    Full Text Available B-cells integrate antigen-specific signals transduced via the B-cell receptor (BCR and antigen non-specific co-stimulatory signals provided by cytokines and CD40 ligation in order to produce IgG antibodies. Toll-like receptors (TLRs also provide co-stimulation, but the requirement for TLRs to generate T-cell independent and T-cell dependent antigen specific antibody responses is debated. Little is known about the role of B-cell expressed TLRs in inducing antigen-specific antibodies to antigens that also activate TLR signaling. We found that mice lacking functional TLR4 or its adaptor molecule MyD88 harbored significantly less IgG3 natural antibodies to LPS, and required higher amounts of LPS to induce anti-LPS IgG3. In vitro, BCR and TLR4 signaling synergized, lowering the threshold for production of T-cell independent IgG3 and IL-10. Moreover, BCR and TLR4 directly associate through the transmembrane domain of TLR4. Thus, in vivo, BCR/TLR synergism could facilitate the induction of IgG3 antibodies against microbial antigens that engage both innate and adaptive B-cell receptors. Vaccines might exploit BCR/TLR synergism to rapidly induce antigen-specific antibodies before significant T-cell responses arise.

  4. Multivalent Soluble Antigen Arrays Exhibit High Avidity Binding and Modulation of B Cell Receptor-Mediated Signaling to Drive Efficacy against Experimental Autoimmune Encephalomyelitis.

    Science.gov (United States)

    Hartwell, Brittany L; Pickens, Chad J; Leon, Martin; Berkland, Cory

    2017-06-12

    A pressing need exists for antigen-specific immunotherapies (ASIT) that induce selective tolerance in autoimmune disease while avoiding deleterious global immunosuppression. Multivalent soluble antigen arrays (SAgA PLP:LABL ), consisting of a hyaluronic acid (HA) linear polymer backbone cografted with multiple copies of autoantigen (PLP) and cell adhesion inhibitor (LABL) peptides, are designed to induce tolerance to a specific multiple sclerosis (MS) autoantigen. Previous studies established that hydrolyzable SAgA PLP:LABL , employing a degradable linker to codeliver PLP and LABL, was therapeutic in experimental autoimmune encephalomyelitis (EAE) in vivo and exhibited antigen-specific binding with B cells, targeted the B cell receptor (BCR), and dampened BCR-mediated signaling in vitro. Our results pointed to sustained BCR engagement as the SAgA PLP:LABL therapeutic mechanism, so we developed a new version of the SAgA molecule using nonhydrolyzable conjugation chemistry, hypothesizing it would enhance and maintain the molecule's action at the cell surface to improve efficacy. "Click SAgA" (cSAgA PLP:LABL ) uses hydrolytically stable covalent conjugation chemistry (Copper-catalyzed Azide-Alkyne Cycloaddition (CuAAC)) rather than a hydrolyzable oxime bond to attach PLP and LABL to HA. We explored cSAgA PLP:LABL B cell engagement and modulation of BCR-mediated signaling in vitro through flow cytometry binding and calcium flux signaling assays. Indeed, cSAgA PLP:LABL exhibited higher avidity B cell binding and greater dampening of BCR-mediated signaling than hydrolyzable SAgA PLP:LABL . Furthermore, cSAgA PLP:LABL exhibited significantly enhanced in vivo efficacy compared to hydrolyzable SAgA PLP:LABL , achieving equivalent efficacy at one-quarter of the dose. These results indicate that nonhydrolyzable conjugation increased the avidity of cSAgA PLP:LABL to drive in vivo efficacy through modulated BCR-mediated signaling.

  5. Continuous signaling of CD79b and CD19 is required for the fitness of Burkitt lymphoma B cells.

    Science.gov (United States)

    He, Xiaocui; Kläsener, Kathrin; Iype, Joseena M; Becker, Martin; Maity, Palash C; Cavallari, Marco; Nielsen, Peter J; Yang, Jianying; Reth, Michael

    2018-04-18

    Expression of the B-cell antigen receptor (BCR) is essential not only for the development but also for the maintenance of mature B cells. Similarly, many B-cell lymphomas, including Burkitt lymphoma (BL), require continuous BCR signaling for their tumor growth. This growth is driven by immunoreceptor tyrosine-based activation motif (ITAM) and PI3 kinase (PI3K) signaling. Here, we employ CRISPR/Cas9 to delete BCR and B-cell co-receptor genes in the human BL cell line Ramos. We find that Ramos B cells require the expression of the BCR signaling component Igβ (CD79b), and the co-receptor CD19, for their fitness and competitive growth in culture. Furthermore, we show that in the absence of any other BCR component, Igβ can be expressed on the B-cell surface, where it is found in close proximity to CD19 and signals in an ITAM-dependent manner. These data suggest that Igβ and CD19 are part of an alternative B-cell signaling module that use continuous ITAM/PI3K signaling to promote the survival of B lymphoma and normal B cells. © 2018 The Authors. Published under the terms of the CC BY 4.0 license.

  6. Monovalent engagement of the BCR activates ovalbumin-specific transnuclear B cells

    NARCIS (Netherlands)

    Avalos, Ana M.; Bilate, Angelina M.; Witte, Martin D.; Tai, Albert K.; He, Jiang; Frushicheva, Maria P.; Thill, Peter D.; Meyer-Wentrup, Friederike; Theile, Christopher S.; Chakraborty, Arup K.; Zhuang, Xiaowei; Ploegh, Hidde L.

    2014-01-01

    Valency requirements for B cell activation upon antigen encounter are poorly understood. OB1 transnuclear B cells express an IgG1 B cell receptor (BCR) specific for ovalbumin (OVA), the epitope of which can be mimicked using short synthetic peptides to allow antigen-specific engagement of the BCR.

  7. CD40 signaling synergizes with TLR-2 in the BCR independent activation of resting B cells.

    Directory of Open Access Journals (Sweden)

    Shweta Jain

    Full Text Available Conventionally, signaling through BCR initiates sequence of events necessary for activation and differentiation of B cells. We report an alternative approach, independent of BCR, for stimulating resting B (RB cells, by involving TLR-2 and CD40--molecules crucial for innate and adaptive immunity. CD40 triggering of TLR-2 stimulated RB cells significantly augments their activation, proliferation and differentiation. It also substantially ameliorates the calcium flux, antigen uptake capacity and ability of B cells to activate T cells. The survival of RB cells was improved and it increases the number of cells expressing activation induced deaminase (AID, signifying class switch recombination (CSR. Further, we also observed increased activation rate and decreased threshold period required for optimum stimulation of RB cells. These results corroborate well with microarray gene expression data. This study provides novel insights into coordination between the molecules of innate and adaptive immunity in activating B cells, in a BCR independent manner. This strategy can be exploited to design vaccines to bolster B cell activation and antigen presenting efficiency, leading to faster and better immune response.

  8. CD40 signaling synergizes with TLR-2 in the BCR independent activation of resting B cells.

    Science.gov (United States)

    Jain, Shweta; Chodisetti, Sathi Babu; Agrewala, Javed N

    2011-01-01

    Conventionally, signaling through BCR initiates sequence of events necessary for activation and differentiation of B cells. We report an alternative approach, independent of BCR, for stimulating resting B (RB) cells, by involving TLR-2 and CD40--molecules crucial for innate and adaptive immunity. CD40 triggering of TLR-2 stimulated RB cells significantly augments their activation, proliferation and differentiation. It also substantially ameliorates the calcium flux, antigen uptake capacity and ability of B cells to activate T cells. The survival of RB cells was improved and it increases the number of cells expressing activation induced deaminase (AID), signifying class switch recombination (CSR). Further, we also observed increased activation rate and decreased threshold period required for optimum stimulation of RB cells. These results corroborate well with microarray gene expression data. This study provides novel insights into coordination between the molecules of innate and adaptive immunity in activating B cells, in a BCR independent manner. This strategy can be exploited to design vaccines to bolster B cell activation and antigen presenting efficiency, leading to faster and better immune response.

  9. Drug Modulators of B Cell Signaling Pathways and Epstein-Barr Virus Lytic Activation.

    Science.gov (United States)

    Kosowicz, John G; Lee, Jaeyeun; Peiffer, Brandon; Guo, Zufeng; Chen, Jianmeng; Liao, Gangling; Hayward, S Diane; Liu, Jun O; Ambinder, Richard F

    2017-08-15

    Epstein-Barr virus (EBV) is a ubiquitous human gammaherpesvirus that establishes a latency reservoir in B cells. In this work, we show that ibrutinib, idelalisib, and dasatinib, drugs that block B cell receptor (BCR) signaling and are used in the treatment of hematologic malignancies, block BCR-mediated lytic induction at clinically relevant doses. We confirm that the immunosuppressive drugs cyclosporine and tacrolimus also inhibit BCR-mediated lytic induction but find that rapamycin does not inhibit BCR-mediated lytic induction. Further investigation shows that mammalian target of rapamycin complex 2 (mTORC2) contributes to BCR-mediated lytic induction and that FK506-binding protein 12 (FKBP12) binding alone is not adequate to block activation. Finally, we show that BCR signaling can activate EBV lytic induction in freshly isolated B cells from peripheral blood mononuclear cells (PBMCs) and that activation can be inhibited by ibrutinib or idelalisib. IMPORTANCE EBV establishes viral latency in B cells. Activation of the B cell receptor pathway activates lytic viral expression in cell lines. Here we show that drugs that inhibit important kinases in the BCR signaling pathway inhibit activation of lytic viral expression but do not inhibit several other lytic activation pathways. Immunosuppressant drugs such as cyclosporine and tacrolimus but not rapamycin also inhibit BCR-mediated EBV activation. Finally, we show that BCR activation of lytic infection occurs not only in tumor cell lines but also in freshly isolated B cells from patients and that this activation can be blocked by BCR inhibitors. Copyright © 2017 American Society for Microbiology.

  10. Recruitment of Cbl-b to B cell antigen receptor couples antigen recognition to Toll-like receptor 9 activation in late endosomes.

    Directory of Open Access Journals (Sweden)

    Margaret Veselits

    Full Text Available Casitas B-lineage lymphoma-b (Cbl-b is a ubiquitin ligase (E3 that modulates signaling by tagging molecules for degradation. It is a complex protein with multiple domains and binding partners that are not involved in ubiquitinating substrates. Herein, we demonstrate that Cbl-b, but not c-Cbl, is recruited to the clustered B cell antigen receptor (BCR and that Cbl-b is required for entry of endocytosed BCRs into late endosomes. The E3 activity of Cbl-b is not necessary for BCR endocytic trafficking. Rather, the ubiquitin associated (UBA domain is required. Furthermore, the Cbl-b UBA domain is sufficient to confer the receptor trafficking functions of Cbl-b on c-Cbl. Cbl-b is also required for entry of the Toll-like receptor 9 (TLR9 into late endosomes and for the in vitro activation of TLR9 by BCR-captured ligands. These data indicate that Cbl-b acts as a scaffolding molecule to coordinate the delivery of the BCR and TLR9 into subcellular compartments required for productively delivering BCR-captured ligands to TLR9.

  11. Endogenous Nur77 Is a Specific Indicator of Antigen Receptor Signaling in Human T and B Cells.

    Science.gov (United States)

    Ashouri, Judith F; Weiss, Arthur

    2017-01-15

    Distinguishing true Ag-stimulated lymphocytes from bystanders activated by the inflammatory milieu has been difficult. Nur77 is an immediate early gene whose expression is rapidly upregulated by TCR signaling in murine T cells and human thymocytes. Nur77-GFP transgenes serve as specific TCR and BCR signaling reporters in murine transgenic models. In this study, we demonstrate that endogenous Nur77 protein expression can serve as a reporter of TCR and BCR specific signaling in human PBMCs. Nur77 protein amounts were assessed by immunofluorescence and flow cytometry in T and B cells isolated from human PBMCs obtained from healthy donors that had been stimulated by their respective Ag receptors. We demonstrate that endogenous Nur77 is a more specific reporter of Ag-specific signaling events than the commonly used CD69 activation marker in both human T and B cells. This is reflective of the disparity in signaling pathways that regulate the expression of Nur77 and CD69. Assessing endogenous Nur77 protein expression has great potential to identify Ag-activated lymphocytes in human disease. Copyright © 2017 by The American Association of Immunologists, Inc.

  12. The Scaffolding Protein Synapse-Associated Protein 97 is Required for Enhanced Signaling Through Isotype-Switched IgG Memory B Cell Receptors

    Science.gov (United States)

    Liu, Wanli; Chen, Elizabeth; Zhao, Xing Wang; Wan, Zheng Peng; Gao, Yi Ren; Davey, Angel; Huang, Eric; Zhang, Lijia; Crocetti, Jillian; Sandoval, Gabriel; Joyce, M. Gordon; Miceli, Carrie; Lukszo, Jan; Aravind, L.; Swat, Wojciech; Brzostowski, Joseph; Pierce, Susan K.

    2012-01-01

    Memory B cells are generated during an individual's first encounter with a foreign antigen and respond to re-encounter with the same antigen through cell surface immunoglobulin G (IgG) B cell receptors (BCRs) resulting in rapid, high-titered IgG antibody responses. Despite a central role for IgG BCRs in B cell memory, our understanding of the molecular mechanism by which IgG BCRs enhance antibody responses is incomplete. Here, we showed that the conserved cytoplasmic tail of the IgG BCR, which contains a putative PDZ-binding motif, associated with synapse-associated protein 97 (SAP97), a member of the PDZ domain–containing, membrane-associated guanylate-kinase family of scaffolding molecules that play key roles in controlling receptor density and signal strength at neuronal synapses. We showed that SAP97 accumulated and bound to IgG BCRs in the immune synapses that formed in response to engagement of the B cell with antigen. Knocking down SAP97 in IgG-expressing B cells or mutating the putative PDZ-binding motif in the tail impaired immune synapse formation, the initiation of IgG BCR signaling, and downstream activation of p38 mitogen-activated protein kinase. Thus, heightened B cell memory responses are encoded, in part, by a mechanism that involves SAP97 serving as a scaffolding protein in the IgG BCR immune synapse. PMID:22855505

  13. The B-cell receptor controls fitness of MYC-driven lymphoma cells via GSK3β inhibition.

    Science.gov (United States)

    Varano, Gabriele; Raffel, Simon; Sormani, Martina; Zanardi, Federica; Lonardi, Silvia; Zasada, Christin; Perucho, Laura; Petrocelli, Valentina; Haake, Andrea; Lee, Albert K; Bugatti, Mattia; Paul, Ulrike; Van Anken, Eelco; Pasqualucci, Laura; Rabadan, Raul; Siebert, Reiner; Kempa, Stefan; Ponzoni, Maurilio; Facchetti, Fabio; Rajewsky, Klaus; Casola, Stefano

    2017-06-08

    Similar to resting mature B cells, where the B-cell antigen receptor (BCR) controls cellular survival, surface BCR expression is conserved in most mature B-cell lymphomas. The identification of activating BCR mutations and the growth disadvantage upon BCR knockdown of cells of certain lymphoma entities has led to the view that BCR signalling is required for tumour cell survival. Consequently, the BCR signalling machinery has become an established target in the therapy of B-cell malignancies. Here we study the effects of BCR ablation on MYC-driven mouse B-cell lymphomas and compare them with observations in human Burkitt lymphoma. Whereas BCR ablation does not, per se, significantly affect lymphoma growth, BCR-negative (BCR - ) tumour cells rapidly disappear in the presence of their BCR-expressing (BCR + ) counterparts in vitro and in vivo. This requires neither cellular contact nor factors released by BCR + tumour cells. Instead, BCR loss induces the rewiring of central carbon metabolism, increasing the sensitivity of receptor-less lymphoma cells to nutrient restriction. The BCR attenuates glycogen synthase kinase 3 beta (GSK3β) activity to support MYC-controlled gene expression. BCR - tumour cells exhibit increased GSK3β activity and are rescued from their competitive growth disadvantage by GSK3β inhibition. BCR - lymphoma variants that restore competitive fitness normalize GSK3β activity after constitutive activation of the MAPK pathway, commonly through Ras mutations. Similarly, in Burkitt lymphoma, activating RAS mutations may propagate immunoglobulin-crippled tumour cells, which usually represent a minority of the tumour bulk. Thus, while BCR expression enhances lymphoma cell fitness, BCR-targeted therapies may profit from combinations with drugs targeting BCR - tumour cells.

  14. Differential regulation of the Rac1 GTPase-activating protein (GAP) BCR during oxygen/glucose deprivation in hippocampal and cortical neurons.

    Science.gov (United States)

    Smith, Katharine R; Rajgor, Dipen; Hanley, Jonathan G

    2017-12-08

    Brain ischemia causes oxygen and glucose deprivation (OGD) in neurons, triggering a cascade of events leading to synaptic accumulation of glutamate. Excessive activation of glutamate receptors causes excitotoxicity and delayed cell death in vulnerable neurons. Following global cerebral ischemia, hippocampal CA1 pyramidal neurons are more vulnerable to injury than their cortical counterparts, but the mechanisms that underlie this difference are unclear. Signaling via Rho-family small GTPases, their upstream guanine nucleotide exchange factors, and GTPase-activating proteins (GAPs) is differentially dysregulated in response to OGD/ischemia in hippocampal and cortical neurons. Increased Rac1 activity caused by OGD/ischemia contributes to neuronal death in hippocampal neurons via diverse effects on NADPH oxidase activity and dendritic spine morphology. The Rac1 guanine nucleotide exchange factor Tiam1 mediates an OGD-induced increase in Rac1 activity in hippocampal neurons; however, the identity of an antagonistic GAP remains elusive. Here we show that the Rac1 GAP breakpoint cluster region (BCR) associates with NMDA receptors (NMDARs) along with Tiam1 and that this protein complex is more abundant in hippocampal compared with cortical neurons. Although total BCR is similar in the two neuronal types, BCR is more active in hippocampal compared with cortical neurons. OGD causes an NMDAR- and Ca 2+ -permeable AMPAR-dependent deactivation of BCR in hippocampal but not cortical neurons. BCR knockdown occludes OGD-induced Rac1 activation in hippocampal neurons. Furthermore, disrupting the Tiam1-NMDAR interaction with a fragment of Tiam1 blocks OGD-induced Tiam1 activation but has no effect on the deactivation of BCR. This work identifies BCR as a critical player in Rac1 regulation during OGD in hippocampal neurons. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. Phospho-specific flow cytometry identifies aberrant signaling in indolent B-cell lymphoma

    Directory of Open Access Journals (Sweden)

    Blix Egil S

    2012-10-01

    Full Text Available Abstract Background Knowledge about signaling pathways in malignant cells may provide prognostic and diagnostic information in addition to identify potential molecular targets for therapy. B-cell receptor (BCR and co-receptor CD40 signaling is essential for normal B cells, and there is increasing evidence that signaling via BCR and CD40 plays an important role in the pathogenesis of B-cell lymphoma. The aim of this study was to investigate basal and induced signaling in lymphoma B cells and infiltrating T cells in single-cell suspensions of biopsies from small cell lymphocytic lymphoma/chronic lymphocytic leukemia (SLL/CLL and marginal zone lymphoma (MZL patients. Methods Samples from untreated SLL/CLL and MZL patients were examined for basal and activation induced signaling by phospho-specific flow cytometry. A panel of 9 stimulation conditions targeting B and T cells, including crosslinking of the B cell receptor (BCR, CD40 ligand and interleukins in combination with 12 matching phospho-protein readouts was used to study signaling. Results Malignant B cells from SLL/CLL patients had higher basal levels of phosphorylated (p-SFKs, p-PLCγ, p-ERK, p-p38, p-p65 (NF-κB, p-STAT5 and p-STAT6, compared to healthy donor B cells. In contrast, anti-BCR induced signaling was highly impaired in SLL/CLL and MZL B cells as determined by low p-SFK, p-SYK and p-PLCγ levels. Impaired anti-BCR-induced p-PLCγ was associated with reduced surface expression of IgM and CD79b. Similarly, CD40L-induced p-ERK and p-p38 were also significantly reduced in lymphoma B cells, whereas p-p65 (NF-κB was equal to that of normal B cells. In contrast, IL-2, IL-7 and IL-15 induced p-STAT5 in tumor-infiltrating T cells were not different from normal T cells. Conclusions BCR signaling and CD40L-induced p-p38 was suppressed in malignant B cells from SLL/CLL and MZL patients. Single-cell phospho-specific flow cytometry for detection of basal as well as activation

  16. Lipid rafts and B cell signaling.

    Science.gov (United States)

    Gupta, Neetu; DeFranco, Anthony L

    2007-10-01

    B cells comprise an essential component of the humoral immune system. They are equipped with the unique ability to synthesize and secrete pathogen-neutralizing antibodies, and share with professional antigen presenting cells the ability to internalize foreign antigens, and process them for presentation to helper T cells. Recent evidence indicates that specialized cholesterol- and glycosphingolipid-rich microdomains in the plasma membrane commonly referred to as lipid rafts, serve to compartmentalize key signaling molecules during the different stages of B cell activation including B cell antigen receptor (BCR)-initiated signal transduction, endocytosis of BCR-antigen complexes, loading of antigenic peptides onto MHC class II molecules, MHC-II associated antigen presentation to helper T cells, and receipt of helper signals via the CD40 receptor. Here we review the recent literature arguing for a role of lipid rafts in the spatial organization of B cell function.

  17. Diverse phosphorylation patterns of B cell receptor-associated signaling in naïve and memory human B cells revealed by phosphoflow, a powerful technique to study signaling at the single cell level

    Directory of Open Access Journals (Sweden)

    Franklin R Toapanta

    2012-10-01

    Full Text Available Following interaction with cognate antigens, B cells undergo cell activation, proliferation and differentiation. Ligation of the B cell receptor (BCR leads to the phosphorylation of BCR-associated signaling proteins within minutes of antigen binding, a process with profound consequences for the fate of the cells and development of effector immunity. Phosphoflow allows a rapid evaluation of various signaling pathways in complex heterogenous cell subsets. This novel technique was used in combination with multi-chromatic flow cytometry and fluorescent-cell barcoding to study phosphorylation of BCR-associated signaling pathways in naïve and memory human B cell subsets. Proteins of the initiation (Syk, propagation (Btk, Akt and integration (p38MAPK and Erk1/2 signaling units were studied. Switched memory (Sm CD27+ and Sm CD27- phosphorylation patterns were similar when stimulated with anti-IgA or -IgG. In contrast, naïve and unswitched memory (Um cells showed significant differences following IgM stimulation. Enhanced phosphorylation of Syk was observed in Um cells, suggesting a lower activation threshold. This is likely the result of higher amounts of IgM on the cell surface, higher pan-Syk levels and enhanced susceptibility to phosphatase inhibition. All other signaling proteins evaluated also showed some degree of enhanced phosphorylation in Um cells. Furthermore, both the PLC-γ2 and PI3K pathways were activated in Um cells, while only the PI3K pathway was activated on naïve cells. Um cells were the only ones that activated signaling pathways when stimulated with fluorescently-labeled S. Typhi and S. pneumoniae. Finally, simultaneous evaluation of signaling proteins at the single cell level (multi-phosphorylated cells revealed that interaction with gram positive and negative bacteria resulted in complex and diverse signaling patterns. Phosphoflow holds great potential to accelerate vaccine development by identifying signaling profiles in good

  18. Dissection of the BCR-ABL signaling network using highly specific monobody inhibitors to the SHP2 SH2 domains.

    Science.gov (United States)

    Sha, Fern; Gencer, Emel Basak; Georgeon, Sandrine; Koide, Akiko; Yasui, Norihisa; Koide, Shohei; Hantschel, Oliver

    2013-09-10

    The dysregulated tyrosine kinase BCR-ABL causes chronic myelogenous leukemia in humans and forms a large multiprotein complex that includes the Src-homology 2 (SH2) domain-containing phosphatase 2 (SHP2). The expression of SHP2 is necessary for BCR-ABL-dependent oncogenic transformation, but the precise signaling mechanisms of SHP2 are not well understood. We have developed binding proteins, termed monobodies, for the N- and C-terminal SH2 domains of SHP2. Intracellular expression followed by interactome analysis showed that the monobodies are essentially monospecific to SHP2. Two crystal structures revealed that the monobodies occupy the phosphopeptide-binding sites of the SH2 domains and thus can serve as competitors of SH2-phosphotyrosine interactions. Surprisingly, the segments of both monobodies that bind to the peptide-binding grooves run in the opposite direction to that of canonical phosphotyrosine peptides, which may contribute to their exquisite specificity. When expressed in cells, monobodies targeting the N-SH2 domain disrupted the interaction of SHP2 with its upstream activator, the Grb2-associated binder 2 adaptor protein, suggesting decoupling of SHP2 from the BCR-ABL protein complex. Inhibition of either N-SH2 or C-SH2 was sufficient to inhibit two tyrosine phosphorylation events that are critical for SHP2 catalytic activity and to block ERK activation. In contrast, targeting the N-SH2 or C-SH2 revealed distinct roles of the two SH2 domains in downstream signaling, such as the phosphorylation of paxillin and signal transducer and activator of transcription 5. Our results delineate a hierarchy of function for the SH2 domains of SHP2 and validate monobodies as potent and specific antagonists of protein-protein interactions in cancer cells.

  19. Pristimerin induces apoptosis in imatinib-resistant chronic myelogenous leukemia cells harboring T315I mutation by blocking NF-κB signaling and depleting Bcr-Abl

    Science.gov (United States)

    2010-01-01

    Background Chronic myelogenous leukemia (CML) is characterized by the chimeric tyrosine kinase Bcr-Abl. Bcr-Abl-T315I is the notorious point mutation that causes resistance to imatinib and the second generation tyrosine kinase inhibitors, leading to poor prognosis. CML blasts have constitutive p65 (RelA NF-κB) transcriptional activity, and NF-κB may be a potential target for molecular therapies in CML that may also be effective against CML cells with Bcr-Abl-T315I. Results In this report, we discovered that pristimerin, a quinonemethide triterpenoid isolated from Celastraceae and Hippocrateaceae, inhibited growth and induced apoptosis in CML cells, including the cells harboring Bcr-Abl-T315I mutation. Additionally, pristimerin inhibited the growth of imatinib-resistant Bcr-Abl-T315I xenografts in nude mice. Pristimerin blocked the TNFα-induced IκBα phosphorylation, translocation of p65, and expression of NF-κB-regulated genes. Pristimerin inhibited two steps in NF-κB signaling: TAK1→IKK and IKK→IκBα. Pristimerin potently inhibited two pairs of CML cell lines (KBM5 versus KBM5-T315I, 32D-Bcr-Abl versus 32D-Bcr-Abl-T315I) and primary cells from a CML patient with acquired resistance to imatinib. The mRNA and protein levels of Bcr-Abl in imatinib-sensitive (KBM5) or imatinib-resistant (KBM5-T315I) CML cells were reduced after pristimerin treatment. Further, inactivation of Bcr-Abl by imatinib pretreatment did not abrogate the TNFα-induced NF-κB activation while silencing p65 by siRNA did not affect the levels of Bcr-Abl, both results together indicating that NF-κB inactivation and Bcr-Abl inhibition may be parallel independent pathways. Conclusion To our knowledge, this is the first report to show that pristimerin is effective in vitro and in vivo against CML cells, including those with the T315I mutation. The mechanisms may involve inhibition of NF-κB and Bcr-Abl. We concluded that pristimerin could be a lead compound for further drug development to

  20. Feedback regulation of mitochondria by caspase-9 in the B cell receptor-mediated apoptosis.

    Science.gov (United States)

    Eeva, J; Nuutinen, U; Ropponen, A; Mättö, M; Eray, M; Pellinen, R; Wahlfors, J; Pelkonen, J

    2009-12-01

    During the germinal centre reaction (GC), B cells with non-functional or self-reactive antigen receptors are negatively selected by apoptosis to generate B cell repertoire with appropriate antigen specificities. We studied the molecular mechanism of Fas/CD95- and B cell receptor (BCR)-induced apoptosis to shed light on the signalling events involved in the negative selection of GC B cells. As an experimental model, we used human follicular lymphoma (FL) cell line HF1A3, which originates from a GC B cell, and transfected HF1A3 cell lines overexpressing Bcl-x(L), c-FLIP(long) or dominant negative (DN) caspase-9. Fas-induced apoptosis was dependent on the caspase-8 activation, since the overexpression of c-FLIP(long), a natural inhibitor of caspase-8 activation, blocked apoptosis induced by Fas. In contrast, caspase-9 activation was not involved in Fas-induced apoptosis. BCR-induced apoptosis showed the typical characteristics of mitochondria-dependent (intrinsic) apoptosis. Firstly, the activation of caspase-9 was involved in BCR-induced DNA fragmentation, while caspase-8 showed only marginal role. Secondly, overexpression of Bcl-x(L) could block all apoptotic changes induced by BCR. As a novel finding, we demonstrate that caspase-9 can enhance the cytochrome-c release and collapse of mitochondrial membrane potential (DeltaPsi(m)) during BCR-induced apoptosis. The requirement of different signalling pathways in apoptosis induced by BCR and Fas may be relevant, since Fas- and BCR-induced apoptosis can thus be regulated independently, and targeted to different subsets of GC B cells.

  1. Targeting the SH2-kinase interface in Bcr-Abl inhibits leukemogenesis.

    Science.gov (United States)

    Grebien, Florian; Hantschel, Oliver; Wojcik, John; Kaupe, Ines; Kovacic, Boris; Wyrzucki, Arkadiusz M; Gish, Gerald D; Cerny-Reiterer, Sabine; Koide, Akiko; Beug, Hartmut; Pawson, Tony; Valent, Peter; Koide, Shohei; Superti-Furga, Giulio

    2011-10-14

    Chronic myelogenous leukemia (CML) is caused by the constitutively active tyrosine kinase Bcr-Abl and treated with the tyrosine kinase inhibitor (TKI) imatinib. However, emerging TKI resistance prevents complete cure. Therefore, alternative strategies targeting regulatory modules of Bcr-Abl in addition to the kinase active site are strongly desirable. Here, we show that an intramolecular interaction between the SH2 and kinase domains in Bcr-Abl is both necessary and sufficient for high catalytic activity of the enzyme. Disruption of this interface led to inhibition of downstream events critical for CML signaling and, importantly, completely abolished leukemia formation in mice. Furthermore, disruption of the SH2-kinase interface increased sensitivity of imatinib-resistant Bcr-Abl mutants to TKI inhibition. An engineered Abl SH2-binding fibronectin type III monobody inhibited Bcr-Abl kinase activity both in vitro and in primary CML cells, where it induced apoptosis. This work validates the SH2-kinase interface as an allosteric target for therapeutic intervention. Copyright © 2011 Elsevier Inc. All rights reserved.

  2. Detection of a rare BCR-ABL tyrosine kinase fusion protein in H929 multiple myeloma cells using immunoprecipitation (IP)-tandem mass spectrometry (MS/MS).

    Science.gov (United States)

    Breitkopf, Susanne B; Yuan, Min; Pihan, German A; Asara, John M

    2012-10-02

    Hypothesis directed proteomics offers higher throughput over global analyses. We show that immunoprecipitation (IP)-tandem mass spectrometry (LC-MS/MS) in H929 multiple myeloma (MM) cancer cells led to the discovery of a rare and unexpected BCR-ABL fusion, informing a therapeutic intervention using imatinib (Gleevec). BCR-ABL is the driving mutation in chronic myeloid leukemia (CML) and is uncommon to other cancers. Three different IP-MS experiments central to cell signaling pathways were sufficient to discover a BCR-ABL fusion in H929 cells: phosphotyrosine (pY) peptide IP, p85 regulatory subunit of phosphoinositide-3-kinase (PI3K) IP, and the GRB2 adaptor IP. The pY peptides inform tyrosine kinase activity, p85 IP informs the activating adaptors and receptor tyrosine kinases (RTKs) involved in AKT activation and GRB2 IP identifies RTKs and adaptors leading to ERK activation. Integration of the bait-prey data from the three separate experiments identified the BCR-ABL protein complex, which was confirmed by biochemistry, cytogenetic methods, and DNA sequencing revealed the e14a2 fusion transcript. The tyrosine phosphatase SHP2 and the GAB2 adaptor protein, important for MAPK signaling, were common to all three IP-MS experiments. The comparative treatment of tyrosine kinase inhibitor (TKI) drugs revealed only imatinib, the standard of care in CML, was inhibitory to BCR-ABL leading to down-regulation of pERK and pS6K and inhibiting cell proliferation. These data suggest a model for directed proteomics from patient tumor samples for selecting the appropriate TKI drug(s) based on IP and LC-MS/MS. The data also suggest that MM patients, in addition to CML patients, may benefit from BCR-ABL diagnostic screening.

  3. Epstein-Barr virus (EBV) LMP2A alters normal transcriptional regulation following B-cell receptor activation

    International Nuclear Information System (INIS)

    Portis, Toni; Longnecker, Richard

    2004-01-01

    The latent membrane protein 2A (LMP2A) of Epstein-Barr virus (EBV) is an important mediator of viral latency in infected B-lymphocytes. LMP2A inhibits B-cell receptor (BCR) signaling in vitro and allows for the survival of BCR-negative B cells in vivo. In this study, we compared gene transcription in BCR-activated B cells from non-transgenic and LMP2A Tg6 transgenic mice. We found that the transcriptional induction and down-regulation of many genes that normally occurs in B cells following BCR activation did not occur in B cells from LMP2A Tg6 transgenic mice. Furthermore, LMP2A induced the expression of various transcription factors and genes associated with DNA/RNA metabolism, which may allow for the altered transcriptional regulation observed in BCR-activated B cells from LMP2A Tg6 mice. These results suggest that LMP2A may inhibit the downstream effects of BCR signaling by directly or indirectly altering gene transcription to ensure EBV persistence in infected B cells

  4. Cooperative ethylene receptor signaling

    OpenAIRE

    Liu, Qian; Wen, Chi-Kuang

    2012-01-01

    The gaseous plant hormone ethylene is perceived by a family of five ethylene receptor members in the dicotyledonous model plant Arabidopsis. Genetic and biochemical studies suggest that the ethylene response is suppressed by ethylene receptor complexes, but the biochemical nature of the receptor signal is unknown. Without appropriate biochemical measures to trace the ethylene receptor signal and quantify the signal strength, the biological significance of the modulation of ethylene responses ...

  5. Dual TORK/DNA-PK inhibition blocks critical signaling pathways in chronic lymphocytic leukemia

    NARCIS (Netherlands)

    Thijssen, Rachel; ter Burg, Johanna; Garrick, Brett; van Bochove, Gregor G. W.; Brown, Jennifer R.; Fernandes, Stacey M.; Rodríguez, María Solé; Michot, Jean-Marie; Hallek, Michael; Eichhorst, Barbara; Reinhardt, Hans Christian; Bendell, Johanna; Derks, Ingrid A. M.; van Kampen, Roel J. W.; Hege, Kristen; Kersten, Marie José; Trowe, Torsten; Filvaroff, Ellen H.; Eldering, Eric; Kater, Arnon P.

    2016-01-01

    Inhibition of B-cell receptor (BCR) signaling pathways in chronic lymphocytic leukemia (CLL) provides significant clinical benefit to patients, mainly by blocking adhesion of CLL cells in the lymph node microenvironment. The currently applied inhibitors ibrutinib and idelalisib have limited capacity

  6. Growth of chronic myeloid leukemia cells is inhibited by infection with Ad-SH2-HA adenovirus that disrupts Grb2-Bcr-Abl complexes.

    Science.gov (United States)

    Peng, Zhi; Luo, Hong-Wei; Yuan, Ying; Shi, Jing; Huang, Shi-Feng; Li, Chun-Li; Cao, Wei-Xi; Huang, Zong-Gan; Feng, Wen-Li

    2011-05-01

    The persistence of Bcr-Abl-positive cells in patients on imatinib therapy indicates that inhibition of the Bcr-Abl kinase activity alone might not be sufficient to eradicate the leukemia cells. Many downstream effectors of Bcr-Abl have been described, including activation of both the Grb2-SoS-Ras-MAPK and Grb2-Gab2-PI3K-Akt pathways. The Bcr-Abl-Grb2 interaction, which is mediated by the direct interaction of the Grb2 SH2 domain with the phospho-Bcr-Abl Y177, is required for activation of these signaling pathways. Therefore, disrupting their interaction represents a potential therapeutic strategy for inhibiting the oncogenic downstream signals of Bcr-Abl. Adenovirus Ad-SH2-HA expressing the Grb2 SH2 domain was constructed and applied in this study. As expected, Ad-SH2-HA efficiently infected CML cells and functioned by binding to the phospho-Bcr-Abl Y177 site, competitively disrupting the Grb2 SH2-phospho-Bcr-Abl Y177 complex. They induced potent anti-proliferation and apoptosis-inducing effects in CML cell lines. Moreover, the Ras, MAPK and Akt activities were significantly reduced in the Ad-SH2-HA treated cells. These were not observed with the point-mutated control adenovirus Ad-Sm-HA with abolished phospho-Bcr-Abl Y177 binding sites. These data indicate that, in addition to the direct targeting of Bcr-Abl, selective inhibition of its downstream signaling pathways may be a therapeutic option for CML, and the Ad-SH2-HA-mediated killing strategy could be explored as a promising anti-leukemia agent in CML.

  7. The proto-oncogene product p120CBL and the adaptor proteins CRKL and c-CRK link c-ABL, p190BCR/ABL and p210BCR/ABL to the phosphatidylinositol-3' kinase pathway.

    Science.gov (United States)

    Sattler, M; Salgia, R; Okuda, K; Uemura, N; Durstin, M A; Pisick, E; Xu, G; Li, J L; Prasad, K V; Griffin, J D

    1996-02-15

    Chronic myelogenous leukemia (CML) and some acute lymphoblastic leukemias (ALL) are caused by the t(9;22) chromosome translocation, which produces the constitutively activated BCR/ABL tyrosine kinase. When introduced into factor dependent hematopoietic cell lines, BCR/ABL induces the tyrosine phosphorylation of many cellular proteins. One prominent BCR/ABL substrate is p120CBL, the cellular homolog of the v-Cbl oncoprotein. In an effort to understand the possible contribution of p120CBL to transformation by BCR/ABL, we looked for cellular proteins which associate with p120CBL in hematopoietic cell lines transformed by BCR/ABL. In addition to p210BCR/ABL and c-ABL, p120CBL coprecipitated with an 85 kDa phosphoprotein, which was identified as the p85 subunit of PI3K. Anti-p120CBL immunoprecipitates from BCR/ABL-transformed, but not from untransformed, cell lines contained PI3K lipid kinase activity. Interestingly, the adaptor proteins CRKL and c-CRK were also found in these complexes. In vitro binding studies indicated that the SH2 domains of CRKL and c-CRK bound directly to p120CBL, while the SH3 domains of c-CRK and CRKL bound to BCR/ABL and c-ABL. The N-terminal and the C-terminal SH2 and the SH3 domain of p85PI3K bound directly in vitro to p120CBL. The ABL-SH2, but not ABL-SH3, could also bind to p120CBL. These data suggest that BCR/ABL may induce the formation of multimeric complexes of signaling proteins which include p120CBL, PI3K, c-CRK or CRKL, c-ABL and BCR/ABL itself.

  8. Purification and molecular cloning of SH2- and SH3-containing inositol polyphosphate-5-phosphatase, which is involved in the signaling pathway of granulocyte-macrophage colony-stimulating factor, erythropoietin, and Bcr-Abl.

    Science.gov (United States)

    Odai, H; Sasaki, K; Iwamatsu, A; Nakamoto, T; Ueno, H; Yamagata, T; Mitani, K; Yazaki, Y; Hirai, H

    1997-04-15

    Grb2/Ash and Shc are the adapter proteins that link tyrosine-kinase receptors to Ras and make tyrosine-kinase functionally associated with receptors and Ras in fibroblasts and hematopoietic cells. Grb2/Ash and Shc have the SH3, SH2, or phosphotyrosine binding domains. These domains bind to proteins containing proline-rich regions or tyrosine-phosphorylated proteins and contribute to the association of Grb2/Ash and Shc with other signaling molecules. However, there could remain unidentified signaling molecules that physically and functionally interact with these adapter proteins and have biologically important roles in the signaling pathways. By using the GST fusion protein including the full length of Grb2/Ash, we have found that c-Cbl and an unidentified 135-kD protein (pp135) are associated with Grb2/Ash. We have also found that they become tyrosine-phosphorylated by treatment of a human leukemia cell line, UT-7, with granulocyte-macrophage colony-stimulating factor (GM-CSF). We have purified the pp135 by using GST-Grb2/Ash affinity column and have isolated the full-length complementary DNA (cDNA) encoding the pp135 using a cDNA probe, which was obtained by the degenerate polymerase chain reaction based on a peptide sequence of the purified pp135. The cloned cDNA has 3,958 nucleotides that contain a single long open reading frame of 3,567 nucleotides, encoding a 1,189 amino acid protein with a predicted molecular weight of approximately 133 kD. The deduced amino acid sequence reveals that pp135 is a protein that has one SH2, one SH3, and one proline-rich domain. The pp135, which contains two motifs conserved among the inositol polyphosphate-5-phosphatase proteins, was shown to have the inositol polyphosphate-5-phosphatase activity. The pp135 was revealed to associate constitutively with Grb2/Ash and inducibly with Shc using UT-7 cells stimulated with GM-CSF. In the cell lines derived from human chronic myelogenous leukemia, pp135 was constitutively tyrosine

  9. Allosteric inhibition enhances the efficacy of ABL kinase inhibitors to target unmutated BCR-ABL and BCR-ABL-T315I

    Directory of Open Access Journals (Sweden)

    Mian Afsar

    2012-09-01

    Full Text Available Abstract Background Chronic myelogenous leukemia (CML and Philadelphia chromosome-positive (Ph+ acute lymphatic leukemia (Ph + ALL are caused by the t(9;22, which fuses BCR to ABL resulting in deregulated ABL-tyrosine kinase activity. The constitutively activated BCR/ABL-kinase “escapes” the auto-inhibition mechanisms of c-ABL, such as allosteric inhibition. The ABL-kinase inhibitors (AKIs Imatinib, Nilotinib or Dasatinib, which target the ATP-binding site, are effective in Ph + leukemia. Another molecular therapy approach targeting BCR/ABL restores allosteric inhibition. Given the fact that all AKIs fail to inhibit BCR/ABL harboring the ‘gatekeeper’ mutation T315I, we investigated the effects of AKIs in combination with the allosteric inhibitor GNF2 in Ph + leukemia. Methods The efficacy of this approach on the leukemogenic potential of BCR/ABL was studied in Ba/F3 cells, primary murine bone marrow cells, and untransformed Rat-1 fibroblasts expressing BCR/ABL or BCR/ABL-T315I as well as in patient-derived long-term cultures (PDLTC from Ph + ALL-patients. Results Here, we show that GNF-2 increased the effects of AKIs on unmutated BCR/ABL. Interestingly, the combination of Dasatinib and GNF-2 overcame resistance of BCR/ABL-T315I in all models used in a synergistic manner. Conclusions Our observations establish a new approach for the molecular targeting of BCR/ABL and its resistant mutants using a combination of AKIs and allosteric inhibitors.

  10. Disrupting BCR-ABL in combination with secondary leukemia-specific pathways in CML cells leads to enhanced apoptosis and decreased proliferation.

    Science.gov (United States)

    Woessner, David W; Lim, Carol S

    2013-01-07

    Chronic myeloid leukemia (CML) is a myeloproliferative disorder caused by expression of the fusion gene BCR-ABL following a chromosomal translocation in the hematopoietic stem cell. Therapeutic management of CML uses tyrosine kinase inhibitors (TKIs), which block ABL-signaling and effectively kill peripheral cells with BCR-ABL. However, TKIs are not curative, and chronic use is required in order to treat CML. The primary failure for TKIs is through the development of a resistant population due to mutations in the TKI binding regions. This led us to develop the mutant coiled-coil, CC(mut2), an alternative method for BCR-ABL signaling inhibition by targeting the N-terminal oligomerization domain of BCR, necessary for ABL activation. In this article, we explore additional pathways that are important for leukemic stem cell survival in K562 cells. Using a candidate-based approach, we test the combination of CC(mut2) and inhibitors of unique secondary pathways in leukemic cells. Transformative potential was reduced following silencing of the leukemic stem cell factor Alox5 by RNA interference. Furthermore, blockade of the oncogenic protein MUC-1 by the novel peptide GO-201 yielded reductions in proliferation and increased cell death. Finally, we found that inhibiting macroautophagy using chloroquine in addition to blocking BCR-ABL signaling with the CC(mut2) was most effective in limiting cell survival and proliferation. This study has elucidated possible combination therapies for CML using novel blockade of BCR-ABL and secondary leukemia-specific pathways.

  11. Mapping of four distinct BCR-related loci to chromosome region 22q11: order of BCR loci relative to chronic myelogenous leukemia and acute lymphoblastic leukemia breakpoints

    International Nuclear Information System (INIS)

    Croce, C.M.; Huebner, K.; Isobe, M.; Fainstain, E.; Lifshitz, B.; Shtivelman, E.; Canaani, E.

    1987-01-01

    A probe derived from the 3' region of the BCR gene (breakpoint cluster region gene) detects four distinct loci in the human genome. One of the loci corresponds to the complete BCR gene, whereas the other contain a 3' segment of the gene. After HindIII cleavage of human DNA, these four loci are detected as 23-, 19-, 13-, and 9-kikobase-pair fragments, designated BCR4, BCR3, BCR2, and BCR1, respectively, with BCR1 deriving from the original complete BCR gene. All four BCR loci segregate 100% concordantly with human chromosome 22 in a rodent-human somatic cell hybrid panel and are located at chromosome region 22q11.2 by chromosomal in situ hybridization. The BCR2 and BCR4 loci are amplified in leukemia cell line K562 cells, indicating that they fall within the amplification unit that includes immunoglobulin λ light chain locus (IGL) and ABL locus on the K562 Philadelphia chromosome (Ph 1 ). Similarly, in mouse-human hybrids retaining a Ph 1 chromosome derived from an acute lymphoblastic leukemia-in the absence of the 9q + and 22, only BCR2 and BCR4 loci are retained. Thus, the order of loci on chromosome 22 is centromere → BCR2, BCR4, and IGL → BCR1 → BCR3 → SIS, possibly eliminating BCR2 and BCR4 loci as candidate targets for juxtaposition to the ABL gene in the acute lymphoblastic leukemia Ph 1 chromosome

  12. Protein tyrosine kinases p53/56lyn and p72syk in MHC class I-mediated signal transduction in B lymphoma cells

    DEFF Research Database (Denmark)

    Pedersen, Anders Elm; Bregenholt, S; Skov, S

    1998-01-01

    syk are among the tyrosine-phosphorylated proteins. The kinetics of phosphorylation of these kinases after MHC-I crosslinking differ from the kinetics observed after crosslinking of the B cell antigen receptor (BCR). Additional experiments were performed with chicken lyn- and syk-negative DT40 B cells...... mobilization of intracellular free calcium compared with MHC-I crosslinking of wild-type DT40 cells. Thus, expression of BCR at the cell surface is likely to be important for the signal cascade initiated by MHC-I crosslinking. Our data suggest that signal transduction initiated through ligation of the MHC...

  13. The interleukin-4 receptor: signal transduction by a hematopoietin receptor.

    Science.gov (United States)

    Keegan, A D; Pierce, J H

    1994-02-01

    Over the last several years, the receptors for numerous cytokines have been molecularly characterized. Analysis of their amino acid sequences shows that some of these receptors bear certain motifs in their extracellular domains that define a family of receptors called the Hematopoietin receptor superfamily. Significant advances in characterizing the structure, function, and mechanisms of signal transduction have been made for several members of this family. The purpose of this review is to discuss the recent advances made for one of the family members, the interleukin (IL) 4 receptor. Other receptor systems have recently been reviewed elsewhere. The IL-4 receptor consists of, at the minimum, the cloned 140 kDa IL-4-binding chain with the potential for associating with other chains. The IL-4 receptor transduces its signal by activating a tyrosine kinase that phosphorylates cellular substrates, including the receptor itself, and the 170 kDa substrate called 4PS. Phosphorylated 4PS interacts with the SH2 domain of the enzyme PI-3'-kinase and increases its enzymatic activity. These early events in the IL-4 receptor initiated signaling pathway may trigger a series of signals that will ultimately lead to an IL-4 specific biologic outcome.

  14. The Role of Mitochondrial DNA Damage and Repair in the Resistance of BCR/ABL-Expressing Cells to Tyrosine Kinase Inhibitors

    Directory of Open Access Journals (Sweden)

    Janusz Blasiak

    2013-08-01

    Full Text Available Chronic myeloid leukemia (CML is a hematological malignancy that arises from the transformation of stem hematopoietic cells by the fusion oncogene BCR/ABL and subsequent clonal expansion of BCR/ABL-positive progenitor leukemic cells. The BCR/ABL protein displays a constitutively increased tyrosine kinase activity that alters many regulatory pathways, leading to uncontrolled growth, impaired differentiation and increased resistance to apoptosis featured by leukemic cells. Current CML therapy is based on tyrosine kinase inhibitors (TKIs, primarily imatinib, which induce apoptosis in leukemic cells. However, some patients show primary resistance to TKIs while others develop it in the course of therapy. In both cases, resistance may be underlined by perturbations in apoptotic signaling in leukemic cells. As mitochondria may play an important role in such signaling, alteration in mitochondrial metabolism may change resistance to pro-apoptotic action of TKIs in BCR/ABL-positive cells. Because BCR/ABL may induce reactive oxygen species and unfaithful DNA repair, it may affect the stability of mitochondrial DNA, influencing mitochondrial apoptotic signaling and in this way change the sensitivity of CML cells to TKIs. Moreover, cancer cells, including BCR/ABL-positive cells, show an increased level of glucose metabolism, resulting from the shift from oxidative phosphorylation to glycolysis to supply ATP for extensive proliferation. Enhanced level of glycolysis may be associated with TKI resistance and requires change in the expression of several genes regulated mostly by hypoxia-inducible factor-1α, HIF-1α. Such regulation may be associated with the impaired mitochondrial respiratory system in CML cells. In summary, mitochondria and mitochondria-associated molecules and pathways may be attractive targets to overcome TKI resistance in CML.

  15. Identification of a new gene regulatory circuit involving B cell receptor activated signaling using a combined analysis of experimental, clinical and global gene expression data

    Science.gov (United States)

    Schrader, Alexandra; Meyer, Katharina; Walther, Neele; Stolz, Ailine; Feist, Maren; Hand, Elisabeth; von Bonin, Frederike; Evers, Maurits; Kohler, Christian; Shirneshan, Katayoon; Vockerodt, Martina; Klapper, Wolfram; Szczepanowski, Monika; Murray, Paul G.; Bastians, Holger; Trümper, Lorenz; Spang, Rainer; Kube, Dieter

    2016-01-01

    To discover new regulatory pathways in B lymphoma cells, we performed a combined analysis of experimental, clinical and global gene expression data. We identified a specific cluster of genes that was coherently expressed in primary lymphoma samples and suppressed by activation of the B cell receptor (BCR) through αIgM treatment of lymphoma cells in vitro. This gene cluster, which we called BCR.1, includes numerous cell cycle regulators. A reduced expression of BCR.1 genes after BCR activation was observed in different cell lines and also in CD10+ germinal center B cells. We found that BCR activation led to a delayed entry to and progression of mitosis and defects in metaphase. Cytogenetic changes were detected upon long-term αIgM treatment. Furthermore, an inverse correlation of BCR.1 genes with c-Myc co-regulated genes in distinct groups of lymphoma patients was observed. Finally, we showed that the BCR.1 index discriminates activated B cell-like and germinal centre B cell-like diffuse large B cell lymphoma supporting the functional relevance of this new regulatory circuit and the power of guided clustering for biomarker discovery. PMID:27166259

  16. Transforming and tumorigenic activity of JAK2 by fusion to BCR: molecular mechanisms of action of a novel BCR-JAK2 tyrosine-kinase.

    Directory of Open Access Journals (Sweden)

    Álvaro Cuesta-Domínguez

    Full Text Available Chromosomal translocations in tumors frequently produce fusion genes coding for chimeric proteins with a key role in oncogenesis. Recent reports described a BCR-JAK2 fusion gene in fatal chronic and acute myeloid leukemia, but the functional behavior of the chimeric protein remains uncharacterized. We used fluorescence in situ hybridization and reverse transcription polymerase chain reaction (RT-PCR assays to describe a BCR-JAK2 fusion gene from a patient with acute lymphoblastic leukemia. The patient has been in complete remission for six years following treatment and autologous transplantation, and minimal residual disease was monitored by real-time RT-PCR. BCR-JAK2 codes for a protein containing the BCR oligomerization domain fused to the JAK2 tyrosine-kinase domain. In vitro analysis of transfected cells showed that BCR-JAK2 is located in the cytoplasm. Transduction of hematopoietic Ba/F3 cells with retroviral vectors carrying BCR-JAK2 induced IL-3-independent cell growth, constitutive activation of the chimeric protein as well as STAT5 phosphorylation and translocation to the nuclei, where Bcl-xL gene expression was elicited. Primary mouse progenitor cells transduced with BCR-JAK2 also showed increased proliferation and survival. Treatment with the JAK2 inhibitor TG101209 abrogated BCR-JAK2 and STAT5 phosphorylation, decreased Bcl-xL expression and triggered apoptosis of transformed Ba/F3 cells. Therefore, BCR-JAK2 is a novel tyrosine-kinase with transforming activity. It deregulates growth factor-dependent proliferation and cell survival, which can be abrogated by the TG101209 inhibitor. Moreover, transformed Ba/F3 cells developed tumors when injected subcutaneously into nude mice, thus proving the tumorigenic capacity of BCR-JAK2 in vivo. Together these findings suggest that adult and pediatric patients with BCR-ABL-negative leukemia and JAK2 overexpression may benefit from targeted therapies.

  17. Modeling of molecular interaction between apoptin, BCR-Abl and CrkL--an alternative approach to conventional rational drug design.

    Directory of Open Access Journals (Sweden)

    Soumya Panigrahi

    Full Text Available In this study we have calculated a 3D structure of apoptin and through modeling and docking approaches, we show its interaction with Bcr-Abl oncoprotein and its downstream signaling components, following which we confirm some of the newly-found interactions by biochemical methods. Bcr-Abl oncoprotein is aberrantly expressed in chronic myelogenous leukaemia (CML. It has several distinct functional domains in addition to the Abl kinase domain. The SH3 and SH2 domains cooperatively play important roles in autoinhibiting its kinase activity. Adapter molecules such as Grb2 and CrkL interact with proline-rich region and activate multiple Bcr-Abl downstream signaling pathways that contribute to growth and survival. Therefore, the oncogenic effect of Bcr-Abl could be inhibited by the interaction of small molecules with these domains. Apoptin is a viral protein with well-documented cancer-selective cytotoxicity. Apoptin attributes such as SH2-like sequence similarity with CrkL SH2 domain, unique SH3 domain binding sequence, presence of proline-rich segments, and its nuclear affinity render the molecule capable of interaction with Bcr-Abl. Despite almost two decades of research, the mode of apoptin's action remains elusive because 3D structure of apoptin is unavailable. We performed in silico three-dimensional modeling of apoptin, molecular docking experiments between apoptin model and the known structure of Bcr-Abl, and the 3D structures of SH2 domains of CrkL and Bcr-Abl. We also biochemically validated some of the interactions that were first predicted in silico. This structure-property relationship of apoptin may help in unlocking its cancer-selective toxic properties. Moreover, such models will guide us in developing of a new class of potent apoptin-like molecules with greater selectivity and potency.

  18. Prostaglandin Receptor Signaling in Disease

    Directory of Open Access Journals (Sweden)

    Toshiyuki Matsuoka

    2007-01-01

    Full Text Available Prostanoids, consisting of the prostaglandins (PGs and the thromboxanes (TXs, are a group of lipid mediators formed in response to various stimuli. They include PGD2, PGE2, PGF2α, PGI2, and TXA2. They are released outside of the cells immediately after synthesis, and exert their actions by binding to a G-protein coupled rhodopsin-type receptor on the surface of target cells. There are eight types of the prostanoid receptors conserved in mammals from mouse to human. They are the PGD receptor (DP, four subtypes of the PGE receptor (EP1, EP2, EP3, and EP4, the PGF receptor (FP, PGI receptor (IP, and TXA receptor (TP. Recently, mice deficient in each of these prostanoid receptors were generated and subjected to various experimental models of disease. These studies have revealed the roles of PG receptor signaling in various pathological conditions, and suggest that selective manipulation of the prostanoid receptors may be beneficial in treatment of the pathological conditions. Here we review these recent findings of roles of prostanoid receptor signaling and their therapeutic implications.

  19. Translocation of the B cell receptor to lipid rafts is inhibited in B cells from BLV-infected, persistent lymphocytosis cattle

    International Nuclear Information System (INIS)

    Hamilton, Valerie T.; Stone, Diana M.; Cantor, Glenn H.

    2003-01-01

    Bovine leukemia virus (BLV) infection causes a significant polyclonal expansion of CD5 + , IgM+ B lymphocytes known as persistent lymphocytosis (PL) in approximately 30% of infected cattle. There is evidence that this expanded B cell population has altered signaling, and resistance to apoptosis has been proposed as one mechanism of B cell expansion. In human and murine B cells, antigen binding initiates movement of the B cell receptor (BCR) into membrane microdomains enriched in sphingolipids and cholesterol, termed lipid rafts. Lipid rafts include members of the Src-family kinases and exclude certain phosphatases. Inclusion of the BCR into lipid rafts plays an important role in regulation of early signaling events and subsequent antigen internalization. Viral proteins may also influence signaling events in lipid rafts. Here we demonstrate that the largely CD5 + B cell population in PL cattle has different mobilization and internalization of the BCR when compared to the largely CD5-negative B cells in BLV-negative cattle. Unlike B cells from BLV-negative cattle, the BCR in B cells of BLV-infected, PL cattle resists movement into lipid rafts upon stimulation and is only weakly internalized. Expression of viral proteins as determined by detection of the BLV transmembrane (TM) envelope glycoprotein gp30 did not alter these events in cells from PL cattle. This exclusion of the BCR from lipid rafts may, in part, explain signaling differences seen between B cells of BLV-infected, PL, and BLV-negative cattle and the resistance to apoptosis speculated to contribute to persistent lymphocytosis

  20. Hypoxia-Like Signatures Induced by BCR-ABL Potentially Alter the Glutamine Uptake for Maintaining Oxidative Phosphorylation.

    Directory of Open Access Journals (Sweden)

    Pallavi Sontakke

    Full Text Available The Warburg effect is probably the most prominent metabolic feature of cancer cells, although little is known about the underlying mechanisms and consequences. Here, we set out to study these features in detail in a number of leukemia backgrounds. The transcriptomes of human CB CD34+ cells transduced with various oncogenes, including BCR-ABL, MLL-AF9, FLT3-ITD, NUP98-HOXA9, STAT5A and KRASG12V were analyzed in detail. Our data indicate that in particular BCR-ABL, KRASG12V and STAT5 could impose hypoxic signaling under normoxic conditions. This coincided with an upregulation of glucose importers SLC2A1/3, hexokinases and HIF1 and 2. NMR-based metabolic profiling was performed in CB CD34+ cells transduced with BCR-ABL versus controls, both cultured under normoxia and hypoxia. Lactate and pyruvate levels were increased in BCR-ABL-expressing cells even under normoxia, coinciding with enhanced glutaminolysis which occurred in an HIF1/2-dependent manner. Expression of the glutamine importer SLC1A5 was increased in BCR-ABL+ cells, coinciding with an increased susceptibility to the glutaminase inhibitor BPTES. Oxygen consumption rates also decreased upon BPTES treatment, indicating a glutamine dependency for oxidative phosphorylation. The current study suggests that BCR-ABL-positive cancer cells make use of enhanced glutamine metabolism to maintain TCA cell cycle activity in glycolytic cells.

  1. BCR-ABL fusion genes are inducible by X-irradiation in vitro

    International Nuclear Information System (INIS)

    Ito, Takashi; Seyama, Toshio; Mizuno, Terumi; Hayashi, Tomonori; Nakamura, Nori; Akiyama, Mitoshi; Dohi, Kiyohiko.

    1992-01-01

    The Philadelphia chromosome consists of a reciprocal translocation between the ABL oncogene at chromosome 9q34 and the BCR gene at chromosome 22q resulting in the expression of chimeric BCR-ABL mRNAs specific to chronic myelogenous leukemia (CML). The presence of the fusion genes can be detected with high specificity and sensitivity by means of reverse transcription and polymerase chain reaction. Using this assay, it was possible to detect BCR-ABL fusion genes induced among HL60 cells after 100 Gy of X-irradiation in vitro. A total of five fusion gene transcripts were obtained. These fusion genes contained not only CML-specific BCR-ABL rearrangements, but also other forms of BCR-ABL fusions. These latter genes had junctions of BCR exon 4/ABL exon 2 intervened by a segment of DNA of unknown origin, BCR exon 5/ABL exon 2, and BCR exon 4/ABL exon 2. The results appear to be the first evidence for the induction of the BCR-ABL fusion gene by X-irradiation. In terms of leukemogenesis, it is suggested that only those cells bearing certain CML-related BCR-ABL fusion genes are positively selected by virtue of a growth advantage in vivo. (author)

  2. Muscarinic Receptor Signaling in Colon Cancer

    Energy Technology Data Exchange (ETDEWEB)

    Rosenvinge, Erik C. von, E-mail: evonrose@medicine.umaryland.edu; Raufman, Jean-Pierre [University of Maryland School of Medicine, Division of Gastroenterology & Hepatology, 22 S. Greene Street, N3W62, Baltimore, MD 21201 (United States); Department of Veterans Affairs, VA Maryland Health Care System, 10 North Greene Street, Baltimore, MD 21201 (United States)

    2011-03-02

    According to the adenoma-carcinoma sequence, colon cancer results from accumulating somatic gene mutations; environmental growth factors accelerate and augment this process. For example, diets rich in meat and fat increase fecal bile acids and colon cancer risk. In rodent cancer models, increased fecal bile acids promote colon dysplasia. Conversely, in rodents and in persons with inflammatory bowel disease, low-dose ursodeoxycholic acid treatment alters fecal bile acid composition and attenuates colon neoplasia. In the course of elucidating the mechanism underlying these actions, we discovered that bile acids interact functionally with intestinal muscarinic receptors. The present communication reviews muscarinic receptor expression in normal and neoplastic colon epithelium, the role of autocrine signaling following synthesis and release of acetylcholine from colon cancer cells, post-muscarinic receptor signaling including the role of transactivation of epidermal growth factor receptors and activation of the ERK and PI3K/AKT signaling pathways, the structural biology and metabolism of bile acids and evidence for functional interaction of bile acids with muscarinic receptors on human colon cancer cells. In murine colon cancer models, deficiency of subtype 3 muscarinic receptors attenuates intestinal neoplasia; a proof-of-concept supporting muscarinic receptor signaling as a therapeutic target for colon cancer.

  3. Muscarinic Receptor Signaling in Colon Cancer

    International Nuclear Information System (INIS)

    Rosenvinge, Erik C. von; Raufman, Jean-Pierre

    2011-01-01

    According to the adenoma-carcinoma sequence, colon cancer results from accumulating somatic gene mutations; environmental growth factors accelerate and augment this process. For example, diets rich in meat and fat increase fecal bile acids and colon cancer risk. In rodent cancer models, increased fecal bile acids promote colon dysplasia. Conversely, in rodents and in persons with inflammatory bowel disease, low-dose ursodeoxycholic acid treatment alters fecal bile acid composition and attenuates colon neoplasia. In the course of elucidating the mechanism underlying these actions, we discovered that bile acids interact functionally with intestinal muscarinic receptors. The present communication reviews muscarinic receptor expression in normal and neoplastic colon epithelium, the role of autocrine signaling following synthesis and release of acetylcholine from colon cancer cells, post-muscarinic receptor signaling including the role of transactivation of epidermal growth factor receptors and activation of the ERK and PI3K/AKT signaling pathways, the structural biology and metabolism of bile acids and evidence for functional interaction of bile acids with muscarinic receptors on human colon cancer cells. In murine colon cancer models, deficiency of subtype 3 muscarinic receptors attenuates intestinal neoplasia; a proof-of-concept supporting muscarinic receptor signaling as a therapeutic target for colon cancer

  4. Muscarinic Receptor Signaling in Colon Cancer

    Directory of Open Access Journals (Sweden)

    Jean-Pierre Raufman

    2011-03-01

    Full Text Available According to the adenoma-carcinoma sequence, colon cancer results from accumulating somatic gene mutations; environmental growth factors accelerate and augment this process. For example, diets rich in meat and fat increase fecal bile acids and colon cancer risk. In rodent cancer models, increased fecal bile acids promote colon dysplasia. Conversely, in rodents and in persons with inflammatory bowel disease, low-dose ursodeoxycholic acid treatment alters fecal bile acid composition and attenuates colon neoplasia. In the course of elucidating the mechanism underlying these actions, we discovered that bile acids interact functionally with intestinal muscarinic receptors. The present communication reviews muscarinic receptor expression in normal and neoplastic colon epithelium, the role of autocrine signaling following synthesis and release of acetylcholine from colon cancer cells, post-muscarinic receptor signaling including the role of transactivation of epidermal growth factor receptors and activation of the ERK and PI3K/AKT signaling pathways, the structural biology and metabolism of bile acids and evidence for functional interaction of bile acids with muscarinic receptors on human colon cancer cells. In murine colon cancer models, deficiency of subtype 3 muscarinic receptors attenuates intestinal neoplasia; a proof-of-concept supporting muscarinic receptor signaling as a therapeutic target for colon cancer.

  5. Activity of the novel BCR kinase inhibitor IQS019 in preclinical models of B-cell non-Hodgkin lymphoma

    Directory of Open Access Journals (Sweden)

    P. Balsas

    2017-03-01

    Full Text Available Abstract Background Pharmacological inhibition of B cell receptor (BCR signaling has recently emerged as an effective approach in a wide range of B lymphoid neoplasms. However, despite promising clinical activity of the first Bruton’s kinase (Btk and spleen tyrosine kinase (Syk inhibitors, a small fraction of patients tend to develop progressive disease after initial response to these agents. Methods We evaluated the antitumor activity of IQS019, a new BCR kinase inhibitor with increased affinity for Btk, Syk, and Lck/Yes novel tyrosine kinase (Lyn, in a set of 34 B lymphoid cell lines and primary cultures, including samples with acquired resistance to the first-in-class Btk inhibitor ibrutinib. Safety and efficacy of the compound were then evaluated in two xenograft mouse models of B cell lymphoma. Results IQS019 simultaneously engaged a rapid and dose-dependent de-phosphorylation of both constitutive and IgM-activated Syk, Lyn, and Btk, leading to impaired cell proliferation, reduced CXCL12-dependent cell migration, and induction of caspase-dependent apoptosis. Accordingly, B cell lymphoma-bearing mice receiving IQS019 presented a reduced tumor outgrowth characterized by a decreased mitotic index and a lower infiltration of malignant cells in the spleen, in tight correlation with downregulation of phospho-Syk, phospho-Lyn, and phospho-Btk. More interestingly, IQS019 showed improved efficacy in vitro and in vivo when compared to the first-in-class Btk inhibitor ibrutinib, and was active in cells with acquired resistance to this latest. Conclusions These results define IQS019 as a potential drug candidate for a variety of B lymphoid neoplasms, including cases with acquired resistance to current BCR-targeting therapies.

  6. Various domains of the B-cell regulatory molecule CD72 has diverged at different rates in mammals

    DEFF Research Database (Denmark)

    Petersen, Cathrine Bie; Hillig, Ann-Britt Nygaard; Fredholm, Merete

    2007-01-01

    72 has been shown to be a negatively regulating BCR co-receptor. We isolated and sequenced three porcine CD72 transcript variants. Using a pig radiation hybrid panel we found the porcien CD72 gene to be located on chromosome 1q21-28 in a region syntenic to human chromosome 9. The porcine CD72 gene......We report the cloning of the porcine B-cell co-receptor CD72, as well as genomic mapping and examination of transcription. The B-cell receptor (BCR) complex mediates signalling upon antigen recognition by the membrane bound BCR. Several co-receptors modulate this signal positively or negatively. CD...

  7. Androgen Receptor Signaling in Bladder Cancer

    OpenAIRE

    Li, Peng; Chen, Jinbo; Miyamoto, Hiroshi

    2017-01-01

    Emerging preclinical findings have indicated that steroid hormone receptor signaling plays an important role in bladder cancer outgrowth. In particular, androgen-mediated androgen receptor signals have been shown to correlate with the promotion of tumor development and progression, which may clearly explain some sex-specific differences in bladder cancer. This review summarizes and discusses the available data, suggesting the involvement of androgens and/or the androgen receptor pathways in u...

  8. Regulatory effects of sestrin 3 (SESN3 in BCR-ABL expressing cells.

    Directory of Open Access Journals (Sweden)

    Eliza Vakana

    Full Text Available Chronic myeloid leukemia (CML and Ph+ acute lymphoblastic leukemia (ALL are characterized by the presence of the BCR-ABL oncoprotein, which leads to activation of a plethora of pro-mitogenic and pro-survival pathways, including the mTOR signaling cascade. We provide evidence that in BCR-ABL expressing cells, treatment with tyrosine kinase inhibitors (TKIs results in upregulation of mRNA levels and protein expression of sestrin3 (SESN3, a unique cellular inhibitor of mTOR complex 1 (mTORC1. Such upregulation appears to be mediated by regulatory effects on mTOR, as catalytic inhibition of the mTOR kinase also induces SESN3. Catalytic mTOR inhibition also results in upregulation of SESN3 expression in cells harboring the TKI-insensitive T315I-BCR-ABL mutant, which is resistant to imatinib mesylate. Overexpression of SESN3 results in inhibitory effects on different Ph+ leukemic cell lines including KT-1-derived leukemic precursors, indicating that SESN3 mediates anti-leukemic responses in Ph+ cells. Altogether, our findings suggest the existence of a novel mechanism for the generation of antileukemic responses in CML cells, involving upregulation of SESN3 expression.

  9. Probing Biased Signaling in Chemokine Receptors

    DEFF Research Database (Denmark)

    Amarandi, Roxana Maria; Hjortø, Gertrud Malene; Rosenkilde, Mette Marie

    2016-01-01

    The chemokine system mediates leukocyte migration during homeostatic and inflammatory processes. Traditionally, it is described as redundant and promiscuous, with a single chemokine ligand binding to different receptors and a single receptor having several ligands. Signaling of chemokine receptors...... of others has been termed signaling bias and can accordingly be grouped into ligand bias, receptor bias, and tissue bias. Bias has so far been broadly overlooked in the process of drug development. The low number of currently approved drugs targeting the chemokine system, as well as the broad range...... of failed clinical trials, reflects the need for a better understanding of the chemokine system. Thus, understanding the character, direction, and consequence of biased signaling in the chemokine system may aid the development of new therapeutics. This review describes experiments to assess G protein...

  10. Blockade of Y177 and Nuclear Translocation of Bcr-Abl Inhibits Proliferation and Promotes Apoptosis in Chronic Myeloid Leukemia Cells.

    Science.gov (United States)

    Li, Qianyin; Huang, Zhenglan; Gao, Miao; Cao, Weixi; Xiao, Qin; Luo, Hongwei; Feng, Wenli

    2017-03-02

    The gradual emerging of resistance to imatinib urgently calls for the development of new therapy for chronic myeloid leukemia (CML). The fusion protein Bcr-Abl, which promotes the malignant transformation of CML cells, is mainly located in the cytoplasm, while the c-Abl protein which is expressed in the nucleus can induce apoptosis. Based on the hetero-dimerization of FKBP (the 12-kDa FK506- and rapamycin-binding protein) and FRB (the FKBP-rapamycin binding domain of the protein kinase, mTOR) mediated by AP21967, we constructed a nuclear transport system to induce cytoplasmic Bcr-Abl into nuclear. In this study, we reported the construction of the nuclear transport system, and we demonstrated that FN3R (three nuclear localization signals were fused to FRBT2098L with a FLAG tag), HF2S (two FKBP domains were in tandem and fused to the SH2 domain of Grb2 with an HA tag) and Bcr-Abl form a complexus upon AP21967. Bcr-Abl was imported into the nucleus successfully by the nuclear transport system. The nuclear transport system inhibited CML cell proliferation through mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription 5 (STAT5) pathways mainly by HF2S. It was proven that nuclear located Bcr-Abl induced CML cell (including imatinib-resistant K562G01 cells) apoptosis by activation of p73 and its downstream molecules. In summary, our study provides a new targeted therapy for the CML patients even with Tyrosine Kinase Inhibitor (TKI)-resistance.

  11. Blockade of Y177 and Nuclear Translocation of Bcr-Abl Inhibits Proliferation and Promotes Apoptosis in Chronic Myeloid Leukemia Cells

    Directory of Open Access Journals (Sweden)

    Qianyin Li

    2017-03-01

    Full Text Available The gradual emerging of resistance to imatinib urgently calls for the development of new therapy for chronic myeloid leukemia (CML. The fusion protein Bcr-Abl, which promotes the malignant transformation of CML cells, is mainly located in the cytoplasm, while the c-Abl protein which is expressed in the nucleus can induce apoptosis. Based on the hetero-dimerization of FKBP (the 12-kDa FK506- and rapamycin-binding protein and FRB (the FKBP-rapamycin binding domain of the protein kinase, mTOR mediated by AP21967, we constructed a nuclear transport system to induce cytoplasmic Bcr-Abl into nuclear. In this study, we reported the construction of the nuclear transport system, and we demonstrated that FN3R (three nuclear localization signals were fused to FRBT2098L with a FLAG tag, HF2S (two FKBP domains were in tandem and fused to the SH2 domain of Grb2 with an HA tag and Bcr-Abl form a complexus upon AP21967. Bcr-Abl was imported into the nucleus successfully by the nuclear transport system. The nuclear transport system inhibited CML cell proliferation through mitogen-activated protein kinase (MAPK and signal transducer and activator of transcription 5 (STAT5 pathways mainly by HF2S. It was proven that nuclear located Bcr-Abl induced CML cell (including imatinib-resistant K562G01 cells apoptosis by activation of p73 and its downstream molecules. In summary, our study provides a new targeted therapy for the CML patients even with Tyrosine Kinase Inhibitor (TKI-resistance.

  12. Next generation sequencing reveals skewing of the T and B cell receptor repertoires in patients with Wiskott Aldrich syndrome

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    Amy E O'Connell

    2014-07-01

    Full Text Available The Wiskott Aldrich syndrome (WAS is due to mutations of the WAS gene encoding for the cytoskeletal WAS protein (WASp, leading to abnormal downstream signaling from the T cell and B cell antigen receptors (TCR, BCR. We hypothesized that the impaired signaling through the TCR and BCR in WAS would subsequently lead to aberrations in the immune repertoire of WAS patients. Using next generation sequencing, the T cell receptor beta (TRB and B cell immunoglobulin heavy chain (IGH repertoires of 8 patients with WAS and 6 controls were sequenced. Clonal expansions were identified within memory CD4+ cells, as well as in total, naïve and memory CD8+ cells from WAS patients. In the B cell compartment, WAS patient IGH repertoires were also clonally expanded and showed skewed usage of IGHV and IGHJ genes, and increased usage of IGHG constant genes, compared with controls. To our knowledge, this is the first study that demonstrates significant abnormalities of the immune repertoire in WAS patients using next generation sequencing.

  13. The Bcr-Abl kinase inhibitor INNO-406 induces autophagy and different modes of cell death execution in Bcr-Abl-positive leukemias.

    Science.gov (United States)

    Kamitsuji, Y; Kuroda, J; Kimura, S; Toyokuni, S; Watanabe, K; Ashihara, E; Tanaka, H; Yui, Y; Watanabe, M; Matsubara, H; Mizushima, Y; Hiraumi, Y; Kawata, E; Yoshikawa, T; Maekawa, T; Nakahata, T; Adachi, S

    2008-11-01

    Bcr-Abl tyrosine kinase (TK) inhibitors are promising therapeutic agents for Bcr-Abl-positive (Bcr-Abl(+)) leukemias. Although they are known to promote caspase-mediated apoptosis, it remains unclear whether caspase-independent cell death-inducing mechanisms are also triggered. Here we demonstrated that INNO-406, a second-generation Bcr-Abl TK inhibitor, induces programmed cell death (PCD) in chronic myelogenous leukemia (CML) cell lines through both caspase-mediated and caspase-independent pathways. The latter pathways include caspase-independent apoptosis (CIA) and necrosis-like cell death (CIND), and the cell lines varied regarding which mechanism was elicited upon INNO-406 treatment. We also observed that the propensity toward CIA or CIND in cells was strongly associated with cellular dependency on apoptosome-mediated caspase activity. Cells that undergo CIND have a high apoptosome activity potential whereas cells that undergo CIA tend to have a lower potential. Moreover, we found that INNO-406 promotes autophagy. When autophagy was inhibited with chloroquine or gene knockdown of beclin1 by shRNA, INNO-406-induced cell death was enhanced, which indicates that the autophagic response of the tumor cells is protective. These findings suggest new insights into the biology and therapy of Bcr-Abl(+) leukemias.

  14. Disrupting BCR-ABL in Combination with Secondary Leukemia-Specific Pathways in CML Cells Leads to Enhanced Apoptosis and Decreased Proliferation

    OpenAIRE

    Woessner, David W.; Lim, Carol S.

    2012-01-01

    Chronic myeloid leukemia (CML) is a myeloproliferative disorder caused by expression of the fusion gene BCR-ABL following a chromosomal translocation in the hematopoietic stem cell.1 Therapeutic management of CML uses tyrosine kinase inhibitors (TKIs), which blocks ABL-signaling and effectively kill peripheral cells with BCR-ABL. However, TKIs are not curative, and chronic use of is required in order to treat CML. The primary failure for TKIs is through development of a resistant population d...

  15. Chronic Lymphocytic Leukemia with Mutated IGHV4-34 Receptors

    DEFF Research Database (Denmark)

    Xochelli, Aliki; Baliakas, Panagiotis; Kavakiotis, Ioannis

    2017-01-01

    Purpose: We sought to investigate whether B cell receptor immunoglobulin (BcR IG) stereotypy is associated with particular clinicobiological features among chronic lymphocytic leukemia (CLL) patients expressing mutated BcR IG (M-CLL) encoded by the IGHV4-34 gene, and also ascertain whether...

  16. Plant cell surface receptor-mediated signaling - a common theme amid diversity.

    Science.gov (United States)

    He, Yunxia; Zhou, Jinggeng; Shan, Libo; Meng, Xiangzong

    2018-01-29

    Sessile plants employ a diverse array of plasma membrane-bound receptors to perceive endogenous and exogenous signals for regulation of plant growth, development and immunity. These cell surface receptors include receptor-like kinases (RLKs) and receptor-like proteins (RLPs) that harbor different extracellular domains for perception of distinct ligands. Several RLK and RLP signaling pathways converge at the somatic embryogenesis receptor kinases (SERKs), which function as shared co-receptors. A repertoire of receptor-like cytoplasmic kinases (RLCKs) associate with the receptor complexes to relay intracellular signaling. Downstream of the receptor complexes, mitogen-activated protein kinase (MAPK) cascades are among the key signaling modules at which the signals converge, and these cascades regulate diverse cellular and physiological responses through phosphorylation of different downstream substrates. In this Review, we summarize the emerging common theme that underlies cell surface receptor-mediated signaling pathways in Arabidopsis thaliana : the dynamic association of RLKs and RLPs with specific co-receptors and RLCKs for signal transduction. We further discuss how signaling specificities are maintained through modules at which signals converge, with a focus on SERK-mediated receptor signaling. © 2018. Published by The Company of Biologists Ltd.

  17. Androgen Receptor Signaling in Bladder Cancer

    Science.gov (United States)

    Li, Peng; Chen, Jinbo; Miyamoto, Hiroshi

    2017-01-01

    Emerging preclinical findings have indicated that steroid hormone receptor signaling plays an important role in bladder cancer outgrowth. In particular, androgen-mediated androgen receptor signals have been shown to correlate with the promotion of tumor development and progression, which may clearly explain some sex-specific differences in bladder cancer. This review summarizes and discusses the available data, suggesting the involvement of androgens and/or the androgen receptor pathways in urothelial carcinogenesis as well as tumor growth. While the precise mechanisms of the functions of the androgen receptor in urothelial cells remain far from being fully understood, current evidence may offer chemopreventive or therapeutic options, using androgen deprivation therapy, in patients with bladder cancer. PMID:28241422

  18. Androgen Receptor Signaling in Bladder Cancer

    Directory of Open Access Journals (Sweden)

    Peng Li

    2017-02-01

    Full Text Available Emerging preclinical findings have indicated that steroid hormone receptor signaling plays an important role in bladder cancer outgrowth. In particular, androgen-mediated androgen receptor signals have been shown to correlate with the promotion of tumor development and progression, which may clearly explain some sex-specific differences in bladder cancer. This review summarizes and discusses the available data, suggesting the involvement of androgens and/or the androgen receptor pathways in urothelial carcinogenesis as well as tumor growth. While the precise mechanisms of the functions of the androgen receptor in urothelial cells remain far from being fully understood, current evidence may offer chemopreventive or therapeutic options, using androgen deprivation therapy, in patients with bladder cancer.

  19. Membrane Trafficking of Death Receptors: Implications on Signalling

    Directory of Open Access Journals (Sweden)

    Wulf Schneider-Brachert

    2013-07-01

    Full Text Available Death receptors were initially recognised as potent inducers of apoptotic cell death and soon ambitious attempts were made to exploit selective ignition of controlled cellular suicide as therapeutic strategy in malignant diseases. However, the complexity of death receptor signalling has increased substantially during recent years. Beyond activation of the apoptotic cascade, involvement in a variety of cellular processes including inflammation, proliferation and immune response was recognised. Mechanistically, these findings raised the question how multipurpose receptors can ensure selective activation of a particular pathway. A growing body of evidence points to an elegant spatiotemporal regulation of composition and assembly of the receptor-associated signalling complex. Upon ligand binding, receptor recruitment in specialized membrane compartments, formation of receptor-ligand clusters and internalisation processes constitute key regulatory elements. In this review, we will summarise the current concepts of death receptor trafficking and its implications on receptor-associated signalling events.

  20. Silencing of BCR/ABL Chimeric Gene in Human Chronic Myelogenous Leukemia Cell Line K562 by siRNA-Nuclear Export Signal Peptide Conjugates.

    Science.gov (United States)

    Shinkai, Yasuhiro; Kashihara, Shinichi; Minematsu, Go; Fujii, Hirofumi; Naemura, Madoka; Kotake, Yojiro; Morita, Yasutaka; Ohnuki, Koichiro; Fokina, Alesya A; Stetsenko, Dmitry A; Filichev, Vyacheslav V; Fujii, Masayuki

    2017-06-01

    Herein we described the synthesis of siRNA-NES (nuclear export signal) peptide conjugates by solid phase fragment coupling and the application of them to silencing of bcr/abl chimeric gene in human chronic myelogenous leukemia cell line K562. Two types of siRNA-NES conjugates were prepared, and both sense strands at 5' ends were covalently linked to a NES peptide derived from TFIIIA and HIV-1 REV, respectively. Significant enhancement of silencing efficiency was observed for both of them. siRNA-TFIIIA NES conjugate suppressed the expression of BCR/ABL gene to 8.3% at 200 nM and 11.6% at 50 nM, and siRNA-HIV-1REV NES conjugate suppressed to 4.0% at 200 nM and 6.3% at 50 nM, whereas native siRNA suppressed to 36.3% at 200 nM and 30.2% at 50 nM. We could also show complex of siRNA-NES conjugate and designed amphiphilic peptide peptideβ7 could be taken up into cells with no cytotoxicity and showed excellent silencing efficiency. We believe that the complex siRNA-NES conjugate and peptideβ7 is a promising candidate for in vivo use and therapeutic applications.

  1. The novel anticancer agent JNJ-26854165 is active in chronic myeloid leukemic cells with unmutated BCR/ABL and T315I mutant BCR/ABL through promoting proteosomal degradation of BCR/ABL proteins.

    Science.gov (United States)

    You, Liangshun; Liu, Hui; Huang, Jian; Xie, Wanzhuo; Wei, Jueying; Ye, Xiujin; Qian, Wenbin

    2017-01-31

    Chronic myeloid leukemia (CML) is a clonal malignant disease caused by the expression of BCR/ABL. MDM2 (human homolog of the murine double minute-2) inhibitors such as Nutlin-3 have been shown to induce apoptosis in a p53-dependent manner in CML cells and sensitize cells to Imatinib. Here, we demonstrate that JNJ-26854165, an inhibitor of MDM2, inhibits proliferation and triggers cell death in a p53-independent manner in various BCR/ABL-expressing cells, which include primary leukemic cells from patients with CML blast crisis and cells expressing the Imatinib-resistant T315I BCR/ABL mutant. The response to JNJ-26854165 is associated with the downregulation of BCR/ABL dependently of proteosome activation. Moreover, in all tested CML cells, with the exception of T315I mutation cells, combining JNJ-26854165 and tyrosine kinase inhibitor (TKI) Imatinib or PD180970 leads to a synergistic effect. In conclusion, our results suggest that JNJ-26854165, used either alone or in combination with TKIs, represents a promising novel targeted approach to overcome TKI resistance and improve patient outcome in CML.

  2. Erythropoietin Receptor Signaling Is Membrane Raft Dependent

    Science.gov (United States)

    McGraw, Kathy L.; Fuhler, Gwenny M.; Johnson, Joseph O.; Clark, Justine A.; Caceres, Gisela C.; Sokol, Lubomir; List, Alan F.

    2012-01-01

    Upon erythropoietin (Epo) engagement, Epo-receptor (R) homodimerizes to activate JAK2 and Lyn, which phosphorylate STAT5. Although recent investigations have identified key negative regulators of Epo-R signaling, little is known about the role of membrane localization in controlling receptor signal fidelity. Here we show a critical role for membrane raft (MR) microdomains in creation of discrete signaling platforms essential for Epo-R signaling. Treatment of UT7 cells with Epo induced MR assembly and coalescence. Confocal microscopy showed that raft aggregates significantly increased after Epo stimulation (mean, 4.3±1.4(SE) vs. 25.6±3.2 aggregates/cell; p≤0.001), accompanied by a >3-fold increase in cluster size (p≤0.001). Raft fraction immunoblotting showed Epo-R translocation to MR after Epo stimulation and was confirmed by fluorescence microscopy in Epo stimulated UT7 cells and primary erythroid bursts. Receptor recruitment into MR was accompanied by incorporation of JAK2, Lyn, and STAT5 and their activated forms. Raft disruption by cholesterol depletion extinguished Epo induced Jak2, STAT5, Akt and MAPK phosphorylation in UT7 cells and erythroid progenitors. Furthermore, inhibition of the Rho GTPases Rac1 or RhoA blocked receptor recruitment into raft fractions, indicating a role for these GTPases in receptor trafficking. These data establish a critical role for MR in recruitment and assembly of Epo-R and signal intermediates into discrete membrane signaling units. PMID:22509308

  3. Receptor downregulation and desensitization enhance the information processing ability of signalling receptors

    Directory of Open Access Journals (Sweden)

    Resat Haluk

    2007-11-01

    Full Text Available Abstract Background In addition to initiating signaling events, the activation of cell surface receptors also triggers regulatory processes that restrict the duration of signaling. Acute attenuation of signaling can be accomplished either via ligand-induced internalization of receptors (endocytic downregulation or via ligand-induced receptor desensitization. These phenomena have traditionally been viewed in the context of adaptation wherein the receptor system enters a refractory state in the presence of sustained ligand stimuli and thereby prevents the cell from over-responding to the ligand. Here we use the epidermal growth factor receptor (EGFR and G-protein coupled receptors (GPCR as model systems to respectively examine the effects of downregulation and desensitization on the ability of signaling receptors to decode time-varying ligand stimuli. Results Using a mathematical model, we show that downregulation and desensitization mechanisms can lead to tight and efficient input-output coupling thereby ensuring synchronous processing of ligand inputs. Frequency response analysis indicates that upstream elements of the EGFR and GPCR networks behave like low-pass filters with the system being able to faithfully transduce inputs below a critical frequency. Receptor downregulation and desensitization increase the filter bandwidth thereby enabling the receptor systems to decode inputs in a wider frequency range. Further, system-theoretic analysis reveals that the receptor systems are analogous to classical mechanical over-damped systems. This analogy enables us to metaphorically describe downregulation and desensitization as phenomena that make the systems more resilient in responding to ligand perturbations thereby improving the stability of the system resting state. Conclusion Our findings suggest that in addition to serving as mechanisms for adaptation, receptor downregulation and desensitization can play a critical role in temporal information

  4. B cell receptor pathway in chronic lymphocytic leukemia: specific role of CC-292

    Directory of Open Access Journals (Sweden)

    Arnason JE

    2014-01-01

    Full Text Available Jon E Arnason,1 Jennifer R Brown21Beth Israel Deaconess Medical Center, 2CLL Center, Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USAAbstract: Chronic lymphocytic leukemia (CLL is the most common adult leukemia. The current treatment paradigm involves the use of chemoimmunotherapy, when patients develop an indication for therapy. With this strategy, a majority of patients will obtain a remission, though cure remains elusive. While treatable, the majority of CLL patients will die of complications of their disease. Recent advances in the understanding of the importance of the B cell receptor (BCR pathway in CLL have led to the development of a number of agents targeting this pathway. In this review, we discuss recent developments in the targeting of the BCR pathway, with a focus on CC-292. CC-292 covalently binds to Bruton's tyrosine kinase, a key mediator of BCR signaling, and has demonstrated preclinical and clinical activity in CLL, with acceptable tolerability. Based on the success of CC-292 and other inhibitors of the BCR pathway, these agents are being investigated in combination with standard therapy, with the hope that they will increase the depth and length of response, without significant toxicity.Keywords: Bruton's tyrosine kinase inhibitor, ibrutinib

  5. Glucocorticoid receptor signaling in health and disease

    Science.gov (United States)

    Kadmiel, Mahita; Cidlowski, John A.

    2013-01-01

    Glucocorticoids are steroid hormones regulated in a circadian and stres-associated manner to maintain various metabolic and homeostatic functions that are necessary for life. Synthetic glucocorticoids are widely prescribed drugs for many conditions including asthma, chronic obstructive pulmonary disease (COPD), and inflammatory disorders of the eye. Research in the last few years has begun to unravel the profound complexity of glucocorticoid signaling and has contributed remarkably to improved therapeutic strategies. Glucocorticoids signal through the glucocorticoid receptor, a member of the superfamily of nuclear receptors, in both genomic and non-genomic ways in almost every tissue in the human body. In this review, we will provide an update on glucocorticoid receptor signaling and highlight the role of GR signaling in physiological and pathophysiological conditions in the major organ systems in the human body. PMID:23953592

  6. Lck is a relevant target in chronic lymphocytic leukaemia cells whose expression variance is unrelated to disease outcome.

    Science.gov (United States)

    Till, Kathleen J; Allen, John C; Talab, Fatima; Lin, Ke; Allsup, David; Cawkwell, Lynn; Bentley, Alison; Ringshausen, Ingo; Duckworth, Andrew D; Pettitt, Andrew R; Kalakonda, Nagesh; Slupsky, Joseph R

    2017-12-01

    Pathogenesis of chronic lymphocytic leukaemia (CLL) is contingent upon antigen receptor (BCR) expressed by malignant cells of this disease. Studies on somatic hypermutation of the antigen binding region, receptor expression levels and signal capacity have all linked BCR on CLL cells to disease prognosis. Our previous work showed that the src-family kinase Lck is a targetable mediator of BCR signalling in CLL cells, and that variance in Lck expression associated with ability of BCR to induce signal upon engagement. This latter finding makes Lck similar to ZAP70, another T-cell kinase whose aberrant expression in CLL cells also associates with BCR signalling capacity, but also different because ZAP70 is not easily pharmacologically targetable. Here we describe a robust method of measuring Lck expression in CLL cells using flow cytometry. However, unlike ZAP70 whose expression in CLL cells predicts prognosis, we find Lck expression and disease outcome in CLL are unrelated despite observations that its inhibition produces effects that biologically resemble the egress phenotype taken on by CLL cells treated with idelalisib. Taken together, our findings provide insight into the pathobiology of CLL to suggest a more complex relationship between expression of molecules within the BCR signalling pathway and disease outcome.

  7. Receptor tyrosine kinase signaling: a view from quantitative proteomics

    DEFF Research Database (Denmark)

    Dengjel, Joern; Kratchmarova, Irina; Blagoev, Blagoy

    2009-01-01

    Growth factor receptor signaling via receptor tyrosine kinases (RTKs) is one of the basic cellular communication principals found in all metazoans. Extracellular signals are transferred via membrane spanning receptors into the cytoplasm, reversible tyrosine phosphorylation being the hallmark of all...

  8. Steroid Hormone Receptor Signals as Prognosticators for Urothelial Tumor

    Directory of Open Access Journals (Sweden)

    Hiroki Ide

    2015-01-01

    Full Text Available There is a substantial amount of preclinical or clinical evidence suggesting that steroid hormone receptor-mediated signals play a critical role in urothelial tumorigenesis and tumor progression. These receptors include androgen receptor, estrogen receptors, glucocorticoid receptor, progesterone receptor, vitamin D receptor, retinoid receptors, peroxisome proliferator-activated receptors, and others including orphan receptors. In particular, studies using urothelial cancer tissue specimens have demonstrated that elevated or reduced expression of these receptors as well as alterations of their upstream or downstream pathways correlates with patient outcomes. This review summarizes and discusses available data suggesting that steroid hormone receptors and related signals serve as biomarkers for urothelial carcinoma and are able to predict tumor recurrence or progression.

  9. Dynamics of the actin cytoskeleton mediates receptor cross talk: An emerging concept in tuning receptor signaling

    Science.gov (United States)

    Mattila, Pieta K.; Batista, Facundo D.

    2016-01-01

    Recent evidence implicates the actin cytoskeleton in the control of receptor signaling. This may be of particular importance in the context of immune receptors, such as the B cell receptor, where dysregulated signaling can result in autoimmunity and malignancy. Here, we discuss the role of the actin cytoskeleton in controlling receptor compartmentalization, dynamics, and clustering as a means to regulate receptor signaling through controlling the interactions with protein partners. We propose that the actin cytoskeleton is a point of integration for receptor cross talk through modulation of protein dynamics and clustering. We discuss the implication of this cross talk via the cytoskeleton for both ligand-induced and low-level constitutive (tonic) signaling necessary for immune cell survival. PMID:26833785

  10. Design principles of nuclear receptor signaling: how complex networking improves signal transduction

    Science.gov (United States)

    Kolodkin, Alexey N; Bruggeman, Frank J; Plant, Nick; Moné, Martijn J; Bakker, Barbara M; Campbell, Moray J; van Leeuwen, Johannes P T M; Carlberg, Carsten; Snoep, Jacky L; Westerhoff, Hans V

    2010-01-01

    The topology of nuclear receptor (NR) signaling is captured in a systems biological graphical notation. This enables us to identify a number of ‘design' aspects of the topology of these networks that might appear unnecessarily complex or even functionally paradoxical. In realistic kinetic models of increasing complexity, calculations show how these features correspond to potentially important design principles, e.g.: (i) cytosolic ‘nuclear' receptor may shuttle signal molecules to the nucleus, (ii) the active export of NRs may ensure that there is sufficient receptor protein to capture ligand at the cytoplasmic membrane, (iii) a three conveyor belts design dissipating GTP-free energy, greatly aids response, (iv) the active export of importins may prevent sequestration of NRs by importins in the nucleus and (v) the unspecific nature of the nuclear pore may ensure signal-flux robustness. In addition, the models developed are suitable for implementation in specific cases of NR-mediated signaling, to predict individual receptor functions and differential sensitivity toward physiological and pharmacological ligands. PMID:21179018

  11. Nonredundant roles of Src-family kinases and Syk in the initiation of B-cell antigen receptor signaling

    Czech Academy of Sciences Publication Activity Database

    Štěpánek, Ondřej; Dráber, Peter; Drobek, Aleš; Hořejší, Václav; Brdička, Tomáš

    2013-01-01

    Roč. 190, č. 4 (2013), s. 1807-1818 ISSN 0022-1767 R&D Projects: GA ČR(CZ) GBP302/12/G101; GA ČR GAP302/12/1712 Institutional support: RVO:68378050 Keywords : BCR signaling * Src family kinases * Syk Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.362, year: 2013

  12. Expression of p190 BCR-ABL fusion gene in a patient with chronic myeloid leukemia Expressão do rearranjo gênico BCR-ABL com ponto de quebra na região menor do gene BCR em um paciente com leucemia mielóide crônica

    Directory of Open Access Journals (Sweden)

    P. V. B. Carvalho

    2003-01-01

    Full Text Available A minority of chronic myeloid leukemia cases have breakpoints in the minor cluster region (m-bcr of the BCR-ABL gene. We report on a patient with Ph-positive and m-bcr breakpoint at diagnosis. She was treated with hydroxyurea and interferon-alpha. Two years later, she developed a lymphoid blast crisis and died shortly after. We discuss herein the different forms of the BCR-ABL oncogene, its products, and the possible influence of them on the clinical outcome of patients with the disease.A leucemia mielóide crônica (LMC é uma doença mieloproliferativa clonal e caracteriza-se pela presença da translocação cromossômica entre os braços longos dos cromossomos 9 e 22, o denominado cromossomo Ph. Esta translocação determina a fusão dos genes BCR e ABL. Os diferentes pontos de quebra no gene BCR determinam a síntese de proteínas com diferentes pesos moleculares pelo gene BCR-ABL. Nós relatamos o caso de uma paciente portadora de LMC com ponto de quebra cromossômico na região menor do gene BCR. Foi tratada com hidroxiuréia e interferon alfa. Dois anos após o diagnóstico desenvolveu crise blástica linfóide e evoluiu rapidamente para o óbito. Nós discutimos nesta apresentação as diferentes formas do gene BCR-ABL e seus produtos e a possível influência dos mesmos na evolução clínica dos pacientes com a doença.

  13. SOCS proteins in regulation of receptor tyrosine kinase signaling

    DEFF Research Database (Denmark)

    Kazi, Julhash U.; Kabir, Nuzhat N.; Flores Morales, Amilcar

    2014-01-01

    Receptor tyrosine kinases (RTKs) are a family of cell surface receptors that play critical roles in signal transduction from extracellular stimuli. Many in this family of kinases are overexpressed or mutated in human malignancies and thus became an attractive drug target for cancer treatment....... The signaling mediated by RTKs must be tightly regulated by interacting proteins including protein-tyrosine phosphatases and ubiquitin ligases. The suppressors of cytokine signaling (SOCS) family proteins are well-known negative regulators of cytokine receptors signaling consisting of eight structurally similar...

  14. Expression of P190 and P210 BCR/ABL1 in normal human CD34(+) cells induces similar gene expression profiles and results in a STAT5-dependent expansion of the erythroid lineage

    DEFF Research Database (Denmark)

    Järås, Marcus; Johnels, Petra; Agerstam, Helena

    2009-01-01

    OBJECTIVE: The P190 and P210 BCR/ABL1 fusion genes are mainly associated with different types of hematologic malignancies, but it is presently unclear whether they are functionally different following expression in primitive human hematopoietic cells. MATERIALS AND METHODS: We investigated...... and systematically compared the effects of retroviral P190 BCR/ABL1 and P210 BCR/ABL1 expression on cell proliferation, differentiation, and global gene expression in human CD34(+) cells from cord blood. RESULTS: Expression of either P190 BCR/ABL1 or P210 BCR/ABL1 resulted in expansion of erythroid cells...... and stimulated erythropoietin-independent burst-forming unit-erythroid colony formation. By using a lentiviral anti-signal transducer and activator of transcription 5 (STAT5) short-hairpin RNA, we found that both P190 BCR/ABL1- and P210 BCR/ABL1-induced erythroid cell expansion were STAT5-dependent. Under...

  15. Cocaine Inhibits Dopamine D2 Receptor Signaling via Sigma-1-D2 Receptor Heteromers

    Science.gov (United States)

    Navarro, Gemma; Moreno, Estefania; Bonaventura, Jordi; Brugarolas, Marc; Farré, Daniel; Aguinaga, David; Mallol, Josefa; Cortés, Antoni; Casadó, Vicent; Lluís, Carmen; Ferre, Sergi

    2013-01-01

    Under normal conditions the brain maintains a delicate balance between inputs of reward seeking controlled by neurons containing the D1-like family of dopamine receptors and inputs of aversion coming from neurons containing the D2-like family of dopamine receptors. Cocaine is able to subvert these balanced inputs by altering the cell signaling of these two pathways such that D1 reward seeking pathway dominates. Here, we provide an explanation at the cellular and biochemical level how cocaine may achieve this. Exploring the effect of cocaine on dopamine D2 receptors function, we present evidence of σ1 receptor molecular and functional interaction with dopamine D2 receptors. Using biophysical, biochemical, and cell biology approaches, we discovered that D2 receptors (the long isoform of the D2 receptor) can complex with σ1 receptors, a result that is specific to D2 receptors, as D3 and D4 receptors did not form heteromers. We demonstrate that the σ1-D2 receptor heteromers consist of higher order oligomers, are found in mouse striatum and that cocaine, by binding to σ1 -D2 receptor heteromers, inhibits downstream signaling in both cultured cells and in mouse striatum. In contrast, in striatum from σ1 knockout animals these complexes are not found and this inhibition is not seen. Taken together, these data illuminate the mechanism by which the initial exposure to cocaine can inhibit signaling via D2 receptor containing neurons, destabilizing the delicate signaling balance influencing drug seeking that emanates from the D1 and D2 receptor containing neurons in the brain. PMID:23637801

  16. BCR CDR3 length distributions differ between blood and spleen and between old and young patients, and TCR distributions can be used to detect myelodysplastic syndrome

    International Nuclear Information System (INIS)

    Pickman, Yishai; Mehr, Ramit; Dunn-Walters, Deborah

    2013-01-01

    Complementarity-determining region 3 (CDR3) is the most hyper-variable region in B cell receptor (BCR) and T cell receptor (TCR) genes, and the most critical structure in antigen recognition and thereby in determining the fates of developing and responding lymphocytes. There are millions of different TCR Vβ chain or BCR heavy chain CDR3 sequences in human blood. Even now, when high-throughput sequencing becomes widely used, CDR3 length distributions (also called spectratypes) are still a much quicker and cheaper method of assessing repertoire diversity. However, distribution complexity and the large amount of information per sample (e.g. 32 distributions of the TCRα chain, and 24 of TCRβ) calls for the use of machine learning tools for full exploration. We have examined the ability of supervised machine learning, which uses computational models to find hidden patterns in predefined biological groups, to analyze CDR3 length distributions from various sources, and distinguish between experimental groups. We found that (a) splenic BCR CDR3 length distributions are characterized by low standard deviations and few local maxima, compared to peripheral blood distributions; (b) healthy elderly people's BCR CDR3 length distributions can be distinguished from those of the young; and (c) a machine learning model based on TCR CDR3 distribution features can detect myelodysplastic syndrome with approximately 93% accuracy. Overall, we demonstrate that using supervised machine learning methods can contribute to our understanding of lymphocyte repertoire diversity. (paper)

  17. BCR CDR3 length distributions differ between blood and spleen and between old and young patients, and TCR distributions can be used to detect myelodysplastic syndrome

    Science.gov (United States)

    Pickman, Yishai; Dunn-Walters, Deborah; Mehr, Ramit

    2013-10-01

    Complementarity-determining region 3 (CDR3) is the most hyper-variable region in B cell receptor (BCR) and T cell receptor (TCR) genes, and the most critical structure in antigen recognition and thereby in determining the fates of developing and responding lymphocytes. There are millions of different TCR Vβ chain or BCR heavy chain CDR3 sequences in human blood. Even now, when high-throughput sequencing becomes widely used, CDR3 length distributions (also called spectratypes) are still a much quicker and cheaper method of assessing repertoire diversity. However, distribution complexity and the large amount of information per sample (e.g. 32 distributions of the TCRα chain, and 24 of TCRβ) calls for the use of machine learning tools for full exploration. We have examined the ability of supervised machine learning, which uses computational models to find hidden patterns in predefined biological groups, to analyze CDR3 length distributions from various sources, and distinguish between experimental groups. We found that (a) splenic BCR CDR3 length distributions are characterized by low standard deviations and few local maxima, compared to peripheral blood distributions; (b) healthy elderly people's BCR CDR3 length distributions can be distinguished from those of the young; and (c) a machine learning model based on TCR CDR3 distribution features can detect myelodysplastic syndrome with approximately 93% accuracy. Overall, we demonstrate that using supervised machine learning methods can contribute to our understanding of lymphocyte repertoire diversity.

  18. Biased and g protein-independent signaling of chemokine receptors

    DEFF Research Database (Denmark)

    Steen, Anne; Larsen, Olav; Thiele, Stefanie

    2014-01-01

    ), different receptors (with the same ligand), or different tissues or cells (for the same ligand-receptor pair). Most often biased signaling is differentiated into G protein-dependent and β-arrestin-dependent signaling. Yet, it may also cover signaling differences within these groups. Moreover, it may...

  19. Nuclear Receptor Signaling Atlas: Opening Access to the Biology of Nuclear Receptor Signaling Pathways.

    Science.gov (United States)

    Becnel, Lauren B; Darlington, Yolanda F; Ochsner, Scott A; Easton-Marks, Jeremy R; Watkins, Christopher M; McOwiti, Apollo; Kankanamge, Wasula H; Wise, Michael W; DeHart, Michael; Margolis, Ronald N; McKenna, Neil J

    2015-01-01

    Signaling pathways involving nuclear receptors (NRs), their ligands and coregulators, regulate tissue-specific transcriptomes in diverse processes, including development, metabolism, reproduction, the immune response and neuronal function, as well as in their associated pathologies. The Nuclear Receptor Signaling Atlas (NURSA) is a Consortium focused around a Hub website (www.nursa.org) that annotates and integrates diverse 'omics datasets originating from the published literature and NURSA-funded Data Source Projects (NDSPs). These datasets are then exposed to the scientific community on an Open Access basis through user-friendly data browsing and search interfaces. Here, we describe the redesign of the Hub, version 3.0, to deploy "Web 2.0" technologies and add richer, more diverse content. The Molecule Pages, which aggregate information relevant to NR signaling pathways from myriad external databases, have been enhanced to include resources for basic scientists, such as post-translational modification sites and targeting miRNAs, and for clinicians, such as clinical trials. A portal to NURSA's Open Access, PubMed-indexed journal Nuclear Receptor Signaling has been added to facilitate manuscript submissions. Datasets and information on reagents generated by NDSPs are available, as is information concerning periodic new NDSP funding solicitations. Finally, the new website integrates the Transcriptomine analysis tool, which allows for mining of millions of richly annotated public transcriptomic data points in the field, providing an environment for dataset re-use and citation, bench data validation and hypothesis generation. We anticipate that this new release of the NURSA database will have tangible, long term benefits for both basic and clinical research in this field.

  20. Nuclear Receptor Signaling Atlas: Opening Access to the Biology of Nuclear Receptor Signaling Pathways.

    Directory of Open Access Journals (Sweden)

    Lauren B Becnel

    Full Text Available Signaling pathways involving nuclear receptors (NRs, their ligands and coregulators, regulate tissue-specific transcriptomes in diverse processes, including development, metabolism, reproduction, the immune response and neuronal function, as well as in their associated pathologies. The Nuclear Receptor Signaling Atlas (NURSA is a Consortium focused around a Hub website (www.nursa.org that annotates and integrates diverse 'omics datasets originating from the published literature and NURSA-funded Data Source Projects (NDSPs. These datasets are then exposed to the scientific community on an Open Access basis through user-friendly data browsing and search interfaces. Here, we describe the redesign of the Hub, version 3.0, to deploy "Web 2.0" technologies and add richer, more diverse content. The Molecule Pages, which aggregate information relevant to NR signaling pathways from myriad external databases, have been enhanced to include resources for basic scientists, such as post-translational modification sites and targeting miRNAs, and for clinicians, such as clinical trials. A portal to NURSA's Open Access, PubMed-indexed journal Nuclear Receptor Signaling has been added to facilitate manuscript submissions. Datasets and information on reagents generated by NDSPs are available, as is information concerning periodic new NDSP funding solicitations. Finally, the new website integrates the Transcriptomine analysis tool, which allows for mining of millions of richly annotated public transcriptomic data points in the field, providing an environment for dataset re-use and citation, bench data validation and hypothesis generation. We anticipate that this new release of the NURSA database will have tangible, long term benefits for both basic and clinical research in this field.

  1. Tyrosine kinase fusion genes in pediatric BCR-ABL1-like acute lymphoblastic leukemia

    Science.gov (United States)

    Boer, Judith M.; Steeghs, Elisabeth M.P.; Marchante, João R.M.; Boeree, Aurélie; Beaudoin, James J.; Berna Beverloo, H.; Kuiper, Roland P.; Escherich, Gabriele; van der Velden, Vincent H.J.; van der Schoot, C. Ellen; de Groot-Kruseman, Hester A.; Pieters, Rob; den Boer, Monique L.

    2017-01-01

    Approximately 15% of pediatric B cell precursor acute lymphoblastic leukemia (BCP-ALL) is characterized by gene expression similar to that of BCR-ABL1-positive disease and unfavorable prognosis. This BCR-ABL1-like subtype shows a high frequency of B-cell development gene aberrations and tyrosine kinase-activating lesions. To evaluate the clinical significance of tyrosine kinase gene fusions in children with BCP-ALL, we studied the frequency of recently identified tyrosine kinase fusions, associated genetic features, and prognosis in a representative Dutch/German cohort. We identified 14 tyrosine kinase fusions among 77 BCR-ABL1-like cases (18%) and none among 76 non-BCR-ABL1-like B-other cases. Novel exon fusions were identified for RCSD1-ABL2 and TERF2-JAK2. JAK2 mutation was mutually exclusive with tyrosine kinase fusions and only occurred in cases with high CRLF2 expression. The non/late response rate and levels of minimal residual disease in the fusion-positive BCR-ABL1-like group were higher than in the non-BCR-ABL1-like B-others (p<0.01), and also higher, albeit not statistically significant, compared with the fusion-negative BCR-ABL1-like group. The 8-year cumulative incidence of relapse in the fusion-positive BCR-ABL1-like group (35%) was comparable with that in the fusion-negative BCR-ABL1-like group (35%), and worse than in the non-BCR-ABL1-like B-other group (17%, p=0.07). IKZF1 deletions, predominantly other than the dominant-negative isoform and full deletion, co-occurred with tyrosine kinase fusions. This study shows that tyrosine kinase fusion-positive cases are a high-risk subtype of BCP-ALL, which warrants further studies with specific kinase inhibitors to improve outcome. PMID:27894077

  2. Translocation of BCR to chromosome 9: A new cytogenetic variant detected by FISH in two Ph-negative, BCR-positive patients with chronic myeloid leukemia

    NARCIS (Netherlands)

    A. Hagemeijer (Anne); A. Buijs (Arjan); E.M.E. Smit (Elisabeth); L.A.J. Janssen (Bart); G.J.M. Creemers (Geert-Jan); D. van der Plas (D.); G.C. Grosveld (Gerard)

    1993-01-01

    textabstractLeukemic cells from two patients with Philadelphia-negative chronic myeloid leukemia (CML) were investigated: I) Cytogenetics showed a normal 46.XY karyotype in both cases, 2) molecular studies revealed rearrangement of the M-BCR region and formation of BCR-ABL fusion mRNA with b2a2

  3. Heterogeneity of BCR-ABL rearrangement in patients with chronic myeloid leukemia in Pakistan.

    Science.gov (United States)

    Tabassum, Najia; Saboor, Mohammad; Ghani, Rubina; Moinuddin, Moinuddin

    2014-07-01

    Breakpoint cluster region-Abelson (BCR-ABL) rearrangement or Philadelphia (Ph) chromosome in Chronic Myeloid Leukemia (CML) is derived from a reciprocal chromosomal translocation between ABL gene on chromosome 9 and BCR gene on chromosome 22. This chimeric protein has various sizes and therefore different clinical behaviour. The purpose of this study was to determine the heterogeneity of BCR-ABL rearrangement in patients with Ph(+)CML in Pakistan. The study was conducted at Civil Hospital and Baqai Institute of Hematology (BIH) Karachi. Blood samples from 25 patients with CML were collected. Multiplex reverse transcription polymerase chain reaction (RT-PCR) was performed to identify various BCR-ABL transcripts. All 25 samples showed BCR-ABL rearrangements. Out of these, 24 (96%) patients expressed p210 BCR-ABL rearrangements i.e. 60% (n=15) had b3a2 and 32% (n=8) had b2a2 rearrangements. Co-expression of b3a2 /b2a2 rearrangement and p190 (e1a3) rearrangement was also identified in two patients. It is apparent that majority of the patients had p210 BCR-ABL rearrangements. Frequency of co-expression and rare fusion transcripts was very low.

  4. Regulation of hTERT by BCR-ABL at multiple levels in K562 cells

    International Nuclear Information System (INIS)

    Chai, Juin Hsien; Zhang, Yong; Tan, Wei Han; Chng, Wee Joo; Li, Baojie; Wang, Xueying

    2011-01-01

    The cytogenetic characteristic of Chronic Myeloid Leukemia (CML) is the formation of the Philadelphia chromosome gene product, BCR-ABL. Given that BCR-ABL is the specific target of Gleevec in CML treatment, we investigated the regulation of the catalytic component of telomerase, hTERT, by BCR-ABL at multiple levels in K562 cells. Molecular techniques such as over expression, knockdown, real-time PCR, immunoprecipitation, western blotting, reporter assay, confocal microscopy, telomerase assays and microarray were used to suggest that hTERT expression and activity is modulated by BCR-ABL at multiple levels. Our results suggest that BCR-ABL plays an important role in regulating hTERT in K562 (BCR-ABL positive human leukemia) cells. When Gleevec inhibited the tyrosine kinase activity of BCR-ABL, phosphorylation of hTERT was downregulated, therefore suggesting a positive correlation between BCR-ABL and hTERT. Gleevec treatment inhibited hTERT at mRNA level and significantly reduced telomerase activity (TA) in K562 cells, but not in HL60 or Jurkat cells (BCR-ABL negative cells). We also demonstrated that the transcription factor STAT5a plays a critical role in hTERT gene regulation in K562 cells. Knockdown of STAT5a, but not STAT5b, resulted in a marked downregulation of hTERT mRNA level, TA and hTERT protein level in K562 cells. Furthermore, translocation of hTERT from nucleoli to nucleoplasm was observed in K562 cells induced by Gleevec. Our data reveal that BCR-ABL can regulate TA at multiple levels, including transcription, post-translational level, and proper localization. Thus, suppression of cell growth and induction of apoptosis by Gleevec treatment may be partially due to TA inhibition. Additionally, we have identified STAT5a as critical mediator of the hTERT gene expression in BCR-ABL positive CML cells, suggesting that targeting STAT5a may be a promising therapeutic strategy for BCR-ABL positive CML patients

  5. Coupling a universal DNA circuit with graphene sheets/polyaniline/AuNPs nanocomposites for the detection of BCR/ABL fusion gene

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Xueping [Key Laboratory of Laboratory Medical Diagnostics of Education, Department of Laboratory Medicine, Chongqing Medical University, Chongqing, 400016 (China); Wang, Li [Key Laboratory of Laboratory Medical Diagnostics of Education, Department of Laboratory Medicine, Chongqing Medical University, Chongqing, 400016 (China); Department of Medical Laboratory, Chongqing Emergency Medical Center (Chongqing The Fourth Hospital), Chongqing, 400016 (China); Sheng, Shangchun [The No.2 Peoples' Hospital of Yibin, Sichuan, 644000 (China); Wang, Teng; Yang, Juan [Key Laboratory of Laboratory Medical Diagnostics of Education, Department of Laboratory Medicine, Chongqing Medical University, Chongqing, 400016 (China); Xie, Guoming, E-mail: guomingxie@cqmu.edu.cn [Key Laboratory of Laboratory Medical Diagnostics of Education, Department of Laboratory Medicine, Chongqing Medical University, Chongqing, 400016 (China); Feng, Wenli, E-mail: fengwlcqmu@sina.com [Key Laboratory of Laboratory Medical Diagnostics of Education, Department of Laboratory Medicine, Chongqing Medical University, Chongqing, 400016 (China)

    2015-08-19

    This article described a novel method by coupling a universal DNA circuit with graphene sheets/polyaniline/AuNPs nanocomposites (GS/PANI/AuNPs) for highly sensitive and specific detection of BCR/ABL fusion gene (bcr/abl) in chronic myeloid leukemia (CML). DNA circuit known as catalyzed hairpin assembly (CHA) is enzyme-free and can be simply operated to achieve exponential amplification, which has been widely employed in biosensing. However, application of CHA has been hindered by the need of specially redesigned sequences for each single-stranded DNA input. Herein, a transducer hairpin (HP) was designed to obtain a universal DNA circuit with favorable signal-to-background ratio. To further improve signal amplification, GS/PANI/AuNPs with excellent conductivity and enlarged effective area were introduced into this DNA circuit. Consequently, by combining the advantages of CHA and GS/PANI/AuNPs, bcr/abl could be detected in a linear range from 10 pM to 20 nM with a detection limit of 1.05 pM. Moreover, this protocol showed excellent specificity, good stability and was successfully applied for the detection of real sample, which demonstrated its great potential in clinical application. - Highlights: • A transducer hairpin was designed to improve the versatility of DNA circuit. • GS/PANI/AuNPs were introduced to the DNA circuit for further signal amplification. • The established biosensor displayed high sensitivity and good specificity.

  6. DMPD: Lysophospholipid receptors: signaling and biology. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 15189145 Lysophospholipid receptors: signaling and biology. Ishii I, Fukushima N, Y...e X, Chun J. Annu Rev Biochem. 2004;73:321-54. (.png) (.svg) (.html) (.csml) Show Lysophospholipid receptors...: signaling and biology. PubmedID 15189145 Title Lysophospholipid receptors: signaling and biology. Authors

  7. Towards the generation of B-cell receptor retrogenic mice.

    Directory of Open Access Journals (Sweden)

    Jenny Freitag

    Full Text Available Transgenic expression of B- and T-cell receptors (BCRs and TCRs, respectively has been a standard tool to study lymphocyte development and function in vivo. The generation of transgenic mice is time-consuming and, therefore, a faster method to study the biology of defined lymphocyte receptors in vivo would be highly welcome. Using 2A peptide-linked multicistronic retroviral vectors to transduce stem cells, TCRs can be expressed rapidly in mice of any background. We aimed at adopting this retrogenic technology to the in vivo expression of BCRs. Using a well characterised BCR specific for hen egg lysozyme (HEL, we achieved surface expression of the retrogenically encoded BCR in a Rag-deficient pro B-cell line in vitro. In vivo, retrogenic BCRs were detectable only intracellularly but not on the surface of B cells from wild type or Rag2-deficient mice. This data, together with the fact that no BCR retrogenic mouse model has been published in the 7 years since the method was originally published for TCRs, strongly suggests that achieving BCR-expression in vivo with retrogenic technology is highly challenging if not impossible.

  8. Nuclear Receptor Signaling Atlas (NURSA)

    Data.gov (United States)

    U.S. Department of Health & Human Services — The Nuclear Receptor Signaling Atlas (NURSA) is designed to foster the development of a comprehensive understanding of the structure, function, and role in disease...

  9. Lack of bcr and abr promotes hypoxia-induced pulmonary hypertension in mice.

    Directory of Open Access Journals (Sweden)

    Min Yu

    Full Text Available Bcr and Abr are GTPase activating proteins that specifically downregulate activity of the small GTPase Rac in restricted cell types in vivo. Rac1 is expressed in smooth muscle cells, a critical cell type involved in the pathogenesis of pulmonary hypertension. The molecular mechanisms that underlie hypoxia-associated pulmonary hypertension are not well-defined.Bcr and abr null mutant mice were compared to wild type controls for the development of pulmonary hypertension after exposure to hypoxia. Also, pulmonary arterial smooth muscle cells from those mice were cultured in hypoxia and examined for proliferation, p38 activation and IL-6 production. Mice lacking Bcr or Abr exposed to hypoxia developed increased right ventricular pressure, hypertrophy and pulmonary vascular remodeling. Perivascular leukocyte infiltration in the lungs was increased, and under hypoxia bcr-/- and abr-/- macrophages generated more reactive oxygen species. Consistent with a contribution of inflammation and oxidative stress in pulmonary hypertension-associated vascular damage, Bcr and Abr-deficient animals showed elevated endothelial leakage after hypoxia exposure. Hypoxia-treated pulmonary arterial smooth muscle cells from Bcr- or Abr-deficient mice also proliferated faster than those of wild type mice. Moreover, activated Rac1, phosphorylated p38 and interleukin 6 were increased in these cells in the absence of Bcr or Abr. Inhibition of Rac1 activation with Z62954982, a novel Rac inhibitor, decreased proliferation, p38 phosphorylation and IL-6 levels in pulmonary arterial smooth muscle cells exposed to hypoxia.Bcr and Abr play a critical role in down-regulating hypoxia-induced pulmonary hypertension by deactivating Rac1 and, through this, reducing both oxidative stress generated by leukocytes as well as p38 phosphorylation, IL-6 production and proliferation of pulmonary arterial smooth muscle cells.

  10. B cell antigen receptor signaling and internalization are mutually exclusive events.

    Directory of Open Access Journals (Sweden)

    Ping Hou

    2006-07-01

    Full Text Available Engagement of the B cell antigen receptor initiates two concurrent processes, signaling and receptor internalization. While both are required for normal humoral immune responses, the relationship between these two processes is unknown. Herein, we demonstrate that following receptor ligation, a small subpopulation of B cell antigen receptors are inductively phosphorylated and selectively retained at the cell surface where they can serve as scaffolds for the assembly of signaling molecules. In contrast, the larger population of non-phosphorylated receptors is rapidly endocytosed. Each receptor can undergo only one of two mutually exclusive fates because the tyrosine-based motifs that mediate signaling when phosphorylated mediate internalization when not phosphorylated. Mathematical modeling indicates that the observed competition between receptor phosphorylation and internalization enhances signaling responses to low avidity ligands.

  11. The Pseudo signal peptide of the corticotropin-releasing factor receptor type 2A prevents receptor oligomerization.

    Science.gov (United States)

    Teichmann, Anke; Rutz, Claudia; Kreuchwig, Annika; Krause, Gerd; Wiesner, Burkhard; Schülein, Ralf

    2012-08-03

    N-terminal signal peptides mediate the interaction of native proteins with the translocon complex of the endoplasmic reticulum membrane and are cleaved off during early protein biogenesis. The corticotropin-releasing factor receptor type 2a (CRF(2(a))R) possesses an N-terminal pseudo signal peptide, which represents a so far unique domain within the large protein family of G protein-coupled receptors (GPCRs). In contrast to a conventional signal peptide, the pseudo signal peptide remains uncleaved and consequently forms a hydrophobic extension at the N terminus of the receptor. The functional consequence of the presence of the pseudo signal peptide is not understood. Here, we have analyzed the significance of this domain for receptor dimerization/oligomerization in detail. To this end, we took the CRF(2(a))R and the homologous corticotropin-releasing factor receptor type 1 (CRF(1)R) possessing a conventional cleaved signal peptide and conducted signal peptide exchange experiments. Using single cell and single molecule imaging methods (fluorescence resonance energy transfer and fluorescence cross-correlation spectroscopy, respectively) as well as biochemical experiments, we obtained two novel findings; we could show that (i) the CRF(2(a))R is expressed exclusively as a monomer, and (ii) the presence of the pseudo signal peptide prevents its oligomerization. Thus, we have identified a novel functional domain within the GPCR protein family, which plays a role in receptor oligomerization and which may be useful to study the functional significance of this process in general.

  12. Lupus risk variants in the PXK locus alter B-cell receptor internalization

    Directory of Open Access Journals (Sweden)

    Samuel E. Vaughn

    2015-01-01

    Full Text Available Genome wide association studies have identified variants in PXK that confer risk for humoral autoimmune diseases, including systemic lupus erythematosus (SLE or lupus, rheumatoid arthritis and more recently systemic sclerosis. While PXK is involved in trafficking of epidermal growth factor Receptor (EGFR in COS-7 cells, mechanisms linking PXK to lupus pathophysiology have remained undefined. In an effort to uncover the mechanism at this locus that increases lupus-risk, we undertook a fine-mapping analysis in a large multi-ancestral study of lupus patients and controls. We define a large (257kb common haplotype that confers lupus risk detected only in European ancestral populations and spans the promoter through the 3’ UTR of PXK. The strongest association was found at rs6445972 with P < 4.62 x 10-10, OR 0.81 (0.75 – 0.86. Using stepwise logistic regression analysis, we demonstrate that one signal drives the genetic association in the region. Bayesian analysis confirms our results, identifying a 95% credible set consisting of 172 variants spanning 200kb.Functionally, we found that PXK operates on the B-cell antigen receptor (BCR; we confirmed that PXK influenced the rate of BCR internalization. Furthermore, we demonstrate that individuals carrying the risk haplotype exhibited a decreased rate of BCR internalization, a process known to impact B cell survival and cell fate. Taken together, these data define a new candidate mechanism for the genetic association of variants around PXK with lupus risk and highlight the regulation of intracellular trafficking as a genetically regulated pathway mediating human autoimmunity.

  13. Peroxisome Proliferator-Activated Receptor and Vitamin D Receptor Signaling Pathways in Cancer Cells

    Directory of Open Access Journals (Sweden)

    Yasuko Kitagishi

    2013-10-01

    Full Text Available Peroxisome proliferator-activated receptors (PPARs are members of the superfamily of nuclear hormone receptors, which respond to specific ligands such as polyunsaturated fatty acids by altering gene expression. Three subtypes of this receptor have been discovered, each evolving to achieve different biological functions. Like other nuclear receptors, the transcriptional activity of PPARs is affected not only by ligand-stimulation, but also by cross-talk with other molecules. For example, both PPARs and the RXRs are ligand-activated transcription factors that coordinately regulate gene expression. In addition, PPARs and vitamin D receptor (VDR signaling pathways regulate a multitude of genes that are of importance for cellular functions including cell proliferation and cell differentiation. Interaction of the PPARs and VDR signaling pathways has been shown at the level of molecular cross-regulation of their transcription factor. A variety of ligands influencing the PPARs and VDR signaling pathways have been shown to reveal chemopreventive potential by mediating tumor suppressive activities in human cancers. Use of these compounds may represent a potential novel strategy to prevent cancers. This review summarizes the roles of the PPARs and the VDR in pathogenesis and progression of cancer.

  14. Peroxisome Proliferator-Activated Receptor and Vitamin D Receptor Signaling Pathways in Cancer Cells

    Energy Technology Data Exchange (ETDEWEB)

    Matsuda, Satoru, E-mail: smatsuda@cc.nara-wu.ac.jp; Kitagishi, Yasuko [Department of Food Science and Nutrition, Nara Women’s University, Kita-Uoya Nishimachi, Nara 630-8506 (Japan)

    2013-10-21

    Peroxisome proliferator-activated receptors (PPARs) are members of the superfamily of nuclear hormone receptors, which respond to specific ligands such as polyunsaturated fatty acids by altering gene expression. Three subtypes of this receptor have been discovered, each evolving to achieve different biological functions. Like other nuclear receptors, the transcriptional activity of PPARs is affected not only by ligand-stimulation, but also by cross-talk with other molecules. For example, both PPARs and the RXRs are ligand-activated transcription factors that coordinately regulate gene expression. In addition, PPARs and vitamin D receptor (VDR) signaling pathways regulate a multitude of genes that are of importance for cellular functions including cell proliferation and cell differentiation. Interaction of the PPARs and VDR signaling pathways has been shown at the level of molecular cross-regulation of their transcription factor. A variety of ligands influencing the PPARs and VDR signaling pathways have been shown to reveal chemopreventive potential by mediating tumor suppressive activities in human cancers. Use of these compounds may represent a potential novel strategy to prevent cancers. This review summarizes the roles of the PPARs and the VDR in pathogenesis and progression of cancer.

  15. Peroxisome Proliferator-Activated Receptor and Vitamin D Receptor Signaling Pathways in Cancer Cells

    International Nuclear Information System (INIS)

    Matsuda, Satoru; Kitagishi, Yasuko

    2013-01-01

    Peroxisome proliferator-activated receptors (PPARs) are members of the superfamily of nuclear hormone receptors, which respond to specific ligands such as polyunsaturated fatty acids by altering gene expression. Three subtypes of this receptor have been discovered, each evolving to achieve different biological functions. Like other nuclear receptors, the transcriptional activity of PPARs is affected not only by ligand-stimulation, but also by cross-talk with other molecules. For example, both PPARs and the RXRs are ligand-activated transcription factors that coordinately regulate gene expression. In addition, PPARs and vitamin D receptor (VDR) signaling pathways regulate a multitude of genes that are of importance for cellular functions including cell proliferation and cell differentiation. Interaction of the PPARs and VDR signaling pathways has been shown at the level of molecular cross-regulation of their transcription factor. A variety of ligands influencing the PPARs and VDR signaling pathways have been shown to reveal chemopreventive potential by mediating tumor suppressive activities in human cancers. Use of these compounds may represent a potential novel strategy to prevent cancers. This review summarizes the roles of the PPARs and the VDR in pathogenesis and progression of cancer

  16. Modulation of β-catenin signaling by glucagon receptor activation.

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    Jiyuan Ke

    Full Text Available The glucagon receptor (GCGR is a member of the class B G protein-coupled receptor family. Activation of GCGR by glucagon leads to increased glucose production by the liver. Thus, glucagon is a key component of glucose homeostasis by counteracting the effect of insulin. In this report, we found that in addition to activation of the classic cAMP/protein kinase A (PKA pathway, activation of GCGR also induced β-catenin stabilization and activated β-catenin-mediated transcription. Activation of β-catenin signaling was PKA-dependent, consistent with previous reports on the parathyroid hormone receptor type 1 (PTH1R and glucagon-like peptide 1 (GLP-1R receptors. Since low-density-lipoprotein receptor-related protein 5 (Lrp5 is an essential co-receptor required for Wnt protein mediated β-catenin signaling, we examined the role of Lrp5 in glucagon-induced β-catenin signaling. Cotransfection with Lrp5 enhanced the glucagon-induced β-catenin stabilization and TCF promoter-mediated transcription. Inhibiting Lrp5/6 function using Dickkopf-1(DKK1 or by expression of the Lrp5 extracellular domain blocked glucagon-induced β-catenin signaling. Furthermore, we showed that Lrp5 physically interacted with GCGR by immunoprecipitation and bioluminescence resonance energy transfer assays. Together, these results reveal an unexpected crosstalk between glucagon and β-catenin signaling, and may help to explain the metabolic phenotypes of Lrp5/6 mutations.

  17. Frequency of BCR-ABL Transcript Types in Syrian CML Patients

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    Sulaf Farhat-Maghribi

    2016-01-01

    Full Text Available Background. In Syria, CML patients are started on tyrosine kinase inhibitors (TKIs and monitored until complete molecular response is achieved. BCR-ABL mRNA transcript type is not routinely identified, contrary to the recommendations. In this study we aimed to identify the frequency of different BCR-ABL transcripts in Syrian CML patients and highlight their significance on monitoring and treatment protocols. Methods. CML patients positive for BCR-ABL transcripts by quantitative RT-PCR were enrolled. BCR-ABL transcript types were investigated using a home-made PCR method that was adapted from published protocols and optimized. The transcript types were then confirmed using a commercially available research kit. Results. Twenty-four transcripts were found in 21 patients. The most common was b2a2, followed by b3a2, b3a3, and e1a3 present solely in 12 (57.1%, 3 (14.3%, 2 (9.5%, and 1 (4.8%, respectively. Three samples (14.3% contained dual transcripts. While b3a2 transcript was apparently associated with warning molecular response to imatinib treatment, b2a2, b3a3, and e1a3 transcripts collectively proved otherwise (P=0.047. Conclusion. It might be advisable to identify the BCR-ABL transcript type in CML patients at diagnosis, using an empirically verified method, in order to link the detected transcript with the clinical findings, possible resistance to treatment, and appropriate monitoring methods.

  18. Inhibitory effect of PTD-OD-HA fusion protein on Bcr-Abl in K562 cells

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    Miao GAO

    2012-10-01

    Full Text Available Objective To study the transduction dynamics, location of PTD-OD-HA fusion protein and its interaction with Bcr-Abl oncoprotein in K562 cell lines, and explore the influence of PTD-OD-HA fusion protein on oligomerization and tyrosine kinase activity of Bcr-Abl. Methods PTD-OD-HA fusion protein was labeled with FITC and co-cultured with K562 cells. The transduction efficiency of labeled PTD-OD-HA at different doses and time intervals was observed under fluorescence microscope. The location of labeled PTD-OD-HA fusion protein in K562 cells was detected by confocal microscopy. The interaction of PTD-OD-HA fusion protein with Bcr-Abl oncoprotein was confirmed by coimmunoprecipitation. The phosphorylation of Bcr-Abl oncoprotein was detected by Western blotting. Results PTD-OD-HA fusion protein labeled with FITC was transduced into K562 cells in a dose- and time-dependent manner. PTD-OD-HA fusion protein was located in the cytoplasm of K562 cells and was consistent with the location of Bcr-Abl oncoprotein. The interaction of PTD-OD-HA fusion protein with Bcr-Abl oncoprotein was proved in K562 cells. This interaction could interrupt the homologous oligomerization of Bcr-Abl oncoprotein and reduce the phosphorylation of Bcr-Abl oncoprotein. Conclusion PTD-OD-HA fusion protein could be transduced into K562 cells efficiently, inhibit the oligomerization and reduce the phosphorylation of Bcr-Abl oncoprotein.

  19. Sweet Taste Receptor Signaling Network: Possible Implication for Cognitive Functioning

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    Menizibeya O. Welcome

    2015-01-01

    Full Text Available Sweet taste receptors are transmembrane protein network specialized in the transmission of information from special “sweet” molecules into the intracellular domain. These receptors can sense the taste of a range of molecules and transmit the information downstream to several acceptors, modulate cell specific functions and metabolism, and mediate cell-to-cell coupling through paracrine mechanism. Recent reports indicate that sweet taste receptors are widely distributed in the body and serves specific function relative to their localization. Due to their pleiotropic signaling properties and multisubstrate ligand affinity, sweet taste receptors are able to cooperatively bind multiple substances and mediate signaling by other receptors. Based on increasing evidence about the role of these receptors in the initiation and control of absorption and metabolism, and the pivotal role of metabolic (glucose regulation in the central nervous system functioning, we propose a possible implication of sweet taste receptor signaling in modulating cognitive functioning.

  20. The Cannabinoid Receptor CB1 Modulates the Signaling Properties of the Lysophosphatidylinositol Receptor GPR55*

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    Kargl, Julia; Balenga, Nariman; Parzmair, Gerald P.; Brown, Andrew J.; Heinemann, Akos; Waldhoer, Maria

    2012-01-01

    The G protein-coupled receptor (GPCR) 55 (GPR55) and the cannabinoid receptor 1 (CB1R) are co-expressed in many tissues, predominantly in the central nervous system. Seven transmembrane spanning (7TM) receptors/GPCRs can form homo- and heteromers and initiate distinct signaling pathways. Recently, several synthetic CB1 receptor inverse agonists/antagonists, such as SR141716A, AM251, and AM281, were reported to activate GPR55. Of these, SR141716A was marketed as a promising anti-obesity drug, but was withdrawn from the market because of severe side effects. Here, we tested whether GPR55 and CB1 receptors are capable of (i) forming heteromers and (ii) whether such heteromers could exhibit novel signaling patterns. We show that GPR55 and CB1 receptors alter each others signaling properties in human embryonic kidney (HEK293) cells. We demonstrate that the co-expression of FLAG-CB1 receptors in cells stably expressing HA-GPR55 specifically inhibits GPR55-mediated transcription factor activation, such as nuclear factor of activated T-cells and serum response element, as well as extracellular signal-regulated kinases (ERK1/2) activation. GPR55 and CB1 receptors can form heteromers, but the internalization of both receptors is not affected. In addition, we observe that the presence of GPR55 enhances CB1R-mediated ERK1/2 and nuclear factor of activated T-cell activation. Our data provide the first evidence that GPR55 can form heteromers with another 7TM/GPCR and that this interaction with the CB1 receptor has functional consequences in vitro. The GPR55-CB1R heteromer may play an important physiological and/or pathophysiological role in tissues endogenously co-expressing both receptors. PMID:23161546

  1. The chimeric ubiquitin ligase SH2-U-box inhibits the growth of imatinib-sensitive and resistant CML by targeting the native and T315I-mutant BCR-ABL.

    Science.gov (United States)

    Ru, Yi; Wang, Qinhao; Liu, Xiping; Zhang, Mei; Zhong, Daixing; Ye, Mingxiang; Li, Yuanchun; Han, Hua; Yao, Libo; Li, Xia

    2016-06-22

    Chronic myeloid leukemia (CML) is characterized by constitutively active fusion protein tyrosine kinase BCR-ABL. Although the tyrosine kinase inhibitor (TKI) against BCR-ABL, imatinib, is the first-line therapy for CML, acquired resistance almost inevitably emerges. The underlying mechanism are point mutations within the BCR-ABL gene, among which T315I is notorious because it resists to almost all currently available inhibitors. Here we took use of a previously generated chimeric ubiquitin ligase, SH2-U-box, in which SH2 from the adaptor protein Grb2 acts as a binding domain for activated BCR-ABL, while U-box from CHIP functions as an E3 ubiquitin ligase domain, so as to target the ubiquitination and degradation of both native and T315I-mutant BCR-ABL. As such, SH2-U-box significantly inhibited proliferation and induced apoptosis in CML cells harboring either the wild-type or T315I-mutant BCR-ABL (K562 or K562R), with BCR-ABL-dependent signaling pathways being repressed. Moreover, SH2-U-box worked in concert with imatinib in K562 cells. Importantly, SH2-U-box-carrying lentivirus could markedly suppress the growth of K562-xenografts in nude mice or K562R-xenografts in SCID mice, as well as that of primary CML cells. Collectively, by degrading the native and T315I-mutant BCR-ABL, the chimeric ubiquitin ligase SH2-U-box may serve as a potential therapy for both imatinib-sensitive and resistant CML.

  2. Signal Diversity of Receptor for Advanced Glycation End Products.

    Science.gov (United States)

    Sakaguchi, Masakiyo; Kinoshita, Rie; Putranto, Endy Widya; Ruma, I Made Winarsa; Sumardika, I Wayan; Youyi, Chen; Tomonobu, Naoko; Yamamoto, Ken-Ichi; Murata, Hitoshi

    2017-12-01

    The receptor for advanced glycation end products (RAGE) is involved in inflammatory pathogenesis. It functions as a receptor to multiple ligands such as AGEs, HMGB1 and S100 proteins, activating multiple intracellular signaling pathways with each ligand binding. The molecular events by which ligand-activated RAGE controls diverse signaling are not well understood, but some progress was made recently. Accumulating evidence revealed that RAGE has multiple binding partners within the cytoplasm and on the plasma membrane. It was first pointed out in 2008 that RAGE's cytoplasmic tail is able to recruit Diaphanous-1 (Dia-1), resulting in the acquisition of increased cellular motility through Rac1/Cdc42 activation. We also observed that within the cytosol, RAGE's cytoplasmic tail behaves similarly to a Toll-like receptor (TLR4)-TIR domain, interacting with TIRAP and MyD88 adaptor molecules that in turn activate multiple downstream signals. Subsequent studies demonstrated the presence of an alternative adaptor molecule, DAP10, on the plasma membrane. The coupling of RAGE with DAP10 is critical for enhancing the RAGE-mediated survival signal. Interestingly, RAGE interaction on the membrane was not restricted to DAP10 alone. The chemotactic G-protein-coupled receptors (GPCRs) formyl peptide receptors1 and 2 (FPR1 and FPR2) also interacted with RAGE on the plasma membrane. Binding interaction between leukotriene B4 receptor 1 (BLT1) and RAGE was also demonstrated. All of the interactions affected the RAGE signal polarity. These findings indicate that functional interactions between RAGE and various molecules within the cytoplasmic area or on the membrane area coordinately regulate multiple ligand-mediated RAGE responses, leading to typical cellular phenotypes in several pathological settings. Here we review RAGE's signaling diversity, to contribute to the understanding of the elaborate functions of RAGE in physiological and pathological contexts.

  3. Combining the ABL1 kinase inhibitor ponatinib and the histone deacetylase inhibitor vorinostat: a potential treatment for BCR-ABL-positive leukemia.

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    Okabe, Seiichi; Tauchi, Tetsuzo; Kimura, Shinya; Maekawa, Taira; Kitahara, Toshihiko; Tanaka, Yoko; Ohyashiki, Kazuma

    2014-01-01

    Resistance to imatinib (Gleevec®) in cancer cells is frequently because of acquired point mutations in the kinase domain of BCR-ABL. Ponatinib, also known as AP24534, is an oral multi-targeted tyrosine kinase inhibitor (TKI), and it has been investigated in a pivotal phase 2 clinical trial. The histone deacetylase inhibitor vorinostat (suberoylanilide hydroxamic acid) has been evaluated for its significant clinical activity in hematological malignancies. Thus, treatments combining ABL TKIs with additional drugs may be a promising strategy in the treatment of leukemia. In the current study, we analyzed the efficacy of ponatinib and vorinostat treatment by using BCR-ABL-positive cell lines. Treatment with ponatinib for 72 h inhibited cell growth and induced apoptosis in K562 cells in a dose-dependent manner. We found that ponatinib potently inhibited the growth of Ba/F3 cells ectopically expressing BCR-ABL T315I mutation. Upon BCR-ABL phosphorylation, Crk-L was decreased, and poly (ADP-ribose) polymerase (PARP) was activated in a dose-dependent manner. Combined treatment of Ba/F3 T315I mutant cells with vorinostat and ponatinib resulted in significantly increased cytotoxicity. Additionally, the intracellular signaling of ponatinib and vorinostat was examined. Caspase 3 and PARP activation increased after combination treatment with ponatinib and vorinostat. Moreover, an increase in the phosphorylation levels of γH2A.X was observed. Previously established ponatinib-resistant Ba/F3 cells were also resistant to imatinib, nilotinib, and dasatinib. We investigated the difference in the efficacy of ponatinib and vorinostat by using ponatinib-resistant Ba/F3 cells. Combined treatment of ponatinib-resistant cells with ponatinib and vorinostat caused a significant increase in cytotoxicity. Thus, combined administration of ponatinib and vorinostat may be a powerful strategy against BCR-ABL mutant cells and could enhance the cytotoxic effects of ponatinib in those BCR

  4. Interphase FISH for BCR-ABL1 rearrangement on neutrophils: A decisive tool to discriminate a lymphoid blast crisis of chronic myeloid leukemia from a de novo BCR-ABL1 positive acute lymphoblastic leukemia.

    Science.gov (United States)

    Balducci, Estelle; Loosveld, Marie; Rahal, Ilhem; Boudjarane, John; Alazard, Emilie; Missirian, Chantal; Lafage-Pochitaloff, Marina; Michel, Gérard; Zattara, Hélène

    2018-02-01

    Discrimination between lymphoid blast crisis of chronic myeloid leukemia (CML) and de novo BCR-ABL1 positive acute lymphoblastic leukemia (ALL) represents a diagnostic challenge because this distinction has a major incidence on the management of patients. Here, we report an uncommon pediatric case of ALL with cryptic ins(22;9)(q11;q34q34) and p190-type BCR-ABL1 transcript. We performed interphase fluorescence in situ hybridization (FISH) for BCR-ABL1 rearrangement on blood neutrophils, which was positive consistent with the diagnosis of lymphoid blast crisis of CML. This case illustrates the major interest of interphase FISH for BCR-ABL1 rearrangement on blood neutrophils as a decisive method to discriminate a lymphoid blast crisis of CML from a de novo BCR-ABL1 positive ALL. Copyright © 2017 John Wiley & Sons, Ltd.

  5. Human GH Receptor-IGF-1 Receptor Interaction: Implications for GH Signaling

    Science.gov (United States)

    Gan, Yujun; Buckels, Ashiya; Liu, Ying; Zhang, Yue; Paterson, Andrew J.; Jiang, Jing; Zinn, Kurt R.

    2014-01-01

    GH signaling yields multiple anabolic and metabolic effects. GH binds the transmembrane GH receptor (GHR) to activate the intracellular GHR-associated tyrosine kinase, Janus kinase 2 (JAK2), and downstream signals, including signal transducer and activator of transcription 5 (STAT5) activation and IGF-1 gene expression. Some GH effects are partly mediated by GH-induced IGF-1 via IGF-1 receptor (IGF-1R), a tyrosine kinase receptor. We previously demonstrated in non-human cells that GH causes formation of a GHR-JAK2-IGF-1R complex and that presence of IGF-1R (even without IGF-1 binding) augments proximal GH signaling. In this study, we use human LNCaP prostate cancer cells as a model system to further study the IGF-1R's role in GH signaling. GH promoted JAK2 and GHR tyrosine phosphorylation and STAT5 activation in LNCaP cells. By coimmunoprecipitation and a new split luciferase complementation assay, we find that GH augments GHR/IGF-1R complex formation, which is inhibited by a Fab of an antagonistic anti-GHR monoclonal antibody. Short hairpin RNA-mediated IGF-1R silencing in LNCaP cells reduced GH-induced GHR, JAK2, and STAT5 phosphorylation. Similarly, a soluble IGF-1R extracellular domain fragment (sol IGF-1R) interacts with GHR in response to GH and blunts GH signaling. Sol IGF-1R also markedly inhibits GH-induced IGF-1 gene expression in both LNCaP cells and mouse primary osteoblast cells. On the basis of these and other findings, we propose a model in which IGF-1R augments GH signaling by allowing a putative IGF-1R-associated molecule that regulates GH signaling to access the activated GHR/JAK2 complex and envision sol IGF-1R as a dominant-negative inhibitor of this IGF-1R-mediated augmentation. Physiological implications of this new model are discussed. PMID:25211187

  6. The Growth Hormone Receptor: Mechanism of Receptor Activation, Cell Signaling, and Physiological Aspects

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    Farhad Dehkhoda

    2018-02-01

    Full Text Available The growth hormone receptor (GHR, although most well known for regulating growth, has many other important biological functions including regulating metabolism and controlling physiological processes related to the hepatobiliary, cardiovascular, renal, gastrointestinal, and reproductive systems. In addition, growth hormone signaling is an important regulator of aging and plays a significant role in cancer development. Growth hormone activates the Janus kinase (JAK–signal transducer and activator of transcription (STAT signaling pathway, and recent studies have provided a new understanding of the mechanism of JAK2 activation by growth hormone binding to its receptor. JAK2 activation is required for growth hormone-mediated activation of STAT1, STAT3, and STAT5, and the negative regulation of JAK–STAT signaling comprises an important step in the control of this signaling pathway. The GHR also activates the Src family kinase signaling pathway independent of JAK2. This review covers the molecular mechanisms of GHR activation and signal transduction as well as the physiological consequences of growth hormone signaling.

  7. Molecular measurement of BCR-ABL transcript variations in chronic myeloid leukemia patients in cytogenetic remission

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    Costa Juliana

    2010-11-01

    Full Text Available Abstract Background The monitoring of BCR-ABL transcript levels by real-time quantitative polymerase chain reaction (RT-qPCR has become important to assess minimal residual disease (MRD and standard of care in the treatment of chronic myeloid leukemia (CML. In this study, we performed a prospective, sequential analysis using RT-qPCR monitoring of BCR-ABL gene rearrangements in blood samples from 91 CML patients in chronic phase (CP who achieved complete cytogenetic remission (CCyR and major molecular remission (MMR throughout imatinib treatment. Methods The absolute level of BCR-ABL transcript from peripheral blood was serially measured every 4 to 12 weeks by RT-qPCR. Only level variations > 0.5%, according to the international scale, was considered positive. Sequential cytogenetic analysis was also performed in bone marrow samples from all patients using standard protocols. Results Based on sequential analysis of BCR-ABL transcripts, the 91 patients were divided into three categories: (A 57 (62.6% had no variation on sequential analysis; (B 30 (32.9% had a single positive variation result obtained in a single sample; and (C 4 (4.39% had variations of BCR-ABL transcripts in at least two consecutive samples. Of the 34 patients who had elevated levels of transcripts (group B and C, 19 (55.8% had a BCR-ABL/BCR ratio, 13 (38.2% patients had a 1% to 10% increase and 2 patients had a >10% increase of RT-qPCR. The last two patients had lost a CCyR, and none of them showed mutations in the ABL gene. Transient cytogenetic alterations in Ph-negative cells were observed in five (5.5% patients, and none of whom lost CCyR. Conclusions Despite an increase levels of BCR-ABL/BCR ratio variations by RT-qPCR, the majority of CML patients with MMR remained in CCyR. Thus, such single variations should neither be considered predictive of subsequent failure and nor an indication for altering imatinib dose or switching to second generation therapy. Changing of

  8. Receptor density balances signal stimulation and attenuation in membrane-assembled complexes of bacterial chemotaxis signaling proteins

    Science.gov (United States)

    Besschetnova, Tatiana Y.; Montefusco, David J.; Asinas, Abdalin E.; Shrout, Anthony L.; Antommattei, Frances M.; Weis, Robert M.

    2008-01-01

    All cells possess transmembrane signaling systems that function in the environment of the lipid bilayer. In the Escherichia coli chemotaxis pathway, the binding of attractants to a two-dimensional array of receptors and signaling proteins simultaneously inhibits an associated kinase and stimulates receptor methylation—a slower process that restores kinase activity. These two opposing effects lead to robust adaptation toward stimuli through a physical mechanism that is not understood. Here, we provide evidence of a counterbalancing influence exerted by receptor density on kinase stimulation and receptor methylation. Receptor signaling complexes were reconstituted over a range of defined surface concentrations by using a template-directed assembly method, and the kinase and receptor methylation activities were measured. Kinase activity and methylation rates were both found to vary significantly with surface concentration—yet in opposite ways: samples prepared at high surface densities stimulated kinase activity more effectively than low-density samples, whereas lower surface densities produced greater methylation rates than higher densities. FRET experiments demonstrated that the cooperative change in kinase activity coincided with a change in the arrangement of the membrane-associated receptor domains. The counterbalancing influence of density on receptor methylation and kinase stimulation leads naturally to a model for signal regulation that is compatible with the known logic of the E. coli pathway. Density-dependent mechanisms are likely to be general and may operate when two or more membrane-related processes are influenced differently by the two-dimensional concentration of pathway elements. PMID:18711126

  9. Receptor activity-independent recruitment of βarrestin2 reveals specific signalling modes

    Science.gov (United States)

    Terrillon, Sonia; Bouvier, Michel

    2004-01-01

    The roles of βarrestins in regulating G protein coupling and receptor endocytosis following agonist stimulation of G protein-coupled receptors are well characterised. However, their ability to act on their own as direct modulators or activators of signalling remains poorly characterised. Here, βarrestin2 intrinsic signalling properties were assessed by forcing the recruitment of this accessory protein to vasopressin V1a or V2 receptors independently of agonist-promoted activation of the receptors. Such induction of a stable interaction with βarrestin2 initiated receptor endocytosis leading to intracellular accumulation of the βarrestin/receptor complexes. Interestingly, βarrestin2 association to a single receptor protomer was sufficient to elicit receptor dimer internalisation. In addition to recapitulating βarrestin2 classical actions on receptor trafficking, the receptor activity-independent recruitment of βarrestin2 activated the extracellular signal-regulated kinases. In the latter case, recruitment to the receptor itself was not required since kinase activation could be mediated by βarrestin2 translocation to the plasma membrane in the absence of any interacting receptor. These data demonstrate that βarrestin2 can act as a ‘bonafide' signalling molecule even in the absence of activated receptor. PMID:15385966

  10. The MEK1/2-ERK Pathway Inhibits Type I IFN Production in Plasmacytoid Dendritic Cells

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    Vaclav Janovec

    2018-02-01

    Full Text Available Recent studies have reported that the crosslinking of regulatory receptors (RRs, such as blood dendritic cell antigen 2 (BDCA-2 (CD303 or ILT7 (CD85g, of plasmacytoid dendritic cells (pDCs efficiently suppresses the production of type I interferons (IFN-I, α/β/ω and other cytokines in response to toll-like receptor 7 and 9 (TLR7/9 ligands. The exact mechanism of how this B cell receptor (BCR-like signaling blocks TLR7/9-mediated IFN-I production is unknown. Here, we stimulated BCR-like signaling by ligation of RRs with BDCA-2 and ILT7 mAbs, hepatitis C virus particles, or BST2 expressing cells. We compared BCR-like signaling in proliferating pDC cell line GEN2.2 and in primary pDCs from healthy donors, and addressed the question of whether pharmacological targeting of BCR-like signaling can antagonize RR-induced pDC inhibition. To this end, we tested the TLR9-mediated production of IFN-I and proinflammatory cytokines in pDCs exposed to a panel of inhibitors of signaling molecules involved in BCR-like, MAPK, NF-ĸB, and calcium signaling pathways. We found that MEK1/2 inhibitors, PD0325901 and U0126 potentiated TLR9-mediated production of IFN-I in GEN2.2 cells. More importantly, MEK1/2 inhibitors significantly increased the TLR9-mediated IFN-I production blocked in both GEN2.2 cells and primary pDCs upon stimulation of BCR-like or phorbol 12-myristate 13-acetate-induced protein kinase C (PKC signaling. Triggering of BCR-like and PKC signaling in pDCs resulted in an upregulation of the expression and phoshorylation of c-FOS, a downstream gene product of the MEK1/2-ERK pathway. We found that the total level of c-FOS was higher in proliferating GEN2.2 cells than in the resting primary pDCs. The PD0325901-facilitated restoration of the TLR9-mediated IFN-I production correlated with the abrogation of MEK1/2-ERK-c-FOS signaling. These results indicate that the MEK1/2-ERK pathway inhibits TLR9-mediated type I IFN production in pDCs and that

  11. A systems toxicology approach identifies Lyn as a key signaling phosphoprotein modulated by mercury in a B lymphocyte cell model

    Energy Technology Data Exchange (ETDEWEB)

    Caruso, Joseph A.; Stemmer, Paul M. [Institute of Environmental Health Sciences, Wayne State University, Detroit, MI (United States); Dombkowski, Alan [Department of Pediatrics, Wayne State University, Detroit, MI (United States); Caruthers, Nicholas J. [Institute of Environmental Health Sciences, Wayne State University, Detroit, MI (United States); Gill, Randall [Department of Immunology and Microbiology, Wayne State University, Detroit, MI (United States); Rosenspire, Allen J., E-mail: arosenspire@wayne.edu [Department of Immunology and Microbiology, Wayne State University, Detroit, MI (United States)

    2014-04-01

    Network and protein–protein interaction analyses of proteins undergoing Hg{sup 2+}-induced phosphorylation and dephosphorylation in Hg{sup 2+}-intoxicated mouse WEHI-231 B cells identified Lyn as the most interconnected node. Lyn is a Src family protein tyrosine kinase known to be intimately involved in the B cell receptor (BCR) signaling pathway. Under normal signaling conditions the tyrosine kinase activity of Lyn is controlled by phosphorylation, primarily of two well known canonical regulatory tyrosine sites, Y-397 and Y-508. However, Lyn has several tyrosine residues that have not yet been determined to play a major role under normal signaling conditions, but are potentially important sites for phosphorylation following mercury exposure. In order to determine how Hg{sup 2+} exposure modulates the phosphorylation of additional residues in Lyn, a targeted MS assay was developed. Initial mass spectrometric surveys of purified Lyn identified 7 phosphorylated tyrosine residues. A quantitative assay was developed from these results using the multiple reaction monitoring (MRM) strategy. WEHI-231 cells were treated with Hg{sup 2+}, pervanadate (a phosphatase inhibitor), or anti-Ig antibody (to stimulate the BCR). Results from these studies showed that the phosphoproteomic profile of Lyn after exposure of the WEHI-231 cells to a low concentration of Hg{sup 2+} closely resembled that of anti-Ig antibody stimulation, whereas exposure to higher concentrations of Hg{sup 2+} led to increases in the phosphorylation of Y-193/Y-194, Y-501 and Y-508 residues. These data indicate that mercury can disrupt a key regulatory signal transduction pathway in B cells and point to phospho-Lyn as a potential biomarker for mercury exposure. - Highlights: • Inorganic mercury (Hg{sup 2+}) induces changes in the WEHI-231 B cell phosphoproteome. • The B cell receptor (BCR) signaling pathway was the pathway most affected by Hg{sup 2+}. • The Src family phosphoprotein kinase Lyn was the

  12. [Evaluation of percentage of lymphocytes B with expression of co-receptors CD 40, CD22 and CD72 in hypertrophied adenoid at children with otitis media with effusion].

    Science.gov (United States)

    Wysocka, Jolanta; Zelazowska-Rutkowska, Beata; Ratomski, Karol; Skotnicka, Bozena; Hassmann-Poznańska, Elzbieta

    2009-01-01

    In hypertrophied adenoid lymphocytes B make up about 60% all lymphocytes. When the lymphocytes B come in interaction with antigens this membranes signal be passed through their receptor (BCR) to interior of cell. This signal affect modulation on gene expression, activation from which depends activation, anergy or apoptosis of lymphocyte B. Accompany BCR co-receptors regulate his functions influence stimulate or inhibitive. To the most important co-receptors stepping out on lymphocyte B belong: CD40, CD22, CD72. The aim of study was evaluation of lymphocytes B (CD19) with co-expression with CD72 and CD40 receptors in hypertrophied adenoid with at children with otitis media with effusion. An investigation was executed in hypertrophied adenoids with or without otitis media with effusion. By flow cytometry percentage of lymphocytes B with co-receptors CD 40, CD22 and CD72 in was analyzed. The percentages of CD19+CD72+ lymphocytes in the group of children with adenoid hypertrophy and exudative otitis media were lower as compared to the reference group. However, the percentages of CD19+CD22+, CD19+CD40+ in the study group was approximate to the reference group. The lower percentage of lymphocytes B CD72 + near approximate percentages of lymphocytes B CD40+ and BCD22+ at children with otitis media with effusion can be the cause of incorrect humoral response in hypertrophied adenoid at children. Maybe it is cause reduced spontaneous production IgA and IgG through lymphocyte at children with otitis media with effusion.

  13. A BCR/ABL-hIL-2 DNA Vaccine Enhances the Immune Responses in BALB/c Mice

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    Yanan Qin

    2013-01-01

    Full Text Available The use of a DNA vaccine encoding the BCR/ABL fusion gene is thought to be a promising approach for patients with chronic myeloid leukemia (CML to eradicate minimal residual disease after treatment with chemotherapy or targeted therapy. In this study, our strategy employs genetic technology to create a DNA vaccine encoding the BCR/ABL fusion and human interleukin-2 (hIL-2 genes. The successfully constructed plasmids BCR/ABL-pIRES-hIL-2, BCR/ABL-pIRES, and pIRES-hIL-2 were delivered intramuscularly to BALB/c mice at 14-day intervals for three cycles. The transcription and expression of the BCR/ABL and hIL-2 genes were found in the injected muscle tissues. The interferon-γ (IFN-γ serum levels were increased, and the splenic CD4+/CD8+ T cell ratio was significantly decreased in the BCR/ABL-pIRES-hIL-2-injected mice. Furthermore, specific antibodies against K562 cells could be detected by indirect immunofluorescence. These results indicate that a DNA vaccine containing BCR/ABL and hIL-2 together may elicit increased in vivo humoral and cellular immune responses in BALB/c mice.

  14. Functionally biased signalling properties of 7TM receptors - opportunities for drug development for the ghrelin receptor

    DEFF Research Database (Denmark)

    Sivertsen, B; Holliday, N; Madsen, A N

    2013-01-01

    UNLABELLED: The ghrelin receptor is a 7 transmembrane (7TM) receptor involved in a variety of physiological functions including growth hormone secretion, increased food intake and fat accumulation as well as modulation of reward and cognitive functions. Because of its important role in metabolism...... and energy expenditure, the ghrelin receptor has become an important therapeutic target for drug design and the development of anti-obesity compounds. However, none of the compounds developed so far have been approved for commercial use. Interestingly, the ghrelin receptor is able to signal through several...... review, we have described how ligands and mutations in the 7TM receptor may bias the receptors to favour either one G-protein over another or to promote G-protein independent signalling pathways rather than G-protein-dependent pathways. For the ghrelin receptor, both agonist and inverse agonists have...

  15. In vivo characterization of high Basal signaling from the ghrelin receptor

    DEFF Research Database (Denmark)

    Petersen, Pia Steen; Woldbye, David P D; Madsen, Andreas Nygaard

    2009-01-01

    The receptor for the orexigenic peptide, ghrelin, is one of the most constitutively active 7TM receptors known, as demonstrated under in vitro conditions. Change in expression of a constitutively active receptor is associated with change in signaling independent of the endogenous ligand. In the f......The receptor for the orexigenic peptide, ghrelin, is one of the most constitutively active 7TM receptors known, as demonstrated under in vitro conditions. Change in expression of a constitutively active receptor is associated with change in signaling independent of the endogenous ligand....... In the following study, we found that the expression of the ghrelin receptor in the hypothalamus was up-regulated approximately 2-fold in rats both during 48-h fasting and by streptozotocin-induced hyperphagia. In a separate experiment, to probe for the effect of the high basal signaling of the ghrelin receptor...... in vivo, we used intracerebroventricular administration by osmotic pumps of a peptide [D-Arg(1), D-Phe(5), D-Trp(7,9), Leu(11)]-substance P. This peptide selectively displays inverse agonism at the ghrelin receptor as compared with an inactive control peptide with just a single amino acid substitution...

  16. PCR detection of a Maell polymorphism in the human major breakpoint cluster region (BCR)

    Energy Technology Data Exchange (ETDEWEB)

    McClure, J.S.; Litz, C.E. (Medical School, Minneapolis, MN (United States))

    1991-09-25

    Nested primer pairs flanking the second intron of the breakpoint cluster region were constructed from the published cDNA sequence. The outer primer pair 5{prime}BCR Exon 2 (5{prime}-GTT TCA GAA GCT TCT CCC TG-3{prime}) and 3{prime}BCR Exon 3 (5{prime}-ACT CTG CTT AAA TCC AGT GG-3{prime}), amplified a fragment of genomic DNA approximately 810 bp in length. The inner primer pair, 3{prime}BCR Exon 2(5{prime}-CGC TGA CCA TCA ATA AGG AA-3{prime}) and 5{prime}BCR Exon 3 (5{prime}-AGA AAC CCA TAG AGC CCC GG-3{prime}), amplified a fragment approximately 730 bp in length. Double stranded DNA amplified with the outer primer pair was subjected to asymmetric PCR using the inner primer pair. Sequencing reactions were performed using the Sequenase dideoxy sequencing kit with S{sup 35}-dATP. Sequences in homozygotes revealed either an A or a G 85 bp 5{prime} of the BCR BamHI site. Heterozygotes demonstrated both bands at the corresponding position.

  17. Testin, a novel binding partner of the calcium-sensing receptor, enhances receptor-mediated Rho-kinase signalling

    International Nuclear Information System (INIS)

    Magno, Aaron L.; Ingley, Evan; Brown, Suzanne J.; Conigrave, Arthur D.; Ratajczak, Thomas; Ward, Bryan K.

    2011-01-01

    Highlights: → A yeast two-hybrid screen revealed testin bound to the calcium-sensing receptor. → The second zinc finger of LIM domain 1 of testin is critical for interaction. → Testin bound to a region of the receptor tail important for cell signalling. → Testin and receptor interaction was confirmed in mammalian (HEK293) cells. → Overexpression of testin enhanced receptor-mediated Rho signalling in HEK293 cells. -- Abstract: The calcium-sensing receptor (CaR) plays an integral role in calcium homeostasis and the regulation of other cellular functions including cell proliferation and cytoskeletal organisation. The multifunctional nature of the CaR is manifested through ligand-dependent stimulation of different signalling pathways that are also regulated by partner binding proteins. Following a yeast two-hybrid library screen using the intracellular tail of the CaR as bait, we identified several novel binding partners including the focal adhesion protein, testin. Testin has not previously been shown to interact with cell surface receptors. The sites of interaction between the CaR and testin were mapped to the membrane proximal region of the receptor tail and the second zinc-finger of LIM domain 1 of testin, the integrity of which was found to be critical for the CaR-testin interaction. The CaR-testin association was confirmed in HEK293 cells by coimmunoprecipitation and confocal microscopy studies. Ectopic expression of testin in HEK293 cells stably expressing the CaR enhanced CaR-stimulated Rho activity but had no effect on CaR-stimulated ERK signalling. These results suggest an interplay between the CaR and testin in the regulation of CaR-mediated Rho signalling with possible effects on the cytoskeleton.

  18. Protein kinase C alpha controls erythropoietin receptor signaling.

    NARCIS (Netherlands)

    M.M. von Lindern (Marieke); M. Parren-Van Amelsvoort (Martine); T.B. van Dijk (Thamar); E. Deiner; B. Löwenberg (Bob); E. van den Akker (Emile); S. van Emst-de Vries (Sjenet); P.J. Willems (Patrick); H. Beug (Hartmut)

    2000-01-01

    textabstractProtein kinase C (PKC) is implied in the activation of multiple targets of erythropoietin (Epo) signaling, but its exact role in Epo receptor (EpoR) signal transduction and in the regulation of erythroid proliferation and differentiation remained elusive. We

  19. Signal Transduction of Sphingosine-1-Phosphate G Protein—Coupled Receptors

    Directory of Open Access Journals (Sweden)

    Nicholas Young

    2006-01-01

    Full Text Available Sphingosine-1-phosphate (S1P is a bioactive lipid capable of eliciting dramatic effects in a variety of cell types. Signaling by this molecule is by a family of five G protein—coupled receptors named S1P1–5 that signal through a variety of pathways to regulate cell proliferation, migration, cytoskeletal organization, and differentiation. These receptors are expressed in a wide variety of tissues and cell types, and their cellular effects contribute to important biological and pathological functions of S1P in many processes, including angiogenesis, vascular development, lymphocyte trafficking, and cancer. This review will focus on the current progress in the field of S1P receptor signaling and biology.

  20. The Role of Cgrp-Receptor Component Protein (Rcp in Cgrp-Mediated Signal Transduction

    Directory of Open Access Journals (Sweden)

    M. A. Prado

    2001-01-01

    Full Text Available The calcitonin gene-related peptide (CGRP-receptor component protein (RCP is a 17-kDa intracellular peripheral membrane protein required for signal transduction at CGRP receptors. To determine the role of RCP in CGRP-mediated signal transduction, RCP was depleted from NIH3T3 cells using antisense strategy. Loss of RCP protein correlated with loss of cAMP production by CGRP in the antisense cells. In contrast, loss of RCP had no effect on CGRP-mediated binding; therefore RCP is not acting as a chaperone for the CGRP receptor. Instead, RCP is a novel signal transduction molecule that couples the CGRP receptor to the cellular signal transduction machinery. RCP thus represents a prototype for a new class of signal transduction proteins that are required for regulation of G protein-coupled receptors.

  1. TAM receptor signaling in development.

    Science.gov (United States)

    Burstyn-Cohen, Tal

    2017-01-01

    TYRO3, AXL and MERTK comprise the TAM family of receptor protein tyrosine kinases. Activated by their ligands, protein S (PROS1) and growth-arrest-specific 6 (GAS6), they mediate numerous cellular functions throughout development and adulthood. Expressed by a myriad of cell types and tissues, they have been implicated in homeostatic regulation of the immune, nervous, vascular, bone and reproductive systems. The loss-of-function of TAM signaling in adult tissues culminates in the destruction of tissue homeostasis and diseased states, while TAM gain-of-function in various tumors promotes cancer phenotypes. Combinatorial ligand-receptor interactions may elicit different molecular and cellular responses. Many of the TAM regulatory functions are essentially developmental, taking place both during embryogenesis and postnatally. This review highlights current knowledge on the role of TAM receptors and their ligands during these developmental processes in the immune, nervous, vascular and reproductive systems.

  2. The Janus face of death receptor signalling during tumour immunoediting.

    Directory of Open Access Journals (Sweden)

    Eimear O' Reilly

    2016-10-01

    Full Text Available Cancer immune-surveillance is essential for the inhibition of carcinogenesis. Malignantly transformed cells can be recognised by both the innate and adaptive immune systems through different mechanisms. Immune effector cells induce extrinsic cell death in the identified tumour cells by expressing death ligand cytokines of the tumour necrosis factor ligand family. However, some tumour cells can escape immune elimination and progress. Acquisition of resistance to the death-ligand induced apoptotic pathway can be obtained through cleavage of effector-cell expressed death-ligands into a poorly active form, mutations or silencing of the death receptors or overexpression of decoy receptors and pro-survival proteins. Although the immune system is highly effective in the elimination of malignantly transformed cells, abnormal/ dysfunctional death-ligand signalling curbs its cytotoxicity. Moreover, death receptors can also transmit pro-survival and pro-migratory signals. Consequently, dysfunctional death receptor-mediated apoptosis/necroptosis signalling does not only give a passive resistance against cell death, but actively drives tumour cell motility, invasion and contributes to consequent metastasis. This dual contribution of the death ligand-death receptor signalling in both the early, elimination phase and then in the late, escape phase of the tumour immunoediting process is discussed in this review. Death receptor agonists still hold potential for cancer therapy since they can execute the tumour-eliminating immune-effector function even in the absence of activation of the immune system against the tumour. The opportunities and challenges of developing death receptor agonists into effective cancer therapeutics are also discussed.

  3. Signal transduction by growth factor receptors: signaling in an instant

    DEFF Research Database (Denmark)

    Dengjel, Joern; Akimov, Vyacheslav; Blagoev, Blagoy

    2007-01-01

    Phosphorylation-based signaling events happening within the first minute of receptor stimulation have so far only been analyzed by classical cell biological approaches like live-cell microscopy. The development of a quench flow system with a time resolution of one second coupled to a read...

  4. Differential modulation of Beta-adrenergic receptor signaling by trace amine-associated receptor 1 agonists.

    Directory of Open Access Journals (Sweden)

    Gunnar Kleinau

    Full Text Available Trace amine-associated receptors (TAAR are rhodopsin-like G-protein-coupled receptors (GPCR. TAAR are involved in modulation of neuronal, cardiac and vascular functions and they are potentially linked with neurological disorders like schizophrenia and Parkinson's disease. Subtype TAAR1, the best characterized TAAR so far, is promiscuous for a wide set of ligands and is activated by trace amines tyramine (TYR, phenylethylamine (PEA, octopamine (OA, but also by thyronamines, dopamine, and psycho-active drugs. Unfortunately, effects of trace amines on signaling of the two homologous β-adrenergic receptors 1 (ADRB1 and 2 (ADRB2 have not been clarified yet in detail. We, therefore, tested TAAR1 agonists TYR, PEA and OA regarding their effects on ADRB1/2 signaling by co-stimulation studies. Surprisingly, trace amines TYR and PEA are partial allosteric antagonists at ADRB1/2, whereas OA is a partial orthosteric ADRB2-antagonist and ADRB1-agonist. To specify molecular reasons for TAAR1 ligand promiscuity and for observed differences in signaling effects on particular aminergic receptors we compared TAAR, tyramine (TAR octopamine (OAR, ADRB1/2 and dopamine receptors at the structural level. We found especially for TAAR1 that the remarkable ligand promiscuity is likely based on high amino acid similarity in the ligand-binding region compared with further aminergic receptors. On the other hand few TAAR specific properties in the ligand-binding site might determine differences in ligand-induced effects compared to ADRB1/2. Taken together, this study points to molecular details of TAAR1-ligand promiscuity and identified specific trace amines as allosteric or orthosteric ligands of particular β-adrenergic receptor subtypes.

  5. Ibrutinib (PCI-32765) in chronic lymphocytic leukemia.

    Science.gov (United States)

    Jain, Nitin; O'Brien, Susan

    2013-08-01

    B-cell receptor (BCR) signaling is essential for chronic lymphocytic leukemia (CLL) cell survival. Many kinases in the BCR signaling pathway are being studied as potential therapeutic targets. Ibrutinib (PCI-32765) is a novel first-in-class selective inhibitor of Bruton tyrosine kinase. Preclinical evidence suggests that ibrutinib inhibits CLL cell survival and proliferation and affects CLL cell migration and homing. Early clinical data in patients with CLL and non-Hodgkin lymphoma is encouraging. It is likely that ibrutinib and other drugs targeting the BCR pathway will become an integral component of CLL therapy. Copyright © 2013 Elsevier Inc. All rights reserved.

  6. Role of protein dynamics in transmembrane receptor signalling

    DEFF Research Database (Denmark)

    Wang, Yong; Bugge, Katrine Østergaard; Kragelund, Birthe Brandt

    2018-01-01

    Cells are dependent on transmembrane receptors to communicate and transform chemical and physical signals into intracellular responses. Because receptors transport 'information', conformational changes and protein dynamics play a key mechanistic role. We here review examples where experiment...... to function. Because the receptors function in a heterogeneous environment and need to be able to switch between distinct functional states, they may be particularly sensitive to small perturbations that complicate studies linking dynamics to function....

  7. Real-time trafficking and signaling of the glucagon-like peptide-1 receptor

    DEFF Research Database (Denmark)

    Roed, Sarah Noerklit; Wismann, Pernille; Underwood, Christina Rye

    2014-01-01

    The glucagon-like peptide-1 incretin receptor (GLP-1R) of family B G protein-coupled receptors (GPCRs) is a major drug target in type-2-diabetes due to its regulatory effect on post-prandial blood-glucose levels. The mechanism(s) controlling GLP-1R mediated signaling are far from fully understood....... A fundamental mechanism controlling the signaling capacity of GPCRs is the post-endocytic trafficking of receptors between recycling and degradative fates. Here, we combined microscopy with novel real-time assays to monitor both receptor trafficking and signaling in living cells. We find that the human GLP-1R...

  8. Imaging of persistent cAMP signaling by internalized G protein-coupled receptors.

    Science.gov (United States)

    Calebiro, Davide; Nikolaev, Viacheslav O; Lohse, Martin J

    2010-07-01

    G protein-coupled receptors (GPCRs) are the largest family of plasma membrane receptors. They mediate the effects of several endogenous cues and serve as important pharmacological targets. Although many biochemical events involved in GPCR signaling have been characterized in great detail, little is known about their spatiotemporal dynamics in living cells. The recent advent of optical methods based on fluorescent resonance energy transfer allows, for the first time, to directly monitor GPCR signaling in living cells. Utilizing these methods, it has been recently possible to show that the receptors for two protein/peptide hormones, the TSH and the parathyroid hormone, continue signaling to cAMP after their internalization into endosomes. This type of intracellular signaling is persistent and apparently triggers specific cellular outcomes. Here, we review these recent data and explain the optical methods used for such studies. Based on these findings, we propose a revision of the current model of the GPCR-cAMP signaling pathway to accommodate receptor signaling at endosomes.

  9. Long-term remission in BCR/ABL-positive AML-M6 patient treated with Imatinib Mesylate.

    Science.gov (United States)

    Pompetti, Franca; Spadano, Antonio; Sau, Antonella; Mennucci, Antonio; Russo, Rosa; Catinella, Virginia; Franchi, Paolo Guanciali; Calabrese, Giuseppe; Palka, Giandomenico; Fioritoni, Giuseppe; Iacone, Antonio

    2007-04-01

    BCR/ABL-positive acute myeloid leukemia (AML) is a rare disease, characterized by a poor prognosis, with resistance to induction chemotherapy and frequent relapses in responsive patients. Here we report a case of BCR/ABL-positive AML-M6 who, after relapse, was treated with Imatinib Mesylate (600 mg/die) and within 4 months achieved a cytogenetic and molecular complete response. After more than 4 years of continuous Imatinib therapy, nested RT-PCR for BCR/ABL is persistently negative. The case reported shows that the response obtained with Imatinib Mesylate in BCR/ABL-positive AML may be long lasting, offering a chance of successful treatment for this poor prognosis group of patients.

  10. Protein kinase C alpha controls erythropoietin receptor signaling

    NARCIS (Netherlands)

    von Lindern, M.; Parren-van Amelsvoort, M.; van Dijk, T.; Deiner, E.; van den Akker, E.; van Emst-de Vries, S.; Willems, P.; Beug, H.; Löwenberg, B.

    2000-01-01

    Protein kinase C (PKC) is implied in the activation of multiple targets of erythropoietin (Epo) signaling, but its exact role in Epo receptor (EpoR) signal transduction and in the regulation of erythroid proliferation and differentiation remained elusive. We analyzed the effect of PKC inhibitors

  11. Distinct and Overlapping Functions of TEC Kinase and BTK in B Cell Receptor Signaling.

    Science.gov (United States)

    de Bruijn, Marjolein J W; Rip, Jasper; van der Ploeg, Esmee K; van Greuningen, Lars W; Ta, Van T B; Kil, Laurens P; Langerak, Anton W; Rimmelzwaan, Guus F; Ellmeier, Wilfried; Hendriks, Rudi W; Corneth, Odilia B J

    2017-04-15

    The Tec tyrosine kinase is expressed in many cell types, including hematopoietic cells, and is a member of the Tec kinase family that also includes Btk. Although the role of Btk in B cells has been extensively studied, the role of Tec kinase in B cells remains largely unclear. It was previously shown that Tec kinase has the ability to partly compensate for loss of Btk activity in B cell differentiation, although the underlying mechanism is unknown. In this study, we confirm that Tec kinase is not essential for normal B cell development when Btk is present, but we also found that Tec-deficient mature B cells showed increased activation, proliferation, and survival upon BCR stimulation, even in the presence of Btk. Whereas Tec deficiency did not affect phosphorylation of phospholipase Cγ or Ca 2+ influx, it was associated with significantly increased activation of the intracellular Akt/S6 kinase signaling pathway upon BCR and CD40 stimulation. The increased S6 kinase phosphorylation in Tec-deficient B cells was dependent on Btk kinase activity, as ibrutinib treatment restored pS6 to wild-type levels, although Btk protein and phosphorylation levels were comparable to controls. In Tec-deficient mice in vivo, B cell responses to model Ags and humoral immunity upon influenza infection were enhanced. Moreover, aged mice lacking Tec kinase developed a mild autoimmune phenotype. Taken together, these data indicate that in mature B cells, Tec and Btk may compete for activation of the Akt signaling pathway, whereby the activating capacity of Btk is limited by the presence of Tec kinase. Copyright © 2017 by The American Association of Immunologists, Inc.

  12. Cirmtuzumab inhibits Wnt5a-induced Rac1 activation in chronic lymphocytic leukemia treated with ibrutinib.

    Science.gov (United States)

    Yu, J; Chen, L; Cui, B; Wu, Christina; Choi, M Y; Chen, Y; Zhang, L; Rassenti, L Z; Widhopf Ii, G F; Kipps, T J

    2017-06-01

    Signaling via the B cell receptor (BCR) plays an important role in the pathogenesis and progression of chronic lymphocytic leukemia (CLL). This is underscored by the clinical effectiveness of ibrutinib, an inhibitor of Bruton's tyrosine kinase (BTK) that can block BCR-signaling. However, ibrutinib cannot induce complete responses (CR) or durable remissions without continued therapy, suggesting alternative pathways also contribute to CLL growth/survival that are independent of BCR-signaling. ROR1 is a receptor for Wnt5a, which can promote activation of Rac1 to enhance CLL-cell proliferation and survival. In this study, we found that CLL cells of patients treated with ibrutinib had activated Rac1. Moreover, Wnt5a could induce Rac1 activation and enhance proliferation of CLL cells treated with ibrutinib at concentrations that were effective in completely inhibiting BTK and BCR-signaling. Wnt5a-induced Rac1 activation could be blocked by cirmtuzumab (UC-961), an anti-ROR1 mAb. We found that treatment with cirmtuzumab and ibrutinib was significantly more effective than treatment with either agent alone in clearing leukemia cells in vivo. This study indicates that cirmtuzumab may enhance the activity of ibrutinib in the treatment of patients with CLL or other ROR1 + B-cell malignancies.

  13. Interplay between TGF-β signaling and receptor tyrosine kinases in tumor development.

    Science.gov (United States)

    Shi, Qiaoni; Chen, Ye-Guang

    2017-10-01

    Transforming growth factor-β (TGF-β) signaling regulates cell proliferation, differentiation, migration and death, and plays a critical role in embryogenesis and tissue homeostasis. Its deregulation results in various diseases including tumor formation. Receptor tyrosine kinases (RTKs), such as epidermal growth factor receptor (EGFR), fibroblast growth factor receptor (FGFR), vascular endothelial growth factor receptor (VEGFR) and platelet-derived growth factor receptor (PDGFR), also play key roles in the development and progression of many types of tumors. It has been realized that TGF-β signaling and RTK pathways interact with each other and their interplay is important for cancer development. They are mutually regulated and cooperatively modulate cell survival and migration, epithelial-mesenchymal transition, and tumor microenvironment to accelerate tumorigenesis and tumor metastasis. RTKs can modulate Smad-dependent transcription or cooperate with TGF-β to potentiate its oncogenic activity, while TGF-β signaling can in turn control RTK signaling by regulating their activities or expression. This review summarizes current understandings of the interplay between TGF-β signaling and RTKs and its influence on tumor development.

  14. P2X receptor-mediated ATP purinergic signaling in health and disease

    Directory of Open Access Journals (Sweden)

    Jiang LH

    2012-09-01

    Full Text Available Lin-Hua JiangSchool of Biomedical Sciences, Faculty of Biological Sciences, University of Leeds, Leeds, United KingdomAbstract: Purinergic P2X receptors are plasma membrane proteins present in a wide range of mammalian cells where they act as a cellular sensor, enabling cells to detect and respond to extracellular adenosine triphosphate (ATP, an important signaling molecule. P2X receptors function as ligand-gated Ca2+-permeable cationic channels that open upon ATP binding to elevate intracellular Ca2+ concentrations and cause membrane depolarization. In response to sustained activation, P2X receptors induce formation of a pore permeable to large molecules. P2X receptors also interact with distinct functional proteins and membrane lipids to form specialized signaling complexes. Studies have provided compelling evidence to show that such P2X receptor-mediated ATP-signaling mechanisms determine and regulate a growing number and diversity of important physiological processes, including neurotransmission, muscle contraction, and cytokine release. There is accumulating evidence to support strong causative relationships of altered receptor expression and function with chronic pain, inflammatory diseases, cancers, and other pathologies or diseases. Numerous high throughput screening drug discovery programs and preclinical studies have thus far demonstrated the proof of concepts that the P2X receptors are druggable targets and selective receptor antagonism is a promising therapeutics approach. This review will discuss the recent progress in understanding the mammalian P2X receptors with respect to the ATP-signaling mechanisms, physiological and pathophysiological roles, and development and preclinical studies of receptor antagonists.Keywords: extracellular ATP, ion channel, large pore, signaling complex, chronic pain, inflammatory diseases

  15. Anaerobic Biochemical Reactor (BCR) Treatment Of Mining-Influenced Water (MIW) - Investigation Of Metal Removal Efficiency and Ecotoxicity

    Science.gov (United States)

    BCR have been successful at removing a high percentage of metals from MIW, while BCR effluent toxicity has not been examined previously in the field. This study examined 4 active pilot BCR systems for removal of metals and toxicity. Removal efficiency for Al, As, Cd, Cu, Ni, Pb...

  16. Cell surface receptors for signal transduction and ligand transport: a design principles study.

    Directory of Open Access Journals (Sweden)

    Harish Shankaran

    2007-06-01

    Full Text Available Receptors constitute the interface of cells to their external environment. These molecules bind specific ligands involved in multiple processes, such as signal transduction and nutrient transport. Although a variety of cell surface receptors undergo endocytosis, the systems-level design principles that govern the evolution of receptor trafficking dynamics are far from fully understood. We have constructed a generalized mathematical model of receptor-ligand binding and internalization to understand how receptor internalization dynamics encodes receptor function and regulation. A given signaling or transport receptor system represents a particular implementation of this module with a specific set of kinetic parameters. Parametric analysis of the response of receptor systems to ligand inputs reveals that receptor systems can be characterized as being: i avidity-controlled where the response control depends primarily on the extracellular ligand capture efficiency, ii consumption-controlled where the ability to internalize surface-bound ligand is the primary control parameter, and iii dual-sensitivity where both the avidity and consumption parameters are important. We show that the transferrin and low-density lipoprotein receptors are avidity-controlled, the vitellogenin receptor is consumption-controlled, and the epidermal growth factor receptor is a dual-sensitivity receptor. Significantly, we show that ligand-induced endocytosis is a mechanism to enhance the accuracy of signaling receptors rather than merely serving to attenuate signaling. Our analysis reveals that the location of a receptor system in the avidity-consumption parameter space can be used to understand both its function and its regulation.

  17. Signal transduction through the IL-4 and insulin receptor families.

    Science.gov (United States)

    Wang, L M; Keegan, A; Frankel, M; Paul, W E; Pierce, J H

    1995-07-01

    Activation of tyrosine kinase-containing receptors and intracellular tyrosine kinases by ligand stimulation is known to be crucial for mediating initial and subsequent events involved in mitogenic signal transduction. Receptors for insulin and insulin-like growth factor 1 (IGF-1) contain cytoplasmic tyrosine kinase domains that undergo autophosphorylation upon ligand stimulation. Activation of these receptors also leads to pronounced and rapid tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1) in cells of connective tissue origin. A related substrate, designated 4PS, is similarly phosphorylated by insulin and IGF-1 stimulation in many hematopoietic cell types. IRS-1 and 4PS possess a number of tyrosine phosphorylation sites that are within motifs that bind specific SH2-containing molecules known to be involved in mitogenic signaling such as PI-3 kinase, SHPTP-2 (Syp) and Grb-2. Thus, they appear to act as docking substrates for a variety of signaling molecules. The majority of hematopoietic cytokines bind to receptors that do not possess intrinsic kinase activity, and these receptors have been collectively termed as members of the hematopoietin receptor superfamily. Despite their lack of tyrosine kinase domains, stimulation of these receptors has been demonstrated to activate intracellular kinases leading to tyrosine phosphorylation of multiple substrates. Recent evidence has demonstrated that activation of different members of the Janus family of tyrosine kinases is involved in mediating tyrosine phosphorylation events by specific cytokines. Stimulation of the interleukin 4 (IL-4) receptor, a member of the hematopoietin receptor superfamily, is thought to result in activation of Jak1, Jak3, and/or Fes tyrosine kinases.(ABSTRACT TRUNCATED AT 250 WORDS)

  18. Optodynamic simulation of β-adrenergic receptor signalling.

    Science.gov (United States)

    Siuda, Edward R; McCall, Jordan G; Al-Hasani, Ream; Shin, Gunchul; Il Park, Sung; Schmidt, Martin J; Anderson, Sonya L; Planer, William J; Rogers, John A; Bruchas, Michael R

    2015-09-28

    Optogenetics has provided a revolutionary approach to dissecting biological phenomena. However, the generation and use of optically active GPCRs in these contexts is limited and it is unclear how well an opsin-chimera GPCR might mimic endogenous receptor activity. Here we show that a chimeric rhodopsin/β2 adrenergic receptor (opto-β2AR) is similar in dynamics to endogenous β2AR in terms of: cAMP generation, MAP kinase activation and receptor internalization. In addition, we develop and characterize a novel toolset of optically active, functionally selective GPCRs that can bias intracellular signalling cascades towards either G-protein or arrestin-mediated cAMP and MAP kinase pathways. Finally, we show how photoactivation of opto-β2AR in vivo modulates neuronal activity and induces anxiety-like behavioural states in both fiber-tethered and wireless, freely moving animals when expressed in brain regions known to contain β2ARs. These new GPCR approaches enhance the utility of optogenetics and allow for discrete spatiotemporal control of GPCR signalling in vitro and in vivo.

  19. BCR ligation induced by IgM stimulation results in gene expression and functional changes only in IgV H unmutated chronic lymphocytic leukemia (CLL) cells.

    Science.gov (United States)

    Guarini, Anna; Chiaretti, Sabina; Tavolaro, Simona; Maggio, Roberta; Peragine, Nadia; Citarella, Franca; Ricciardi, Maria Rosaria; Santangelo, Simona; Marinelli, Marilisa; De Propris, Maria Stefania; Messina, Monica; Mauro, Francesca Romana; Del Giudice, Ilaria; Foà, Robert

    2008-08-01

    Chronic lymphocytic leukemia (CLL) patients exhibit a variable clinical course. To investigate the association between clinicobiologic features and responsiveness of CLL cells to anti-IgM stimulation, we evaluated gene expression changes and modifications in cell-cycle distribution, proliferation, and apoptosis of IgV(H) mutated (M) and unmutated (UM) samples upon BCR cross-linking. Unsupervised analysis highlighted a different response profile to BCR stimulation between UM and M samples. Supervised analysis identified several genes modulated exclusively in the UM cases upon BCR cross-linking. Functional gene groups, including signal transduction, transcription, cell-cycle regulation, and cytoskeleton organization, were up-regulated upon stimulation in UM cases. Cell-cycle and proliferation analyses confirmed that IgM cross-linking induced a significant progression into the G(1) phase and a moderate increase of proliferative activity exclusively in UM patients. Moreover, we observed only a small reduction in the percentage of subG(0/1) cells, without changes in apoptosis, in UM cases; contrariwise, a significant increase of apoptotic levels was observed in stimulated cells from M cases. These results document that a differential genotypic and functional response to BCR ligation between IgV(H) M and UM cases is operational in CLL, indicating that response to antigenic stimulation plays a pivotal role in disease progression.

  20. β1-adrenergic receptors activate two distinct signaling pathways in striatal neurons

    Science.gov (United States)

    Meitzen, John; Luoma, Jessie I.; Stern, Christopher M.; Mermelstein, Paul G.

    2010-01-01

    Monoamine action in the dorsal striatum and nucleus accumbens plays essential roles in striatal physiology. Although research often focuses on dopamine and its receptors, norepinephrine and adrenergic receptors are also crucial in regulating striatal function. While noradrenergic neurotransmission has been identified in the striatum, little is known regarding the signaling pathways activated by β-adrenergic receptors in this brain region. Using cultured striatal neurons, we characterized a novel signaling pathway by which activation of β1-adrenergic receptors leads to the rapid phosphorylation of cAMP Response Element Binding Protein (CREB), a transcription-factor implicated as a molecular switch underlying long-term changes in brain function. Norepinephrine-mediated CREB phosphorylation requires β1-adrenergic receptor stimulation of a receptor tyrosine kinase, ultimately leading to the activation of a Ras/Raf/MEK/MAPK/MSK signaling pathway. Activation of β1-adrenergic receptors also induces CRE-dependent transcription and increased c-fos expression. In addition, stimulation of β1-adrenergic receptors produces cAMP production, but surprisingly, β1-adrenergic receptor activation of adenylyl cyclase was not functionally linked to rapid CREB phosphorylation. These findings demonstrate that activation of β1-adrenergic receptors on striatal neurons can stimulate two distinct signaling pathways. These adrenergic actions can produce long-term changes in gene expression, as well as rapidly modulate cellular physiology. By elucidating the mechanisms by which norepinephrine and β1-adrenergic receptor activation affects striatal physiology, we provide the means to more fully understand the role of monoamines in modulating striatal function, specifically how norepinephrine and β1-adrenergic receptors may affect striatal physiology. PMID:21143600

  1. Complement's participation in acquired immunity

    DEFF Research Database (Denmark)

    Nielsen, Claus Henrik; Leslie, Robert Graham Quinton

    2002-01-01

    of the B cell receptor for antigen (BCR), a complex composed of the iC3b/C3d fragment-binding complement type 2 receptor (CR2, CD21) and its signaling element CD19 and the IgG-binding receptor FcgammaRIIb (CD32). The positive or negative outcome of signaling through this triad is determined by the context...

  2. Multiple roles and therapeutic implications of Akt signaling in cancer

    Directory of Open Access Journals (Sweden)

    Emiliano Calvo

    2009-06-01

    Full Text Available Emiliano Calvo1, Victoria Bolós2, Enrique Grande21Centro Integral Oncológico Clara Campal (CiOCC, Madrid. Spain; 2Pfizer Oncology, Alcobendas-Madrid, SpainAbstract: The prominence of the PI3K-Akt signaling pathway in several tumors indicates a relationship with tumor grade and proliferation. Critical cellular processes are driven through this pathway. More detailed knowledge of the pathogenesis of tumors would enable us to design targeted drugs to block both membrane tyrosine kinase receptors and the intracellular kinases involved in the transmission of the signal. The newly approved molecular inhibitors sunitinib (an inhibitor of vascular endothelial growth factor receptor, platelet-derived growth factor receptor, and other tyrosine kinase receptors, sorafenib (a serine–threonine kinase inhibitor that acts against B-Raf and temsirolimus (an mTOR inhibitor shown clinical activity in advanced kidney cancer. Chronic myeloid leukemia has changed its natural history thanks to imatinib and dasatinib, both of which inhibit the intracellular bcr/abl protein derived from the alteration in the Philadelphia chromosome. Intracellular pathways are still important in cancer development and their blockade directly affects outcome. Cross-talk has been observed but is not well understood. Vertical and horizontal pathway blockade are promising anticancer strategies. Indeed, preclinical and early clinical data suggest that combining superficial and intracellular blocking agents can synergize and leverage single-agent activity. The implication of the Akt signaling pathway in cancer is well established and has led to the development of new anticancer agents that block its activation.Keywords: Akt, cancer, therapeutic target, Akt inhibitors

  3. DMPD: TGF-beta signaling from receptors to the nucleus. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 10611754 TGF-beta signaling from receptors to the nucleus. Roberts AB. Microbes Inf...ect. 1999 Dec;1(15):1265-73. (.png) (.svg) (.html) (.csml) Show TGF-beta signaling from receptors to the nucleus.... PubmedID 10611754 Title TGF-beta signaling from receptors to the nucleus. Authors Roberts AB. Publicat

  4. Autophagy induction by Bcr-Abl-expressing cells facilitates their recovery from a targeted or nontargeted treatment.

    LENUS (Irish Health Repository)

    Crowley, Lisa C

    2012-01-31

    Although Imatinib has transformed the treatment of chronic myeloid leukemia (CML), it is not curative due to the persistence of resistant cells that can regenerate the disease. We have examined how Bcr-Abl-expressing cells respond to two mechanistically different therapeutic agents, etoposide and Imatinib. We also examined Bcr-Abl expression at low and high levels as elevated expression has been associated with treatment failure. Cells expressing low levels of Bcr-Abl undergo apoptosis in response to the DNA-targeting agent (etoposide), whereas high-Bcr-Abl-expressing cells primarily induce autophagy. Autophagic populations engage a delayed nonapoptotic death; however, sufficient cells evade this and repopulate following the withdrawal of the drug. Non-Bcr-Abl-expressing 32D or Ba\\/F3 cells induce both apoptosis and autophagy in response to etoposide and can recover. Imatinib treatment induces both apoptosis and autophagy in all Bcr-Abl-expressing cells and populations rapidly recover. Inhibition of autophagy with ATG7 and Beclin1 siRNA significantly reduced the recovery of Imatinib-treated K562 cells, indicating the importance of autophagy for the recovery of treated cells. Combination regimes incorporating agents that disrupt Imatinib-induced autophagy would remain primarily targeted and may improve response to the treatment in CML.

  5. Negative Regulation of Receptor Tyrosine Kinase (RTK Signaling: A Developing Field

    Directory of Open Access Journals (Sweden)

    Fernanda Ledda

    2007-01-01

    Full Text Available ophic factors control cellular physiology by activating specific receptor tyrosine kinases (RTKs. While the over activation of RTK signaling pathways is associated with cell growth and cancer, recent findings support the concept that impaired down-regulation or deactivation of RTKs may also be a mechanism involved in tumor formation. Under this perspective, the molecular determinants of RTK signaling inhibition may act as tumor-suppressor genes and have a potential role as tumor markers to monitor and predict disease progression. Here, we review the current understanding of the physiological mechanisms that attenuate RTK signaling and discuss evidence that implicates deregulation of these events in cancer.Abbreviations: BDP1: Brain-derived phosphatase 1; Cbl: Casitas B-lineage lymphoma; CIN-85: Cbl-interacting protein of 85 kDa; DER: Drosophila EGFR; EGFR: Epidermal growth factor receptor; ERK 1/2: Extracellular signal-regulated kinase 1/2; Grb2: Growth factor receptor-bound protein 2; HER2: Human epidermal growth factor receptor 2; LRIG: Leucine-rich repeats and immunoglobulin-like domain 1; MAPK: Mitogen-activated protein kinase; Mig 6: Mitogen-inducible gene 6; PTEN: Phosphatase and tensin homologue; RET: Rearranged in transformation; RTK: Receptor tyrosine kinase. SH2 domain: Src-homology 2 domain; SH3 domain: Src-homology 3 domain; Spry: Sprouty.

  6. Distinct Phosphorylation Clusters Determine the Signaling Outcome of Free Fatty Acid Receptor 4/G Protein-Coupled Receptor 120

    DEFF Research Database (Denmark)

    Prihandoko, Rudi; Alvarez-Curto, Elisa; Hudson, Brian D

    2016-01-01

    of these phosphoacceptor sites to alanine completely prevented phosphorylation of mFFA4 but did not limit receptor coupling to extracellular signal regulated protein kinase 1 and 2 (ERK1/2) activation. Rather, an inhibitor of Gq/11proteins completely prevented receptor signaling to ERK1/2. By contrast, the recruitment...... activation. These unique observations define differential effects on signaling mediated by phosphorylation at distinct locations. This hallmark feature supports the possibility that the signaling outcome of mFFA4 activation can be determined by the pattern of phosphorylation (phosphorylation barcode...

  7. Pattern-recognition receptors: signaling pathways and dysregulation in canine chronic enteropathies-brief review.

    Science.gov (United States)

    Heilmann, Romy M; Allenspach, Karin

    2017-11-01

    Pattern-recognition receptors (PRRs) are expressed by innate immune cells and recognize pathogen-associated molecular patterns (PAMPs) as well as endogenous damage-associated molecular pattern (DAMP) molecules. With a large potential for synergism or convergence between their signaling pathways, PRRs orchestrate a complex interplay of cellular mediators and transcription factors, and thus play a central role in homeostasis and host defense. Aberrant activation of PRR signaling, mutations of the receptors and/or their downstream signaling molecules, and/or DAMP/PAMP complex-mediated receptor signaling can potentially lead to chronic auto-inflammatory diseases or development of cancer. PRR signaling pathways appear to also present an interesting new avenue for the modulation of inflammatory responses and to serve as potential novel therapeutic targets. Evidence for a dysregulation of the PRR toll-like receptor (TLR)2, TLR4, TLR5, and TLR9, nucleotide-binding oligomerization domain-containing protein (NOD)2, and the receptor of advanced glycation end products (RAGE) exists in dogs with chronic enteropathies. We describe the TLR, NOD2, and RAGE signaling pathways and evaluate the current veterinary literature-in comparison to human medicine-to determine the role of TLRs, NOD2, and RAGE in canine chronic enteropathies.

  8. DMPD: Signals and receptors involved in recruitment of inflammatory cells. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 7744810 Signals and receptors involved in recruitment of inflammatory cells. Ben-Ba...ow Signals and receptors involved in recruitment of inflammatory cells. PubmedID 7744810 Title Signals and receptors involved in recr...uitment of inflammatory cells. Authors Ben-Baruch A, Mic

  9. A comprehensive target selectivity survey of the BCR-ABL kinase inhibitor INNO-406 by kinase profiling and chemical proteomics in chronic myeloid leukemia cells.

    Science.gov (United States)

    Rix, U; Remsing Rix, L L; Terker, A S; Fernbach, N V; Hantschel, O; Planyavsky, M; Breitwieser, F P; Herrmann, H; Colinge, J; Bennett, K L; Augustin, M; Till, J H; Heinrich, M C; Valent, P; Superti-Furga, G

    2010-01-01

    Resistance to the BCR-ABL tyrosine kinase inhibitor imatinib poses a pressing challenge in treating chronic myeloid leukemia (CML). This resistance is often caused by point mutations in the ABL kinase domain or by overexpression of LYN. The second-generation BCR-ABL inhibitor INNO-406 is known to inhibit most BCR-ABL mutants and LYN efficiently. Knowledge of its full target spectrum would provide the molecular basis for potential side effects or suggest novel therapeutic applications and possible combination therapies. We have performed an unbiased chemical proteomics native target profile of INNO-406 in CML cells combined with functional assays using 272 recombinant kinases thereby identifying several new INNO-406 targets. These include the kinases ZAK, DDR1/2 and various ephrin receptors. The oxidoreductase NQO2, inhibited by both imatinib and nilotinib, is not a relevant target of INNO-406. Overall, INNO-406 has an improved activity over imatinib but a slightly broader target profile than both imatinib and nilotinib. In contrast to dasatinib and bosutinib, INNO-406 does not inhibit all SRC kinases and most TEC family kinases and is therefore expected to elicit fewer side effects. Altogether, these properties may make INNO-406 a valuable component in the drug arsenal against CML.

  10. β1-adrenergic receptor stimulation by agonist Compound 49b restores insulin receptor signal transduction in vivo

    Science.gov (United States)

    Jiang, Youde; Zhang, Qiuhua; Ye, Eun-Ah

    2014-01-01

    Purpose Determine whether Compound 49b treatment ameliorates retinal changes due to the lack of β2-adrenergic receptor signaling. Methods Using retinas from 3-month-old β2-adrenergic receptor-deficient mice, we treated mice with our novel β1-/β2-adrenergic receptor agonist, Compound 49b, to assess the effects of adrenergic agonists acting only on β1-adrenergic receptors due to the absence of β2-adrenergic receptors. Western blotting or enzyme-linked immunosorbent assay (ELISA) analyses were performed for β1- and β2-adrenergic receptors, as well as key insulin resistance proteins, including TNF-α, SOCS3, IRS-1Ser307, and IRTyr960. Analyses were also performed on key anti- and proapoptotic proteins: Akt, Bcl-xL, Bax, and caspase 3. Electroretinogram analyses were conducted to assess functional changes, while histological assessment was conducted for changes in retinal thickness. Results A 2-month treatment of β2-adrenergic receptor-deficient mice with daily eye drops of 1 mM Compound 49b, a novel β1- and β2-adrenergic receptor agonist, reversed the changes in insulin resistance markers (TNF-α and SOCS3) observed in untreated β2-adrenergic receptor-deficient mice, and concomitantly increased morphological integrity (retinal thickness) and functional responses (electroretinogram amplitude). These results suggest that stimulating β1-adrenergic receptors on retinal endothelial cells or Müller cells can compensate for the loss of β2-adrenergic receptor signaling on Müller cells, restore insulin signal transduction, reduce retinal apoptosis, and enhance retinal function. Conclusions Since our previous studies with β1-adrenergic receptor knockout mice confirmed that the reverse also occurs (β2-adrenergic receptor stimulation can compensate for the loss of β1-adrenergic receptor activity), it appears that increased activity in either of these pathways alone is sufficient to block insulin resistance–based retinal cell apoptosis. PMID:24966659

  11. Clinical relevance of the breakpoint sites within the M-BCR in 50 patients from Argentina with chronic myeloid leukemia.

    Science.gov (United States)

    Giere, I A; Larripa, I B

    1996-08-01

    Fifty patients from Argentina with chronic myeloid leukemia (CML) were studied in order to characterize the breakpoint site within the major breakpoint cluster region (M-BCR) and its relationship with the duration of the chronic phase (CP). The DNA digestion with the restriction enzymes: Bgl II, BAM HI and Hind III and hybridization with the 1.2Kb Hind III-Bgl II bcr probe showed that 56% of cases had the breakpoint in 5'M-bcr region and the remaining 44% in 3'M-bcr region. The duration of chronic phase from diagnosis to the onset of the blast crisis (BC) was correlated with the location of the breakpoint within the M-bcr and no statistical differences were observed between the 5' and the 3' groups. These data indicate that the breakpoint site within the bcr gene is not a prognostic indicator of the duration of CP of the disease.

  12. TAM Receptor Signaling in Immune Homeostasis

    Science.gov (United States)

    Rothlin, Carla V.; Carrera-Silva, Eugenio A.; Bosurgi, Lidia; Ghosh, Sourav

    2015-01-01

    The TAM receptor tyrosine kinases (RTKs)—TYRO3, AXL, and MERTK—together with their cognate agonists GAS6 and PROS1 play an essential role in the resolution of inflammation. Deficiencies in TAM signaling have been associated with chronic inflammatory and autoimmune diseases. Three processes regulated by TAM signaling may contribute, either independently or collectively, to immune homeostasis: the negative regulation of the innate immune response, the phagocytosis of apoptotic cells, and the restoration of vascular integrity. Recent studies have also revealed the function of TAMs in infectious diseases and cancer. Here, we review the important milestones in the discovery of these RTKs and their ligands and the studies that underscore the functional importance of this signaling pathway in physiological immune settings and disease. PMID:25594431

  13. Mechanism of inhibition of growth hormone receptor signaling by suppressor of cytokine signaling proteins

    DEFF Research Database (Denmark)

    Hansen, J A; Lindberg, K; Hilton, D J

    1999-01-01

    In this study we have investigated the role of suppressor of cytokine signaling (SOCS) proteins in GH receptor-mediated signaling. GH-induced transcription was inhibited by SOCS-1 and SOCS-3, while SOCS-2 and cytokine inducible SH2-containing protein (CIS) had no effect By using chimeric SOCS pro...

  14. Death receptor Fas (CD95) signaling in the central nervous system: tuning neuroplasticity?

    Science.gov (United States)

    Reich, Arno; Spering, Christopher; Schulz, Jörg B

    2008-09-01

    For over a decade, neuroscientific research has focused on processes of apoptosis and its contribution to the pathophysiology of neurological diseases. In the central nervous system, the degree of intrinsic mitochondrial-mediated apoptotic signaling expresses a cell's individual metabolic stress, whereas activation of the extrinsic death receptor-induced cascade is regarded as a sign of imbalanced cellular networks. Under physiological conditions, most neurons possess death receptors without being sensitive to receptor-mediated apoptosis. This paradox raises two questions: what is the evolutionary advantage of expressing potentially harmful proteins? How is their signaling controlled? This review summarizes the functional relevance of FasL-Fas signaling--a quintessential death ligand/receptor system--in different neurological disease models ranging from traumatic, inflammatory and ischemic to neurodegenerative processes. Furthermore, it outlines alternative non-apoptotic Fas signaling, shedding new light on its neuroplastic capacity. Finally, receptor-proximal regulatory proteins are introduced and identified as potential protagonists of disease-modifying neurological therapies.

  15. Ric-8A, a Gα protein guanine nucleotide exchange factor potentiates taste receptor signaling

    Directory of Open Access Journals (Sweden)

    Claire J Fenech

    2009-10-01

    Full Text Available Taste receptors for sweet, bitter and umami tastants are G-protein coupled receptors (GPCRs. While much effort has been devoted to understanding G-protein-receptor interactions and identifying the components of the signalling cascade downstream of these receptors, at the level of the G-protein the modulation of receptor signal transduction remains relatively unexplored. In this regard a taste-specific regulator of G-protein signaling (RGS, RGS21, has recently been identified. To study whether guanine nucleotide exchange factors (GEFs are involved in the transduction of the signal downstream of the taste GPCRs we investigated the expression of Ric-8A and Ric-8B in mouse taste cells and their interaction with G-protein subunits found in taste buds. Mammalian Ric-8 proteins were initially identified as potent GEFs for a range of Gα subunits and Ric-8B has recently been shown to amplify olfactory signal transduction. We find that both Ric-8A and Ric-8B are expressed in a large portion of taste bud cells and that most of these cells contain IP3R-3 a marker for sweet, umami and bitter taste receptor cells. Ric-8A interacts with Gα-gustducin and Gαi2 through which it amplifies the signal transduction of hTas2R16, a receptor for bitter compounds. Overall, these findings are consistent with a role for Ric-8 in mammalian taste signal transduction.

  16. Erythropoietin receptor signaling is membrane raft dependent

    NARCIS (Netherlands)

    K.L. McGraw (Kathy); G.M. Fuhler (Gwenny); J.O. Johnson (Joseph); J.A. Clark (Justine); G.C. Caceres (Gisela); L. Sokol (Lubomir); A.F. List (Alan)

    2012-01-01

    textabstractUpon erythropoietin (Epo) engagement, Epo-receptor (R) homodimerizes to activate JAK2 and Lyn, which phosphorylate STAT5. Although recent investigations have identified key negative regulators of Epo-R signaling, little is known about the role of membrane localization in controlling

  17. Nuclear receptor TLX inhibits TGF-β signaling in glioblastoma

    International Nuclear Information System (INIS)

    Johansson, Erik; Zhai, Qiwei; Zeng, Zhao-jun; Yoshida, Takeshi; Funa, Keiko

    2016-01-01

    TLX (also called NR2E1) is an orphan nuclear receptor that maintains stemness of neuronal stem cells. TLX is highly expressed in the most malignant form of glioma, glioblastoma multiforme (GBM), and is important for the proliferation and maintenance of the stem/progenitor cells of the tumor. Transforming Growth Factor-β (TGF-β) is a cytokine regulating many different cellular processes such as differentiation, migration, adhesion, cell death and proliferation. TGF-β has an important function in cancer where it can work as either a tumor suppressor or oncogene, depending on the cancer type and stage of tumor development. Since glioblastoma often have dysfunctional TGF-β signaling we wanted to find out if there is any interaction between TLX and TGF-β in glioblastoma cells. We demonstrate that knockdown of TLX enhances the canonical TGF-β signaling response in glioblastoma cell lines. TLX physically interacts with and stabilizes Smurf1, which can ubiquitinate and target TGF-β receptor II for degradation, whereas knockdown of TLX leads to stabilization of TGF-β receptor II, increased nuclear translocation of Smad2/3 and enhanced expression of TGF-β target genes. The interaction between TLX and TGF-β may play an important role in the regulation of proliferation and tumor-initiating properties of glioblastoma cells. - Highlights: • TLX knockdown enhances TGF-β dependent Smad signaling in glioblastoma cells • TLX knockdown increases the protein level of TGF-β receptor II. • TLX stabilizes and retains Smurf1 in the cytoplasm. • TLX enhances Smurf1-dependent ubiquitination and degradation of TGF-β receptor II.

  18. Nuclear receptor TLX inhibits TGF-β signaling in glioblastoma

    Energy Technology Data Exchange (ETDEWEB)

    Johansson, Erik; Zhai, Qiwei [Sahlgrenska Cancer Center at the Sahlgrenska Academy, University of Gothenburg, Box 425, SE 405 30 Gothenburg (Sweden); Zeng, Zhao-jun [Sahlgrenska Cancer Center at the Sahlgrenska Academy, University of Gothenburg, Box 425, SE 405 30 Gothenburg (Sweden); Molecular Biology Research Center, School of Life Sciences, Central South University, 110, Xiangya Road, Changsha, Hunan 410078 (China); Yoshida, Takeshi [Sahlgrenska Cancer Center at the Sahlgrenska Academy, University of Gothenburg, Box 425, SE 405 30 Gothenburg (Sweden); Funa, Keiko, E-mail: keiko.funa@gu.se [Sahlgrenska Cancer Center at the Sahlgrenska Academy, University of Gothenburg, Box 425, SE 405 30 Gothenburg (Sweden)

    2016-05-01

    TLX (also called NR2E1) is an orphan nuclear receptor that maintains stemness of neuronal stem cells. TLX is highly expressed in the most malignant form of glioma, glioblastoma multiforme (GBM), and is important for the proliferation and maintenance of the stem/progenitor cells of the tumor. Transforming Growth Factor-β (TGF-β) is a cytokine regulating many different cellular processes such as differentiation, migration, adhesion, cell death and proliferation. TGF-β has an important function in cancer where it can work as either a tumor suppressor or oncogene, depending on the cancer type and stage of tumor development. Since glioblastoma often have dysfunctional TGF-β signaling we wanted to find out if there is any interaction between TLX and TGF-β in glioblastoma cells. We demonstrate that knockdown of TLX enhances the canonical TGF-β signaling response in glioblastoma cell lines. TLX physically interacts with and stabilizes Smurf1, which can ubiquitinate and target TGF-β receptor II for degradation, whereas knockdown of TLX leads to stabilization of TGF-β receptor II, increased nuclear translocation of Smad2/3 and enhanced expression of TGF-β target genes. The interaction between TLX and TGF-β may play an important role in the regulation of proliferation and tumor-initiating properties of glioblastoma cells. - Highlights: • TLX knockdown enhances TGF-β dependent Smad signaling in glioblastoma cells • TLX knockdown increases the protein level of TGF-β receptor II. • TLX stabilizes and retains Smurf1 in the cytoplasm. • TLX enhances Smurf1-dependent ubiquitination and degradation of TGF-β receptor II.

  19. Susceptibility of Ph-positive all to TKI therapy associated with Bcr-Abl rearrangement patterns: a retrospective analysis.

    Directory of Open Access Journals (Sweden)

    Yu Jing

    Full Text Available BACKGROUND: Tyrosine kinase inhibitors (TKIs have demonstrated success in the treatment of acute lymphoblastic leukemia (ALL in patients that express BCR-ABL rearrangements (Philadelphia chromosome [Ph]. The current study aimed to assess the efficacy of TKIs and prognostic factors in the treatment of adults with Ph+-ALL. METHODS: In this multicenter retrospective study, the relationship between Ph+-ALL and treatment outcomes among Chinese patients receiving TKI-containing induction/consolidation chemotherapy was examined. A total of 86 Ph+-ALL patients were included and followed for 3.85 (0.43-9.30 years. Overall survival (OS and event-free survival (EFS were analyzed. RESULTS: A total of 86 Ph+-ALL patients (40 females and 46 males; median age: 34.0 years were enrolled, including those with BCR/ABL transcripts 190 (n = 52, 210 (n = 25, and 230 (n = 2; BCR/ABL isoform determination was not available for 7 patients. Mortality was influenced by variable BCR/ABL transcripts and TKI administration, and BCR/ABL transcripts, hematopoietic stem cell transplantation (HSCT, and TKI administration were associated with the occurrence of events. The OS rate in the TKI administration group during steady state was significantly higher compared with those patients who did not receive TKI administration (P = 0.008, the EFS rate in the TKI administration group during steady state was significantly higher compared with those patients who did not receive TKIs (P = 0.012, and also higher than those with TKI salvage administration (P = 0.004. BCR/ABL transcripts 210 showed preferable OS and EFS compared with BCR/ABL transcripts 190 and 230 (P<0.05 for each. CONCLUSIONS: The susceptibility of Ph+-ALL to TKI associated with the patterns of BCR-ABL rearrangement is demonstrated for the first time, thus adding another risk-stratifying molecular prognostic tool for the management of patients with Ph+-ALL.

  20. IgM and IgD B cell receptors differentially respond to endogenous antigens and control B cell fate

    Science.gov (United States)

    Noviski, Mark; Mueller, James L; Satterthwaite, Anne; Garrett-Sinha, Lee Ann; Brombacher, Frank

    2018-01-01

    Naive B cells co-express two BCR isotypes, IgM and IgD, with identical antigen-binding domains but distinct constant regions. IgM but not IgD is downregulated on autoreactive B cells. Because these isotypes are presumed to be redundant, it is unknown how this could impose tolerance. We introduced the Nur77-eGFP reporter of BCR signaling into mice that express each BCR isotype alone. Despite signaling strongly in vitro, IgD is less sensitive than IgM to endogenous antigen in vivo and developmental fate decisions are skewed accordingly. IgD-only Lyn−/− B cells cannot generate autoantibodies and short-lived plasma cells (SLPCs) in vivo, a fate thought to be driven by intense BCR signaling induced by endogenous antigens. Similarly, IgD-only B cells generate normal germinal center, but impaired IgG1+ SLPC responses to T-dependent immunization. We propose a role for IgD in maintaining the quiescence of autoreactive B cells and restricting their differentiation into autoantibody secreting cells. PMID:29521626

  1. DMPD: Is HIV infection a TNF receptor signalling-driven disease? [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 18178131 Is HIV infection a TNF receptor signalling-driven disease? Herbein G, Khan... KA. Trends Immunol. 2008 Feb;29(2):61-7. (.png) (.svg) (.html) (.csml) Show Is HIV infection a TNF receptor signalling-driven dise...ase? PubmedID 18178131 Title Is HIV infection a TNF receptor signalling-driven diseas

  2. Metformin Effects on Biochemical Recurrence and Metabolic Signaling in the Prostate

    Science.gov (United States)

    Winters, Brian; Plymate, Stephen; Zeliadt, Steven B; Holt, Sarah; Zhang, Xiaotun; Hu, Elaine; Lin, Daniel W.; Morrissey, Colm; Wooldridge, Bryan; Gore, John L; Porter, Michael P; Wright, Jonathan L

    2015-01-01

    Background Metformin has received considerable attention as a potential anti-cancer agent. Animal and in-vitro prostate cancer (PCa) models have demonstrated decreased tumor growth with metformin, however the precise mechanisms are unknown. We examine the effects of metformin on PCa biochemical recurrence (BCR) in a large clinical database followed by evaluating metabolic signaling changes in a cohort of men undergoing prostate needle biopsy (PNB). Methods Men treated for localized PCa were identified in a comprehensive clinical database between 2001 and 2010. Cox regression was performed to determine association with BCR relative to metformin use. We next identified a separate case-control cohort of men undergoing prostate needle biopsy (PNB) stratified by metformin use. Differences in mean IHC scores were compared with linear regression for phosphorylated IR, IGF-IR, AKT, and AMPK. Results 1,734 men were evaluated for BCR with mean follow up of 41 months (range 1-121 months). ‘Ever’ metformin use was not associated with BCR (HR 1.12, 0.77-1.65), however men reporting both pre/post-treatment metformin use had a 45% reduction in BCR (HR=0.55 (0.31-0.96)). For the tissue-based study, 48 metformin users and 42 controls underwent PNB. Significantly greater staining in phosphorylated nuclear (p-IR, p-AKT) and cytoplasmic (p-IR, p-IGF-1R) insulin signaling proteins were seen in patients with PCa detected compared to those with negative PNB (p-values all < 0.006). When stratified by metformin use, IGF-1R remained significantly elevated (p=0.01) in men with PCa detected whereas p-AMPK (p=0.05) was elevated only in those without PCa. Conclusion Metformin use is associated with reduced BCR after treatment of localized PCa when considering pre-diagnostic and cumulative dosing. In men with cancer detected on PNB, insulin signaling markers were significantly elevated compared to negative PNB patients. The finding of IGF-1R elevation in positive PNBs versus p-AMPK elevation

  3. Targeting Bruton Tyrosine Kinase: A novel strategy in the treatment of B-cell lymphomas

    Directory of Open Access Journals (Sweden)

    Sklavenitis-Pistofidis R.

    2017-06-01

    Full Text Available In normal B-cells, Bruton tyrosine kinase (Btk, a non-receptor tyrosine kinase involved in B-cell receptor (BCR signalling, is essential for cell survival and maturation. Not surprisingly, Btk is also implicated in the pathogenesis of B-cell lymphomas, like Chronic Lymphocytic Leukaemia/Small Lymphocytic Lymphoma (CLL/SLL, Mantle Cell Lymphoma (MCL and Waldenström’s Macroglobulinemia (WM, which are driven by aberrant BCR signalling. Thus, targeting Btk represents a promising therapeutic strategy in the treatment of B-cell lymphoma patients. Ibrutinib, a selective Btk inhibitor, has already been approved as second-line treatment of CLL/SLL, MCL and WM patients, while more clinical studies of ibrutinib and novel Btk inhibitors are currently under way. In light of results of the RESONATE-2 trial, the approval of ibrutinib as a first-line treatment of CLL/SLL may well be approaching. Herein, we review Btk’s role in normal and malignant BCR signalling, as well as ibrutinib’s performance in B-cell lymphoma treatment and prognosis.

  4. Unexpected heterogeneity of BCR-ABL fusion mRNA detected by polymerase chain reaction in Philadelphia chromosome-positive acute lymphoblastic leukemia

    International Nuclear Information System (INIS)

    Hooberman, A.L.; Carrino, J.J.; Leibowitz, D.; Rowley, J.D.; Le Beau, M.M.; Arlin, Z.A.; Westbrook, C.A.

    1989-01-01

    The Philadelphia (Ph 1 ) chromosome results in a fusion of portions of the BCR gene from chromosome 22 and the ABL gene from chromosome 9, producing a chimeric BCR-ABL mRNA and protein. In lymphoblastic leukemias, there are two molecular subtypes of the Ph 1 chromosome, one with a rearrangement of the breakpoint cluster region (bcr) of the BCR gene, producing the same 8.5-kilobase BCR-ABL fusion mRNA seen in chronic myelogenous leukemia (CML), and the other, without a bcr rearrangement, producing a 7.0-kilobase BCR-ABL fusion mRNA that is seen only in acute lymphoblastic leukemia (ALL). The authors studied the molecular subtype of the Ph 1 chromosome in 11 cases of Ph 1 -positive ALL, including 2 with a previous diagnosis of CML, using a sensitive method to analyze the mRNA species based on the polymerase chain reaction (PCR). They observed unexpected heterogeneity in BCR-ABL mRNA in this population. They conclude that the PCR gives additional information about the Ph 1 chromosome gene products that cannot be obtained by genomic analysis, but that it cannot be used as the sole means of detection of this chromosomal abnormality in ALL because of the high incidence of false negative results

  5. Phenobarbital indirectly activates the constitutive active androstane receptor (CAR) by inhibition of epidermal growth factor receptor signaling.

    Science.gov (United States)

    Mutoh, Shingo; Sobhany, Mack; Moore, Rick; Perera, Lalith; Pedersen, Lee; Sueyoshi, Tatsuya; Negishi, Masahiko

    2013-05-07

    Phenobarbital is a central nervous system depressant that also indirectly activates nuclear receptor constitutive active androstane receptor (CAR), which promotes drug and energy metabolism, as well as cell growth (and death), in the liver. We found that phenobarbital activated CAR by inhibiting epidermal growth factor receptor (EGFR) signaling. Phenobarbital bound to EGFR and potently inhibited the binding of EGF, which prevented the activation of EGFR. This abrogation of EGFR signaling induced the dephosphorylation of receptor for activated C kinase 1 (RACK1) at Tyr(52), which then promoted the dephosphorylation of CAR at Thr(38) by the catalytic core subunit of protein phosphatase 2A. The findings demonstrated that the phenobarbital-induced mechanism of CAR dephosphorylation and activation is mediated through its direct interaction with and inhibition of EGFR.

  6. CD38 is a signaling molecule in B-cell chronic lymphocytic leukemia cells.

    Science.gov (United States)

    Deaglio, Silvia; Capobianco, Andrea; Bergui, Luciana; Dürig, Jan; Morabito, Fortunato; Dührsen, Ulrich; Malavasi, Fabio

    2003-09-15

    The prognosis for patients with B-cell chronic lymphocytic leukemia (B-CLL) is generally less favorable for those expressing CD38. Our working hypothesis is that CD38 is not merely a marker in B-CLL, but that it plays a receptor role with pathogenetic potential ruling the proliferation of the malignant clone. CD38 levels were generally low in the patients examined and monoclonal antibody (mAb) ligation was inefficient in signaling. Other cellular models indicated that molecular density and surface organization are critical for CD38 functionality. Interleukin 2 (IL-2) induced a marked up-modulation and surface rearrangement of CD38 in all the patients studied. On reaching a specific expression threshold, CD38 becomes an efficient receptor in purified B-CLL cells. Indeed, mAb ligation is followed by Ca2+ fluxes and by a markedly increased proliferation. The unsuitability of CD38 to perform as a receptor is obviated through close interaction with the B-cell-receptor (BCR) complex and CD19. On mAb binding, CD38 translocates to the membrane lipid microdomains, as shown by a colocalization with the GM1 ganglioside and with CD81, a raft-resident protein. Finally, CD38 signaling in IL-2-treated B-CLL cells prolonged survival and induced the appearance of plasmablasts, providing a pathogenetic hypothesis for the occurrence of Richter syndrome.

  7. A critical role of CDKN3 in Bcr-Abl-mediated tumorigenesis.

    Directory of Open Access Journals (Sweden)

    Qinghuang Chen

    Full Text Available CDKN3 (cyclin-dependent kinase inhibitor 3, a dual specificity protein phosphatase, dephosphorylates cyclin-dependent kinases (CDKs and thus functions as a key negative regulator of cell cycle progression. Deregulation or mutations of CDNK3 have been implicated in various cancers. However, the role of CDKN3 in Bcr-Abl-mediated chronic myelogenous leukemia (CML remains unknown. Here we found that CDKN3 acts as a tumor suppressor in Bcr-Abl-mediated leukemogenesis. Overexpression of CDKN3 sensitized the K562 leukemic cells to imanitib-induced apoptosis and dramatically inhibited K562 xenografted tumor growth in nude mouse model. Ectopic expression of CDKN3 significantly reduced the efficiency of Bcr-Abl-mediated transformation of FDCP1 cells to growth factor independence. In contrast, depletion of CDKN3 expression conferred resistance to imatinib-induced apoptosis in the leukemic cells and accelerated the growth of xenograph leukemia in mice. In addition, we found that CDKN3 mutant (CDKN3-C140S devoid of the phosphatase activity failed to affect the K562 leukemic cell survival and xenografted tumor growth, suggesting that the phosphatase of CDKN3 was required for its tumor suppressor function. Furthermore, we observed that overexpression of CDKN3 reduced the leukemic cell survival by dephosphorylating CDK2, thereby inhibiting CDK2-dependent XIAP expression. Moreover, overexpression of CDKN3 delayed G1/S transition in K562 leukemic cells. Our results highlight the importance of CDKN3 in Bcr-Abl-mediated leukemogenesis, and provide new insights into diagnostics and therapeutics of the leukemia.

  8. Signal transduction by the platelet-derived growth factor receptor

    International Nuclear Information System (INIS)

    Williams, L.T.; Escobedo, J.A.; Keating, M.T.; Coughlin, S.R.

    1988-01-01

    The mitogenic effects of platelet-derived growth factor (PDGF) are mediated by the PDGF receptor. The mouse PDGF receptor was recently purified on the basis of its ability to become tyrosine phosphorylated in response to the A-B human platelet form of PDGF, and the receptor amino acid sequence was determined from a full-length cDNA clone. Both the human and mouse receptor cDNA sequences have been expressed in Chinese hamster ovary fibroblast (CHO) cells that normally lack PDGF receptors. This paper summarizes recent results using this system to study signal transduction by the PDGF receptor. Some of the findings show that the KI domain of the PDGF receptor plays an important role in the stimulation of DNA synthesis by PDGF. Surprisingly, the kinase insert region is not essential for PDGF stimulation of PtdIns turnover, pH change, increase in cellular calcium, and receptor autophosphorylation. In addition, PDGF stimulates a conformational change in the receptor

  9. ABA receptors: The START of a new paradigm in phytohormone signalling

    KAUST Repository

    Klingler, John

    2010-06-03

    The phytohormone abscisic acid (ABA) plays a central role in plant development and in plant adaptation to both biotic and abiotic stressors. In recent years, knowledge of ABA metabolism and signal transduction has advanced rapidly to provide detailed glimpses of the hormone\\'s activities at the molecular level. Despite this progress, many gaps in understanding have remained, particularly at the early stages of ABA perception by the plant cell. The search for an ABA receptor protein has produced multiple candidates, including GCR2, GTG1, and GTG2, and CHLH. In addition to these candidates, in 2009 several research groups converged on a novel family of Arabidopsis proteins that bind ABA, and thereby interact directly with a class of protein phosphatases that are well known as critical players in ABA signal transduction. The PYR/PYL/RCAR receptor family is homologous to the Bet v 1-fold and START domain proteins. It consists of 14 members, nearly all of which appear capable of participating in an ABA receptor-signal complex that responds to the hormone by activating the transcription of ABA-responsive genes. Evidence is provided here that PYR/PYL/RCAR receptors can also drive the phosphorylation of the slow anion channel SLAC1 to provide a fast and timely response to the ABA signal. Crystallographic studies have vividly shown the mechanics of ABA binding to PYR/PYL/RCAR receptors, presenting a model that bears some resemblance to the binding of gibberellins to GID1 receptors. Since this ABA receptor family is highly conserved in crop species, its discovery is likely to usher a new wave of progress in the elucidation and manipulation of plant stress responses in agricultural settings. © 2010 The Author(s).

  10. Taste Receptor Signaling-- From Tongues to Lungs

    Science.gov (United States)

    Kinnamon, Sue C.

    2013-01-01

    Taste buds are the transducing endorgans of gustation. Each taste bud comprises 50–100 elongated cells, which extend from the basal lamina to the surface of the tongue, where their apical microvilli encounter taste stimuli in the oral cavity. Salts and acids utilize apically located ion channels for transduction, while bitter, sweet and umami (glutamate) stimuli utilize G protein coupled receptors (GPCRs) and second messenger signaling mechanisms. This review will focus on GPCR signaling mechanisms. Two classes of taste GPCRs have been identified, the T1Rs for sweet and umami (glutamate) stimuli, and the T2Rs for bitter stimuli. These low affinity GPCRs all couple to the same downstream signaling effectors that include Gβγ activation of PLCβ2, IP3-mediated release of Ca2+ from intracellular stores, and Ca2+-dependent activation of the monovalent selective cation channel, TrpM5. These events lead to membrane depolarization, action potentials, and release of ATP as a transmitter to activate gustatory afferents. The Gα subunit, α-gustducin, activates a phosphodiesterase to decrease intracellular cAMP levels, although the precise targets of cAMP have not been identified. With the molecular identification of the taste GPCRs, it has become clear that taste signaling is not limited to taste buds, but occurs in many cell types of the airways. These include solitary chemosensory cells, ciliated epithelial cells, and smooth muscle cells. Bitter receptors are most abundantly expressed in the airways, where they respond to irritating chemicals and promote protective airway reflexes, utilizing the same downstream signaling effectors as taste cells. PMID:21481196

  11. Proliferative signaling initiated in ACTH receptors

    Directory of Open Access Journals (Sweden)

    C.F.P. Lotfi

    2000-10-01

    Full Text Available This article reviews recent results of studies aiming to elucidate modes of integrating signals initiated in ACTH receptors and FGF2 receptors, within the network system of signal transduction found in Y1 adrenocortical cells. These modes of signal integration should be central to the mechanisms underlying the regulation of the G0->G1->S transition in the adrenal cell cycle. FGF2 elicits a strong mitogenic response in G0/G1-arrested Y1 adrenocortical cells, that includes a rapid and transient activation of extracellular signal-regulated kinases-mitogen-activated protein kinases (ERK-MAPK (2 to 10 min, b transcription activation of c-fos, c-jun and c-myc genes (10 to 30 min, c induction of c-Fos and c-Myc proteins by 1 h and cyclin D1 protein by 5 h, and d onset of DNA synthesis stimulation within 8 h. ACTH, itself a weak mitogen, interacts with FGF2 in a complex manner, blocking the FGF2 mitogenic response during the early and middle G1 phase, keeping ERK-MAPK activation and c-Fos and cyclin D1 induction at maximal levels, but post-transcriptionally inhibiting c-Myc expression. c-Fos and c-Jun proteins are mediators in both the strong and the weak mitogenic responses respectively triggered by FGF2 and ACTH. Induction of c-Fos and stimulation of DNA synthesis by ACTH are independent of PKA and are inhibited by the PKC inhibitor GF109203X. In addition, ACTH is a poor activator of ERK-MAPK, but c-Fos induction and DNA synthesis stimulation by ACTH are strongly inhibited by the inhibitor of MEK1 PD98059.

  12. The orphan receptor ALK7 and the Activin receptor ALK4 mediate signaling by Nodal proteins during vertebrate development

    Science.gov (United States)

    Reissmann, Eva; Jörnvall, Henrik; Blokzijl, Andries; Andersson, Olov; Chang, Chenbei; Minchiotti, Gabriella; Persico, M. Graziella; Ibáñez, Carlos F.; Brivanlou, Ali H.

    2001-01-01

    Nodal proteins have crucial roles in mesendoderm formation and left–right patterning during vertebrate development. The molecular mechanisms of signal transduction by Nodal and related ligands, however, are not fully understood. In this paper, we present biochemical and functional evidence that the orphan type I serine/threonine kinase receptor ALK7 acts as a receptor for mouse Nodal and Xenopus Nodal-related 1 (Xnr1). Receptor reconstitution experiments indicate that ALK7 collaborates with ActRIIB to confer responsiveness to Xnr1 and Nodal. Both receptors can independently bind Xnr1. In addition, Cripto, an extracellular protein genetically implicated in Nodal signaling, can independently interact with both Xnr1 and ALK7, and its expression greatly enhances the ability of ALK7 and ActRIIB to respond to Nodal ligands. The Activin receptor ALK4 is also able to mediate Nodal signaling but only in the presence of Cripto, with which it can also interact directly. A constitutively activated form of ALK7 mimics the mesendoderm-inducing activity of Xnr1 in Xenopus embryos, whereas a dominant-negative ALK7 specifically blocks the activities of Nodal and Xnr1 but has little effect on other related ligands. In contrast, a dominant-negative ALK4 blocks all mesoderm-inducing ligands tested, including Nodal, Xnr1, Xnr2, Xnr4, and Activin. In agreement with a role in Nodal signaling, ALK7 mRNA is localized to the ectodermal and organizer regions of Xenopus gastrula embryos and is expressed during early stages of mouse embryonic development. Therefore, our results indicate that both ALK4 and ALK7 can mediate signal transduction by Nodal proteins, although ALK7 appears to be a receptor more specifically dedicated to Nodal signaling. PMID:11485994

  13. Stat5 Exerts Distinct, Vital Functions in the Cytoplasm and Nucleus of Bcr-Abl+ K562 and Jak2(V617F)+ HEL Leukemia Cells

    International Nuclear Information System (INIS)

    Weber, Axel; Borghouts, Corina; Brendel, Christian; Moriggl, Richard; Delis, Natalia; Brill, Boris; Vafaizadeh, Vida; Groner, Bernd

    2015-01-01

    Signal transducers and activators of transcription (Stats) play central roles in the conversion of extracellular signals, e.g., cytokines, hormones and growth factors, into tissue and cell type specific gene expression patterns. In normal cells, their signaling potential is strictly limited in extent and duration. The persistent activation of Stat3 or Stat5 is found in many human tumor cells and contributes to their growth and survival. Stat5 activation plays a pivotal role in nearly all hematological malignancies and occurs downstream of oncogenic kinases, e.g., Bcr-Abl in chronic myeloid leukemias (CML) and Jak2(V617F) in other myeloproliferative diseases (MPD). We defined the mechanisms through which Stat5 affects growth and survival of K562 cells, representative of Bcr-Abl positive CML, and HEL cells, representative for Jak2(V617F) positive acute erythroid leukemia. In our experiments we suppressed the protein expression levels of Stat5a and Stat5b through shRNA mediated downregulation and demonstrated the dependence of cell survival on the presence of Stat5. Alternatively, we interfered with the functional capacities of the Stat5 protein through the interaction with a Stat5 specific peptide ligand. This ligand is a Stat5 specific peptide aptamer construct which comprises a 12mer peptide integrated into a modified thioredoxin scaffold, S5-DBD-PA. The peptide sequence specifically recognizes the DNA binding domain (DBD) of Stat5. Complex formation of S5-DBD-PA with Stat5 causes a strong reduction of P-Stat5 in the nuclear fraction of Bcr-Abl-transformed K562 cells and a suppression of Stat5 target genes. Distinct Stat5 mediated survival mechanisms were detected in K562 and Jak2(V617F)-transformed HEL cells. Stat5 is activated in the nuclear and cytosolic compartments of K562 cells and the S5-DBD-PA inhibitor most likely affects the viability of Bcr-Abl + K562 cells through the inhibition of canonical Stat5 induced target gene transcription. In HEL cells, Stat5

  14. Hypocretin/Orexin regulation of dopamine signaling and cocaine self-administration is mediated predominantly by hypocretin receptor 1.

    Science.gov (United States)

    Prince, Courtney D; Rau, Andrew R; Yorgason, Jordan T; España, Rodrigo A

    2015-01-21

    Extensive evidence suggests that the hypocretins/orexins influence cocaine reinforcement and dopamine signaling via actions at hypocretin receptor 1. By comparison, the involvement of hypocretin receptor 2 in reward and reinforcement processes has received relatively little attention. Thus, although there is some evidence that hypocretin receptor 2 regulates intake of some drugs of abuse, it is currently unclear to what extent hypocretin receptor 2 participates in the regulation of dopamine signaling or cocaine self-administration, particularly under high effort conditions. To address this, we examined the effects of hypocretin receptor 1, and/or hypocretin receptor 2 blockade on dopamine signaling and cocaine reinforcement. We used in vivo fast scan cyclic voltammetry to test the effects of hypocretin antagonists on dopamine signaling in the nucleus accumbens core and a progressive ratio schedule to examine the effects of these antagonists on cocaine self-administration. Results demonstrate that blockade of either hypocretin receptor 1 or both hypocretin receptor 1 and 2 significantly reduces the effects of cocaine on dopamine signaling and decreases the motivation to take cocaine. In contrast, blockade of hypocretin receptor 2 alone had no significant effects on dopamine signaling or self-administration. These findings suggest a differential involvement of the two hypocretin receptors, with hypocretin receptor 1 appearing to be more involved than hypocretin receptor 2 in the regulation of dopamine signaling and cocaine self-administration. When considered with the existing literature, these data support the hypothesis that hypocretins exert a permissive influence on dopamine signaling and motivated behavior via preferential actions on hypocretin receptor 1.

  15. DMPD: Modulation of Toll-interleukin 1 receptor mediated signaling. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 15662540 Modulation of Toll-interleukin 1 receptor mediated signaling. Li X, Qin J.... J Mol Med. 2005 Apr;83(4):258-66. Epub 2005 Jan 21. (.png) (.svg) (.html) (.csml) Show Modulation of Toll-i...nterleukin 1 receptor mediated signaling. PubmedID 15662540 Title Modulation of Toll-interleukin 1 receptor

  16. Calmodulin protects cells from death under normal growth conditions and mitogenic starvation but plays a mediating role in cell death upon B-cell receptor stimulation

    DEFF Research Database (Denmark)

    Schmalzigaug, R; Ye, Q; Berchtold, M W

    2001-01-01

    stimulation of the B-cell receptor (BCR), the resting Ca2+ levels remain elevated after the initial transient in CaMII-/- cells. Despite higher Ca2+ resting levels, the CaMII-/- cells are partially protected from BCR induced apoptosis indicating that CaM plays a dual role in apoptotic processes....

  17. Detection of BCR-ABL Fusion mRNA Using Reverse Transcriptase Loop-mediated Isothermal Amplification

    Energy Technology Data Exchange (ETDEWEB)

    Dugan, L C; Hall, S; Kohlgruber, A; Urbin, S; Torres, C; Wilson, P

    2011-12-08

    RT-PCR is commonly used for the detection of Bcr-Abl fusion transcripts in patients diagnosed with chronic myelogenous leukemia, CML. Two fusion transcripts predominate in CML, Br-Abl e13a2 and e14a2. They have developed reverse transcriptase isothermal loop-mediated amplification (RT-LAMP) assays to detect these two fusion transcripts along with the normal Bcr transcript.

  18. High fructose-mediated attenuation of insulin receptor signaling does not affect PDGF-induced proliferative signaling in vascular smooth muscle cells.

    Science.gov (United States)

    Osman, Islam; Poulose, Ninu; Ganapathy, Vadivel; Segar, Lakshman

    2016-11-15

    Insulin resistance is associated with accelerated atherosclerosis. Although high fructose is known to induce insulin resistance, it remains unclear as to how fructose regulates insulin receptor signaling and proliferative phenotype in vascular smooth muscle cells (VSMCs), which play a major role in atherosclerosis. Using human aortic VSMCs, we investigated the effects of high fructose treatment on insulin receptor substrate-1 (IRS-1) serine phosphorylation, insulin versus platelet-derived growth factor (PDGF)-induced phosphorylation of Akt, S6 ribosomal protein, and extracellular signal-regulated kinase (ERK), and cell cycle proteins. In comparison with PDGF (a potent mitogen), neither fructose nor insulin enhanced VSMC proliferation and cyclin D1 expression. d-[ 14 C(U)]fructose uptake studies revealed a progressive increase in fructose uptake in a time-dependent manner. Concentration-dependent studies with high fructose (5-25mM) showed marked increases in IRS-1 serine phosphorylation, a key adapter protein in insulin receptor signaling. Accordingly, high fructose treatment led to significant diminutions in insulin-induced phosphorylation of downstream signaling components including Akt and S6. In addition, high fructose significantly diminished insulin-induced ERK phosphorylation. Nevertheless, high fructose did not affect PDGF-induced key proliferative signaling events including phosphorylation of Akt, S6, and ERK and expression of cyclin D1 protein. Together, high fructose dysregulates IRS-1 phosphorylation state and proximal insulin receptor signaling in VSMCs, but does not affect PDGF-induced proliferative signaling. These findings suggest that systemic insulin resistance rather than VSMC-specific dysregulation of insulin receptor signaling by high fructose may play a major role in enhancing atherosclerosis and neointimal hyperplasia. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. Evidence for cooperative signal triggering at the extracellular loops of the TSH receptor.

    Science.gov (United States)

    Kleinau, Gunnar; Jaeschke, Holger; Mueller, Sandra; Raaka, Bruce M; Neumann, Susanne; Paschke, Ralf; Krause, Gerd

    2008-08-01

    The mechanisms governing transition of the thyroid stimulating hormone (TSH) receptor (TSHR) from basal to active conformations are poorly understood. Considering that constitutively activating mutations (CAMs) and inactivating mutations in each of the extracellular loops (ECLs) trigger only partial TSHR activation or inactivation, respectively, we hypothesized that full signaling occurs via multiple extracellular signal propagation events. Therefore, individual CAMs in the extracellular region were combined to create double and triple mutants. In support of our hypothesis, combinations of mutants in the ECLs are in some cases additive, while in others they are even synergistic, with triple mutant I486A/I568V/V656F exhibiting a 70-fold increase in TSH-independent signaling. The proximity but likely different spatial orientation of the residues of activating and inactivating mutations in each ECL supports a dual functionality to facilitate signal induction and conduction, respectively. This is the first report for G-protein coupled receptors, suggesting that multiple and cooperative signal propagating events at all three ECLs are required for full receptor activation. Our findings provide new insights concerning molecular signal transmission from extracellular domains toward the transmembrane helix bundle of the glycoprotein hormone receptors.

  20. Glycosylation as a Main Regulator of Growth and Death Factor Receptors Signaling

    Directory of Open Access Journals (Sweden)

    Inês Gomes Ferreira

    2018-02-01

    Full Text Available Glycosylation is a very frequent and functionally important post-translational protein modification that undergoes profound changes in cancer. Growth and death factor receptors and plasma membrane glycoproteins, which upon activation by extracellular ligands trigger a signal transduction cascade, are targets of several molecular anti-cancer drugs. In this review, we provide a thorough picture of the mechanisms bywhich glycosylation affects the activity of growth and death factor receptors in normal and pathological conditions. Glycosylation affects receptor activity through three non-mutually exclusive basic mechanisms: (1 by directly regulating intracellular transport, ligand binding, oligomerization and signaling of receptors; (2 through the binding of receptor carbohydrate structures to galectins, forming a lattice thatregulates receptor turnover on the plasma membrane; and (3 by receptor interaction with gangliosides inside membrane microdomains. Some carbohydrate chains, for example core fucose and β1,6-branching, exert a stimulatory effect on all receptors, while other structures exert opposite effects on different receptors or in different cellular contexts. In light of the crucial role played by glycosylation in the regulation of receptor activity, the development of next-generation drugs targeting glyco-epitopes of growth factor receptors should be considered a therapeutically interesting goal.

  1. Estradiol-induced desensitization of 5-HT1A receptor signaling in the paraventricular nucleus of the hypothalamus is independent of estrogen receptor-beta.

    Science.gov (United States)

    Rossi, Dania V; Dai, Ying; Thomas, Peter; Carrasco, Gonzalo A; DonCarlos, Lydia L; Muma, Nancy A; Li, Qian

    2010-08-01

    Estradiol regulates serotonin 1A (5-HT(1A)) receptor signaling. Since desensitization of 5-HT(1A) receptors may be an underlying mechanism by which selective serotonin reuptake inhibitors (SSRIs) mediate their therapeutic effects and combining estradiol with SSRIs enhances the efficacy of the SSRIs, it is important to determine which estrogen receptors are capable of desensitizating 5-HT(1A) receptor function. We previously demonstrated that selective activation of the estrogen receptor, GPR30, desensitizes 5-HT(1A) receptor signaling in rat hypothalamic paraventricular nucleus (PVN). However, since estrogen receptor-beta (ERbeta), is highly expressed in the PVN, we investigated the role of ERbeta in estradiol-induced desensitization of 5-HT(1A) receptor signaling. We first showed that a selective ERbeta agonist, diarylpropionitrile (DPN) has a 100-fold lower binding affinity than estradiol for GPR30. Administration of DPN did not desensitize 5-HT(1A) receptor signaling in rat PVN as demonstrated by agonist-stimulated hormone release. Second, we used a recombinant adenovirus containing ERbeta siRNAs to decrease ERbeta expression in the PVN. Reductions in ERbeta did not alter the estradiol-induced desensitization of 5-HT(1A) receptor signaling in oxytocin cells. In contrast, in animals with reduced ERbeta, estradiol administration, instead of producing desensitization, augmented the ACTH response to a 5-HT(1A) agonist. Combined with the results from the DPN treatment experiments, desensitization of 5-HT(1A) receptor signaling does not appear to be mediated by ERbeta in oxytocin cells, but that ERbeta, together with GPR30, may play a complex role in central regulation of 5-HT(1A)-mediated ACTH release. Determining the mechanisms by which estrogens induce desensitization may aid in the development of better treatments for mood disorders. Copyright 2010 Elsevier Ltd. All rights reserved.

  2. A model for the biosynthesis and transport of plasma membrane-associated signaling receptors to the cell surface

    Directory of Open Access Journals (Sweden)

    Sorina Claudia Popescu

    2012-04-01

    Full Text Available Intracellular protein transport is emerging as critical in determining the outcome of receptor-activated signal transduction pathways. In plants, relatively little is known about the nature of the molecular components and mechanisms involved in coordinating receptor synthesis and transport to the cell surface. Recent advances in this field indicate that signaling pathways and intracellular transport machinery converge and coordinate to render receptors competent for signaling at their plasma membrane activity sites. The biogenesis and transport to the cell surface of signaling receptors appears to require both general trafficking and receptor-specific factors. Several molecular determinants, residing or associated with compartments of the secretory pathway and known to influence aspects in receptor biogenesis, are discussed and integrated into a predictive cooperative model for the functional expression of signaling receptors at the plasma membrane.

  3. Predictive value of minimal residual disease in Philadelphia-chromosome-positive acute lymphoblastic leukemia treated with imatinib in the European intergroup study of post-induction treatment of Philadelphia-chromosome-positive acute lymphoblastic leukemia, based on immunoglobulin/T-cell receptor and BCR/ABL1 methodologies

    Science.gov (United States)

    Cazzaniga, Giovanni; De Lorenzo, Paola; Alten, Julia; Röttgers, Silja; Hancock, Jeremy; Saha, Vaskar; Castor, Anders; Madsen, Hans O.; Gandemer, Virginie; Cavé, Hélène; Leoni, Veronica; Köhler, Rolf; Ferrari, Giulia M.; Bleckmann, Kirsten; Pieters, Rob; van der Velden, Vincent; Stary, Jan; Zuna, Jan; Escherich, Gabriele; zur Stadt, Udo; Aricò, Maurizio; Conter, Valentino; Schrappe, Martin; Valsecchi, Maria Grazia; Biondi, Andrea

    2018-01-01

    The prognostic value of minimal residual disease (MRD) in Philadelphia-chromosome-positive (Ph+) childhood acute lymphoblastic leukemia (ALL) treated with tyrosine kinase inhibitors is not fully established. We detected MRD by real-time quantitative polymerase chain reaction (RQ-PCR) of rearranged immunoglobulin/T-cell receptor genes (IG/TR) and/or BCR/ABL1 fusion transcript to investigate its predictive value in patients receiving Berlin-Frankfurt-Münster (BFM) high-risk (HR) therapy and post-induction intermittent imatinib (the European intergroup study of post-induction treatment of Philadelphia-chromosome-positive acute lymphoblastic leukemia (EsPhALL) study). MRD was monitored after induction (time point (TP)1), consolidation Phase IB (TP2), HR Blocks, reinductions, and at the end of therapy. MRD negativity progressively increased over time, both by IG/TR and BCR/ABL1. Of 90 patients with IG/TR MRD at TP1, nine were negative and none relapsed, while 11 with MRD<5×10−4 and 70 with MRD≥5×10−4 had a comparable 5-year cumulative incidence of relapse of 36.4 (15.4) and 35.2 (5.9), respectively. Patients who achieved MRD negativity at TP2 had a low relapse risk (5-yr cumulative incidence of relapse (CIR)=14.3[9.8]), whereas those who attained MRD negativity at a later date showed higher CIR, comparable to patients with positive MRD at any level. BCR/ABL1 MRD negative patients at TP1 had a relapse risk similar to those who were IG/TR MRD negative (1/8 relapses). The overall concordance between the two methods is 69%, with significantly higher positivity by BCR/ABL1. In conclusion, MRD monitoring by both methods may be functional not only for measuring response but also for guiding biological studies aimed at investigating causes for discrepancies, although from our data IG/TR MRD monitoring appears to be more reliable. Early MRD negativity is highly predictive of favorable outcome. The earlier MRD negativity is achieved, the better the prognosis. PMID

  4. BCR-701: A review of 10-years of sequential extraction analyses

    International Nuclear Information System (INIS)

    Sutherland, Ross A.

    2010-01-01

    A detailed quantitative analysis was performed on data presented in the literature that focused on the sequential extraction of cadmium (Cd), chromium (Cr), copper (Cu), nickel (Ni), lead (Pb) and zinc (Zn) from the certified reference material BCR-701 (lake sediment) using the three-step harmonized BCR procedure. The accuracy of data reported in the literature, including precision and different measures of trueness, was assessed relative to the certified values for BCR-701. Forty data sets were accepted following extreme outlier removal, and statistically summarized with measures of central tendency, dispersion, and distribution form. In general, literature data were similar in their measurement precision to the expert laboratories used to certify the trace element contents in BCR-701. The overall median precision for literature reported data was 10% (range 6-19%), compared to certifying laboratories of 9% (range 4-33%). One measure of literature data trueness was assessed via a confirmatory approach using a robust bootstrap method. Only 22% of the comparisons indicated significantly different (all were lower) concentrations reported in the literature compared to certified values. The question of whether the differences are practically significant for environmental studies is raised. Bias was computed as a measure of trueness, and literature data were more frequently negatively biased, indicating lower concentrations reported in the literature for the six trace elements for the three-step sequential procedure compared to the certified values. However, 95% confidence intervals about the average bias for the 18 comparisons indicated only four instances when a mean bias of 0 (i.e., measured = certified) was not incorporated-suggesting statistical difference. Finally, Z-scores incorporating a Horwitz-type function were used to assess the general trueness of laboratory data. Of the 468 laboratory Z-score values computed, 92% were considered to be satisfactory, 5% were

  5. Non-genomic actions of aldosterone: From receptors and signals to membrane targets.

    LENUS (Irish Health Repository)

    2012-02-01

    In tissues which express the mineralocorticoid receptor (MR), aldosterone modulates the expression of membrane targets such as the subunits of the epithelial Na(+) channel, in combination with important signalling intermediates such as serum and glucocorticoid-regulated kinase-1. In addition, the rapid \\'non-genomic\\' activation of protein kinases and secondary messenger signalling cascades has also been detected in aldosterone-sensitive tissues of the nephron, distal colon and cardiovascular system. These rapid actions are variously described as being coupled to MR or to an as yet unidentified, membrane-associated aldosterone receptor. The rapidly activated signalling cascades add a level of fine-tuning to the activity of aldosterone-responsive membrane transporters and also modulate the aldosterone-induced changes in gene expression through receptor and transcription factor phosphorylation.

  6. Non-genomic actions of aldosterone: From receptors and signals to membrane targets.

    LENUS (Irish Health Repository)

    Dooley, Ruth

    2011-07-26

    In tissues which express the mineralocorticoid receptor (MR), aldosterone modulates the expression of membrane targets such as the subunits of the epithelial Na(+) channel, in combination with important signalling intermediates such as serum and glucocorticoid-regulated kinase-1. In addition, the rapid \\'non-genomic\\' activation of protein kinases and secondary messenger signalling cascades has also been detected in aldosterone-sensitive tissues of the nephron, distal colon and cardiovascular system. These rapid actions are variously described as being coupled to MR or to an as yet unidentified, membrane-associated aldosterone receptor. The rapidly activated signalling cascades add a level of fine-tuning to the activity of aldosterone-responsive membrane transporters and also modulate the aldosterone-induced changes in gene expression through receptor and transcription factor phosphorylation.

  7. New insights into how trafficking regulates T cell receptor signaling

    Directory of Open Access Journals (Sweden)

    Jieqiong Lou

    2016-07-01

    Full Text Available AbstractThere is emerging evidence that exocytosis plays an important role in regulating T cell receptor (TCR signaling. The trafficking molecules involved in lytic granule (LG secretion in cytotoxic T lymphocytes (CTL have been well studied due to the immune disorder known as familial hemophagocytic lymphohisiocytosis (FHLH. However, the knowledge of trafficking machineries regulating the exocytosis of receptors and signaling molecules remains quite limited. In this review, we summarize the reported trafficking molecules involved in the transport of the TCR and downstream signaling molecules to the cell surface. By combining this information with the known knowledge of LG exocytosis and general exocytic trafficking machinery, we attempt to draw a more complete picture of how the TCR signaling network and exocytic trafficking matrix are interconnected to facilitate T cell activation. This also highlights how membrane compartmentalization facilitates the spatiotemporal organization of cellular responses that are essential for immune functions.

  8. Activation of GABAB receptors inhibits protein kinase B /Glycogen Synthase Kinase 3 signaling

    Directory of Open Access Journals (Sweden)

    Lu Frances Fangjia

    2012-11-01

    Full Text Available Abstract Accumulated evidence has suggested that potentiation of cortical GABAergic inhibitory neurotransmission may be a key mechanism in the treatment of schizophrenia. However, the downstream molecular mechanisms related to GABA potentiation remain unexplored. Recent studies have suggested that dopamine D2 receptor antagonists, which are used in the clinical treatment of schizophrenia, modulate protein kinase B (Akt/glycogen synthase kinase (GSK-3 signaling. Here we report that activation of GABAB receptors significantly inhibits Akt/GSK-3 signaling in a β-arrestin-dependent pathway. Agonist stimulation of GABAB receptors enhances the phosphorylation of Akt (Thr-308 and enhances the phosphorylation of GSK-3α (Ser-21/β (Ser-9 in both HEK-293T cells expressing GABAB receptors and rat hippocampal slices. Furthermore, knocking down the expression of β-arrestin2 using siRNA abolishes the GABAB receptor-mediated modulation of GSK-3 signaling. Our data may help to identify potentially novel targets through which GABAB receptor agents may exert therapeutic effects in the treatment of schizophrenia.

  9. Sphingosine-1-phosphate (S1P) displays sustained S1P1 receptor agonism and signaling through S1P lyase-dependent receptor recycling.

    Science.gov (United States)

    Gatfield, John; Monnier, Lucile; Studer, Rolf; Bolli, Martin H; Steiner, Beat; Nayler, Oliver

    2014-07-01

    The sphingosine-1-phosphate (S1P) type 1 receptor (S1P1R) is a novel therapeutic target in lymphocyte-mediated autoimmune diseases. S1P1 receptor desensitization caused by synthetic S1P1 receptor agonists prevents T-lymphocyte egress from secondary lymphoid organs into the circulation. The selective S1P1 receptor agonist ponesimod, which is in development for the treatment of autoimmune diseases, efficiently reduces peripheral lymphocyte counts and displays efficacy in animal models of autoimmune disease. Using ponesimod and the natural ligand S1P, we investigated the molecular mechanisms leading to different signaling, desensitization and trafficking behavior of S1P1 receptors. In recombinant S1P1 receptor-expressing cells, ponesimod and S1P triggered Gαi protein-mediated signaling and β-arrestin recruitment with comparable potency and efficiency, but only ponesimod efficiently induced intracellular receptor accumulation. In human umbilical vein endothelial cells (HUVEC), ponesimod and S1P triggered translocation of the endogenous S1P1 receptor to the Golgi compartment. However, only ponesimod treatment caused efficient surface receptor depletion, receptor accumulation in the Golgi and degradation. Impedance measurements in HUVEC showed that ponesimod induced only short-lived Gαi protein-mediated signaling followed by resistance to further stimulation, whereas S1P induced sustained Gαi protein-mediated signaling without desensitization. Inhibition of S1P lyase activity in HUVEC rendered S1P an efficient S1P1 receptor internalizing compound and abrogated S1P-mediated sustained signaling. This suggests that S1P lyase - by facilitating S1P1 receptor recycling - is essential for S1P-mediated sustained signaling, and that synthetic agonists are functional antagonists because they are not S1P lyase substrates. Copyright © 2014 Elsevier Inc. All rights reserved.

  10. Molecular and functional profiling of histamine receptor-mediated calcium ion signals in different cell lines.

    Science.gov (United States)

    Meisenberg, Annika; Kaschuba, Dagmar; Balfanz, Sabine; Jordan, Nadine; Baumann, Arnd

    2015-10-01

    Calcium ions (Ca(2+)) play a pivotal role in cellular physiology. Often Ca(2+)-dependent processes are studied in commonly available cell lines. To induce Ca(2+) signals on demand, cells may need to be equipped with additional proteins. A prominent group of membrane proteins evoking Ca(2+) signals are G-protein coupled receptors (GPCRs). These proteins register external signals such as photons, odorants, and neurotransmitters and convey ligand recognition into cellular responses, one of which is Ca(2+) signaling. To avoid receptor cross-talk or cross-activation with introduced proteins, the repertoire of cell-endogenous receptors must be known. Here we examined the presence of histamine receptors in six cell lines frequently used as hosts to study cellular signaling processes. In a concentration-dependent manner, histamine caused a rise in intracellular Ca(2+) in HeLa, HEK 293, and COS-1 cells. The concentration for half-maximal activation (EC50) was in the low micromolar range. In individual cells, transient Ca(2+) signals and Ca(2+) oscillations were uncovered. The results show that (i) HeLa, HEK 293, and COS-1 cells express sufficient amounts of endogenous receptors to study cellular Ca(2+) signaling processes directly and (ii) these cell lines are suitable for calibrating Ca(2+) biosensors in situ based on histamine receptor evoked responses. Copyright © 2015 Elsevier Inc. All rights reserved.

  11. Kinetics in Signal Transduction Pathways Involving Promiscuous Oligomerizing Receptors Can Be Determined by Receptor Specificity : Apoptosis Induction by TRAIL

    NARCIS (Netherlands)

    Szegezdi, Eva; van der Sloot, Almer M.; Mahalingam, Devalingam; O'Leary, Lynda; Cool, Robbert H.; Munoz, Ines G.; Montoya, Guillermo; Quax, Wim J.; de Jong, Steven; Samali, Afshin; Serrano, Luis

    Here we show by computer modeling that kinetics and outcome of signal transduction in case of hetero-oligomerizing receptors of a promiscuous ligand largely depend on the relative amounts of its receptors. Promiscuous ligands can trigger the formation of nonproductive receptor complexes, which slows

  12. Molecular, pharmacological, and signaling properties of octopamine receptors from honeybee (Apis mellifera) brain.

    Science.gov (United States)

    Balfanz, Sabine; Jordan, Nadine; Langenstück, Teresa; Breuer, Johanna; Bergmeier, Vera; Baumann, Arnd

    2014-04-01

    G protein-coupled receptors are important regulators of cellular signaling processes. Within the large family of rhodopsin-like receptors, those binding to biogenic amines form a discrete subgroup. Activation of biogenic amine receptors leads to transient changes of intracellular Ca²⁺-([Ca²⁺](i)) or 3',5'-cyclic adenosine monophosphate ([cAMP](i)) concentrations. Both second messengers modulate cellular signaling processes and thereby contribute to long-lasting behavioral effects in an organism. In vivo pharmacology has helped to reveal the functional effects of different biogenic amines in honeybees. The phenolamine octopamine is an important modulator of behavior. Binding of octopamine to its receptors causes elevation of [Ca²⁺](i) or [cAMP](i). To date, only one honeybee octopamine receptor that induces Ca²⁺ signals has been molecularly and pharmacologically characterized. Here, we examined the pharmacological properties of four additional honeybee octopamine receptors. When heterologously expressed, all receptors induced cAMP production after binding to octopamine with EC₅₀(s) in the nanomolar range. Receptor activity was most efficiently blocked by mianserin, a substance with antidepressant activity in vertebrates. The rank order of inhibitory potency for potential receptor antagonists was very similar on all four honeybee receptors with mianserin > cyproheptadine > metoclopramide > chlorpromazine > phentolamine. The subroot of octopamine receptors activating adenylyl cyclases is the largest that has so far been characterized in arthropods, and it should now be possible to unravel the contribution of individual receptors to the physiology and behavior of honeybees. © 2013 International Society for Neurochemistry.

  13. Guidelines for molecular monitoring of BCR-ABL1 in chronic myeloid leukemia patients by RT-qPCR

    Directory of Open Access Journals (Sweden)

    Irene Larripa

    2017-02-01

    Full Text Available Current clinical guidelines for managing chronic myeloid leukemia include molecular monitoring of BCR-ABL1 transcript quantitative reverse-transcription PCR. Despite the proven prognostic significance of molecular response, it is not widely appreciated that quantitative reverse-transcription PCR potentially produces highly variable data, which may affect the validity of results, making comparability between different laboratories difficult. Therefore, standardized reporting of BCR-ABL1 measurements is needed for optimal clinical management. An approach to achieve comparable BCR-ABL1 values is the use of an international reporting scale. Conversion to the international scale is achieved by the application of laboratory specific conversion factor that is obtained by using validated secondary reference calibrators. Moreover, with the aim to mitigate the interlaboratory imprecision of quantitative BCR-ABL1 measurements and to facilitate local laboratory results interpretation and reporting, we decide to prepare laboratory guidelines that will further facilitate interlaboratory comparative studies and independent quality-assessment programs, which are of paramount importance for worldwide standardization of BCR-ABL1 monitoring results, in particular for those most isolated laboratories, with not easy access to commercial kits or sample interchange programs

  14. Combined inhibition of β-catenin and Bcr-Abl synergistically targets tyrosine kinase inhibitor-resistant blast crisis chronic myeloid leukemia blasts and progenitors in vitro and in vivo.

    Science.gov (United States)

    Zhou, H; Mak, P Y; Mu, H; Mak, D H; Zeng, Z; Cortes, J; Liu, Q; Andreeff, M; Carter, B Z

    2017-10-01

    Tyrosine kinase inhibitor (TKI) resistance and progression to blast crisis (BC), both related to persistent β-catenin activation, remain formidable challenges for chronic myeloid leukemia (CML). We observed overexpression of β-catenin in BC-CML stem/progenitor cells, particularly in granulocyte-macrophage progenitors, and highest among a novel CD34 + CD38 + CD123 hi Tim-3 hi subset as determined by CyTOF analysis. Co-culture with mesenchymal stromal cells (MSCs) induced the expression of β-catenin and its target CD44 in CML cells. A novel Wnt/β-catenin signaling modulator, C82, and nilotinib synergistically killed KBM5 T315I and TKI-resistant primary BC-CML cells with or without BCR-ABL kinase mutations even under leukemia/MSC co-culture conditions. Silencing of β-catenin by short interfering RNA restored sensitivity of primary BCR-ABL T315I/E255V BC-CML cells to nilotinib. Combining the C82 pro-drug, PRI-724, with nilotinib significantly prolonged the survival of NOD/SCID/IL2Rγ null mice injected with primary BCR-ABL T315I/E255V BC-CML cells. The combined treatment selectively targeted CML progenitors and inhibited CD44, c-Myc, survivin, p-CRKL and p-STAT5 expression. In addition, pretreating primary BC-CML cells with C82, or the combination, but not with nilotinib alone, significantly impaired their engraftment potential in NOD/SCID/IL2Rγ-null-3/GM/SF mice and significantly prolonged survival. Our data suggest potential benefit of concomitant β-catenin and Bcr-Abl inhibition to prevent or overcome Bcr-Abl kinase-dependent or -independent TKI resistance in BC-CML.

  15. Quercetin suppresses insulin receptor signaling through inhibition of the insulin ligand–receptor binding and therefore impairs cancer cell proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Feng [Department of Gastroenterology, The Tenth People’s Hospital of Shanghai, Tongji University, Shanghai 200072 (China); Department of Nanomedicine, Houston Methodist Research Institute, Houston, TX 77030 (United States); Yang, Yong, E-mail: yyang@houstonmethodist.org [Department of Nanomedicine, Houston Methodist Research Institute, Houston, TX 77030 (United States); Department of Medicine, Weill Cornell Medical College, New York, NY 10065 (United States)

    2014-10-03

    Graphical abstract: - Highlights: • Quercetin inhibits insulin ligand–receptor interactions. • Quercetin reduces downstream insulin receptor signaling. • Quercetin blocks insulin induced glucose uptake. • Quercetin suppresses insulin stimulated cancer cell proliferation and tumor growth. - Abstract: Although the flavonoid quercetin is known to inhibit activation of insulin receptor signaling, the inhibitory mechanism is largely unknown. In this study, we demonstrate that quercetin suppresses insulin induced dimerization of the insulin receptor (IR) through interfering with ligand–receptor interactions, which reduces the phosphorylation of IR and Akt. This inhibitory effect further inhibits insulin stimulated glucose uptake due to decreased cell membrane translocation of glucose transporter 4 (GLUT4), resulting in impaired cancer cell proliferation. The effect of quercetin in inhibiting tumor growth was also evident in an in vivo model, indicating a potential future application for quercetin in the treatment of cancers.

  16. Quercetin suppresses insulin receptor signaling through inhibition of the insulin ligand–receptor binding and therefore impairs cancer cell proliferation

    International Nuclear Information System (INIS)

    Wang, Feng; Yang, Yong

    2014-01-01

    Graphical abstract: - Highlights: • Quercetin inhibits insulin ligand–receptor interactions. • Quercetin reduces downstream insulin receptor signaling. • Quercetin blocks insulin induced glucose uptake. • Quercetin suppresses insulin stimulated cancer cell proliferation and tumor growth. - Abstract: Although the flavonoid quercetin is known to inhibit activation of insulin receptor signaling, the inhibitory mechanism is largely unknown. In this study, we demonstrate that quercetin suppresses insulin induced dimerization of the insulin receptor (IR) through interfering with ligand–receptor interactions, which reduces the phosphorylation of IR and Akt. This inhibitory effect further inhibits insulin stimulated glucose uptake due to decreased cell membrane translocation of glucose transporter 4 (GLUT4), resulting in impaired cancer cell proliferation. The effect of quercetin in inhibiting tumor growth was also evident in an in vivo model, indicating a potential future application for quercetin in the treatment of cancers

  17. Chronic myeloid leukemia may be associated with several bcr-abl transcripts including the acute lymphoid leukemia-type 7 kb transcript

    NARCIS (Netherlands)

    Selleri, L.; von Lindern, M.; Hermans, A.; Meijer, D.; Torelli, G.; Grosveld, G.

    1990-01-01

    In the majority of Philadelphia (Ph)-positive chronic myeloid leukemia (CML) patients, the c-abl gene is fused to the bcr gene, resulting in the transcription of an 8.5 kb chimeric bcr-abl mRNA, which is translated into a p210bcr-abl fusion protein. In about 50% of the Ph-positive acute lymphoid

  18. c-Myb and its target Bmi1 are required for p190BCR/ABL leukemogenesis in mouse and human cells.

    Science.gov (United States)

    Waldron, T; De Dominici, M; Soliera, A R; Audia, A; Iacobucci, I; Lonetti, A; Martinelli, G; Zhang, Y; Martinez, R; Hyslop, T; Bender, T P; Calabretta, B

    2012-04-01

    Expression of c-Myb is required for normal hematopoiesis and for proliferation of myeloid leukemia blasts and a subset of T-cell leukemia, but its role in B-cell leukemogenesis is unknown. We tested the role of c-Myb in p190(BCR/ABL)-dependent B-cell leukemia in mice transplanted with p190(BCR/ABL)-transduced marrow cells with a c-Myb allele (Myb(f/d)) and in double transgenic p190(BCR/ABL)/Myb(w/d) mice. In both models, loss of a c-Myb allele caused a less aggressive B-cell leukemia. In p190(BCR/ABL)-expressing human B-cell leukemia lines, knockdown of c-Myb expression suppressed proliferation and colony formation. Compared with c-Myb(w/f) cells, expression of Bmi1, a regulator of stem cell proliferation and maintenance, was decreased in pre-B cells from Myb(w/d) p190(BCR/ABL) transgenic mice. Ectopic expression of a mutant c-Myb or Bmi1 enhanced the proliferation and colony formation of Myb(w/d) p190(BCR/ABL) B-cells; by contrast, Bmi1 downregulation inhibited colony formation of p190(BCR/ABL)-expressing murine B cells and human B-cell leukemia lines. Moreover, c-Myb interacted with a segment of the human Bmi1 promoter and enhanced its activity. In blasts from 19 Ph(1) adult acute lymphoblastic leukemia patients, levels of c-Myb and Bmi1 showed a positive correlation. Together, these findings support the existence of a c-Myb-Bmi1 transcription-regulatory pathway required for p190(BCR/ABL) leukemogenesis.

  19. Tyrosine Phosphorylation in Toll-Like Receptor Signaling

    Science.gov (United States)

    Chattopadhyay, Saurabh; Sen, Ganes C.

    2014-01-01

    There is a wealth of knowledge about how different Ser/Thr protein kinases participate in Toll-like receptor (TLR) signaling. In many cases, we know the identities of the Ser/Thr residues of various components of the TLR-signaling pathways that are phosphorylated, the functional consequences of the phosphorylation and the responsible protein kinases. In contrast, the analysis of Tyr-phosphorylation of TLRs and their signaling proteins is currently incomplete, because several existing analyses are not systematic or they do not rely on robust experimental data. Nevertheless, it is clear that many TLRs require, for signaling, ligand-dependent phosphorylation of specific Tyr residues in their cytoplasmic domains; the list includes TLR2, TLR3, TLR4, TLR5, TLR8 and TLR9. In this article, we discuss the current status of knowledge on the effect of Tyr-phosphorylation of TLRs and their signaling proteins on their biochemical and biological functions, the possible identities of the relevant protein tyrosine kinases (PTKs) and the nature of regulations of PTK-mediated activation of TLR signaling pathways. PMID:25022196

  20. Dissection of Signaling Events Downstream of the c-Mpl Receptor in Murine Hematopoietic Stem Cells Via Motif-Engineered Chimeric Receptors.

    Science.gov (United States)

    Saka, Koichiro; Lai, Chen-Yi; Nojima, Masanori; Kawahara, Masahiro; Otsu, Makoto; Nakauchi, Hiromitsu; Nagamune, Teruyuki

    2018-02-01

    Hematopoietic stem cells (HSCs) are a valuable resource in transplantation medicine. Cytokines are often used to culture HSCs aiming at better clinical outcomes through enhancement of HSC reconstitution capability. Roles for each signal molecule downstream of receptors in HSCs, however, remain puzzling due to complexity of the cytokine-signaling network. Engineered receptors that are non-responsive to endogenous cytokines represent an attractive tool for dissection of signaling events. We here tested a previously developed chimeric receptor (CR) system in primary murine HSCs, target cells that are indispensable for analysis of stem cell activity. Each CR contains tyrosine motifs that enable selective activation of signal molecules located downstream of the c-Mpl receptor upon stimulation by an artificial ligand. Signaling through a control CR with a wild-type c-Mpl cytoplasmic tail sufficed to enhance HSC proliferation and colony formation in cooperation with stem cell factor (SCF). Among a series of CRs, only one compatible with selective Stat5 activation showed similar positive effects. The HSCs maintained ex vivo in these environments retained long-term reconstitution ability following transplantation. This ability was also demonstrated in secondary recipients, indicating effective transmission of stem cell-supportive signals into HSCs via these artificial CRs during culture. Selective activation of Stat5 through CR ex vivo favored preservation of lymphoid potential in long-term reconstituting HSCs, but not of myeloid potential, exemplifying possible dissection of signals downstream of c-Mpl. These CR systems therefore offer a useful tool to scrutinize complex signaling pathways in HSCs.

  1. Ethylene Regulates Levels of Ethylene Receptor/CTR1 Signaling Complexes in Arabidopsis thaliana*

    Science.gov (United States)

    Shakeel, Samina N.; Gao, Zhiyong; Amir, Madiha; Chen, Yi-Feng; Rai, Muneeza Iqbal; Haq, Noor Ul; Schaller, G. Eric

    2015-01-01

    The plant hormone ethylene is perceived by a five-member family of receptors in Arabidopsis thaliana. The receptors function in conjunction with the Raf-like kinase CTR1 to negatively regulate ethylene signal transduction. CTR1 interacts with multiple members of the receptor family based on co-purification analysis, interacting more strongly with receptors containing a receiver domain. Levels of membrane-associated CTR1 vary in response to ethylene, doing so in a post-transcriptional manner that correlates with ethylene-mediated changes in levels of the ethylene receptors ERS1, ERS2, EIN4, and ETR2. Interactions between CTR1 and the receptor ETR1 protect ETR1 from ethylene-induced turnover. Kinetic and dose-response analyses support a model in which two opposing factors control levels of the ethylene receptor/CTR1 complexes. Ethylene stimulates the production of new complexes largely through transcriptional induction of the receptors. However, ethylene also induces turnover of receptors, such that levels of ethylene receptor/CTR1 complexes decrease at higher ethylene concentrations. Implications of this model for ethylene signaling are discussed. PMID:25814663

  2. Stat5 Exerts Distinct, Vital Functions in the Cytoplasm and Nucleus of Bcr-Abl{sup +} K562 and Jak2(V617F){sup +} HEL Leukemia Cells

    Energy Technology Data Exchange (ETDEWEB)

    Weber, Axel [Georg-Speyer-Haus, Institute for Tumor Biology and Experimental Therapy, Frankfurt am Main 60596 (Germany); Borghouts, Corina [Ganymed Pharmaceuticals AG, Mainz 55131 (Germany); Brendel, Christian [Boston Children’s Hospital, Division of Hematology/Oncology, Boston, MA 02115 (United States); Moriggl, Richard [Ludwig Boltzmann Institute for Cancer Research (LBI-CR), Vienna 1090 (Austria); Delis, Natalia; Brill, Boris; Vafaizadeh, Vida; Groner, Bernd, E-mail: Groner@em.uni-frankfurt.de [Georg-Speyer-Haus, Institute for Tumor Biology and Experimental Therapy, Frankfurt am Main 60596 (Germany)

    2015-03-19

    Signal transducers and activators of transcription (Stats) play central roles in the conversion of extracellular signals, e.g., cytokines, hormones and growth factors, into tissue and cell type specific gene expression patterns. In normal cells, their signaling potential is strictly limited in extent and duration. The persistent activation of Stat3 or Stat5 is found in many human tumor cells and contributes to their growth and survival. Stat5 activation plays a pivotal role in nearly all hematological malignancies and occurs downstream of oncogenic kinases, e.g., Bcr-Abl in chronic myeloid leukemias (CML) and Jak2(V617F) in other myeloproliferative diseases (MPD). We defined the mechanisms through which Stat5 affects growth and survival of K562 cells, representative of Bcr-Abl positive CML, and HEL cells, representative for Jak2(V617F) positive acute erythroid leukemia. In our experiments we suppressed the protein expression levels of Stat5a and Stat5b through shRNA mediated downregulation and demonstrated the dependence of cell survival on the presence of Stat5. Alternatively, we interfered with the functional capacities of the Stat5 protein through the interaction with a Stat5 specific peptide ligand. This ligand is a Stat5 specific peptide aptamer construct which comprises a 12mer peptide integrated into a modified thioredoxin scaffold, S5-DBD-PA. The peptide sequence specifically recognizes the DNA binding domain (DBD) of Stat5. Complex formation of S5-DBD-PA with Stat5 causes a strong reduction of P-Stat5 in the nuclear fraction of Bcr-Abl-transformed K562 cells and a suppression of Stat5 target genes. Distinct Stat5 mediated survival mechanisms were detected in K562 and Jak2(V617F)-transformed HEL cells. Stat5 is activated in the nuclear and cytosolic compartments of K562 cells and the S5-DBD-PA inhibitor most likely affects the viability of Bcr-Abl{sup +} K562 cells through the inhibition of canonical Stat5 induced target gene transcription. In HEL cells

  3. The receptor kinase CERK1 has dual functions in symbiosis and immunity signalling.

    Science.gov (United States)

    Zhang, Xiaowei; Dong, Wentao; Sun, Jongho; Feng, Feng; Deng, Yiwen; He, Zuhua; Oldroyd, Giles E D; Wang, Ertao

    2015-01-01

    The establishment of symbiotic interactions between mycorrhizal fungi, rhizobial bacteria and their legume hosts involves a common symbiosis signalling pathway. This signalling pathway is activated by Nod factors produced by rhizobia and these are recognised by the Nod factor receptors NFR1/LYK3 and NFR5/NFP. Mycorrhizal fungi produce lipochitooligosaccharides (LCOs) similar to Nod factors, as well as short-chain chitin oligomers (CO4/5), implying commonalities in signalling during mycorrhizal and rhizobial associations. Here we show that NFR1/LYK3, but not NFR5/NFP, is required for the establishment of the mycorrhizal interaction in legumes. NFR1/LYK3 is necessary for the recognition of mycorrhizal fungi and the activation of the symbiosis signalling pathway leading to induction of calcium oscillations and gene expression. Chitin oligosaccharides also act as microbe associated molecular patterns that promote plant immunity via similar LysM receptor-like kinases. CERK1 in rice has the highest homology to NFR1 and we show that this gene is also necessary for the establishment of the mycorrhizal interaction as well as for resistance to the rice blast fungus. Our results demonstrate that NFR1/LYK3/OsCERK1 represents a common receptor for chitooligosaccharide-based signals produced by mycorrhizal fungi, rhizobial bacteria (in legumes) and fungal pathogens. It would appear that mycorrhizal recognition has been conserved in multiple receptors across plant species, but additional diversification in certain plant species has defined other signals that this class of receptors can perceive. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.

  4. BCR SIGNALING INHIBITORS: AN OVERVIEW OF TOXICITIES ASSOCIATED WITH IBRUTINIB AND IDELALISIB IN PATIENTS WITH CHRONIC LYMPHOCYTIC LEUKEMIA

    OpenAIRE

    Lorenzo Falchi; Jessica M Baron; Carrie Anne Orlikowski; Alessandra Ferrajoli

    2016-01-01

    The B-cell receptor signaling inhibitors ibrutinib and idelalisib are revolutionizing the treatment landscape of chronic lymphocytic leukemia (CLL) and other B-cell malignancies. These oral agents, both alone and in combination with other drugs, have shown remarkable clinical activity in relapsed or refractory CLL across all risk groups, and have been approved by the Food and Drug Administration for this indication. Preliminary data suggest that an even greater benefit can be expected in trea...

  5. [Early monitoring of BCR-ABL transcript levels and cytogenetic in assessing the prognosis of chronic myeloid leukemia].

    Science.gov (United States)

    Huang, Qin; Zhang, Xiao-yan; Li, Yan; Wang, Xiao-min

    2013-10-15

    To explore the prognostic significance of early monitoring of BCR-ABL transcript levels and cytogenetic evaluations for chronic myeloid leukemia in chronic phase (CML-CP). From July 2007 to May 2012, 56 CML-CP patients received oral imatinib 400 mg/d. The BCR-ABL transcript levels were monitored and cytogenetic examinations performed after 3 and 6 months respectively. The median follow-up time was 48 months. The 3-month BCR-ABL transcript levels ≤ 10% of patients 5-year overall survival (OS) and progression-free survival (PFS) were better than BCR-ABL transcript levels >10% of patients (OS: 100% vs 84.6%, P = 0.011; PFS: 94.6% vs 67.7%, P = 0.045); cytogenetics: Ph(+) ≤ 35 % of patients 5-year OS and PFS better than Ph(+) > 35% of patients (OS: 100% vs 76.2%, P = 0.001; PFS: 95.2% vs 38.1%, P = 0.001); the 6-month BCR-ABL transcripts level ≤ 1% of patients 5-year OS and PFS also better than BCR-ABL transcript levels> 1% of patients (OS: 100% vs 71.4%, P = 0.000; PFS: 95.2% vs 47.6%, P = 0.001); Ph(+) = 0% and Ph(+)> 0% patients, 5-year OS and PFS were significantly different (OS: 100% vs 68.6%, P = 0.000; PFS: 95.3% vs 45.7%, P = 0.000). Early molecular biology and cytogenetics monitoring have some significance in the prognostic assessment of CML-CP. And individualized treatment strategies should be based upon the monitoring results in conjunctions with comprehensive judgments.

  6. Role of the B-cell receptor in chronic lymphocytic leukemia: where do we stand?

    Science.gov (United States)

    Fais, Franco; Bruno, Silvia; Ghiotto, Fabio

    2010-01-01

    The past 15 years have witnessed an enormous effort in studying B-cell Chronic Lymphocytic Leukemia. A great number of researches brought significant novel information and a better understanding of the natural history of this disease. This mini review will focus on the studies related to the Immunoglobulin variable (IgV) genes rearrangements that compose the B-cell receptor (BcR) of the leukemic clones. These studies have defined a role for the antigen(s) in the paths that lead to leukemic clone generation/expansion and underscore the informative value represented by BcR analyses.

  7. DMPD: G-protein-coupled receptor expression, function, and signaling in macrophages. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 17456803 G-protein-coupled receptor expression, function, and signaling in macropha...2007 Apr 24. (.png) (.svg) (.html) (.csml) Show G-protein-coupled receptor expression, function, and signali...ng in macrophages. PubmedID 17456803 Title G-protein-coupled receptor expression, function

  8. Rapid phospho-turnover by receptor tyrosine kinases impacts downstream signaling and drug binding.

    Science.gov (United States)

    Kleiman, Laura B; Maiwald, Thomas; Conzelmann, Holger; Lauffenburger, Douglas A; Sorger, Peter K

    2011-09-02

    Epidermal growth factor receptors (ErbB1-4) are oncogenic receptor tyrosine kinases (RTKs) that regulate diverse cellular processes. In this study, we combine measurement and mathematical modeling to quantify phospho-turnover at ErbB receptors in human cells and to determine the consequences for signaling and drug binding. We find that phosphotyrosine residues on ErbB1 have half-lives of a few seconds and therefore turn over 100-1000 times in the course of a typical immediate-early response to ligand. Rapid phospho-turnover is also observed for EGF-activated ErbB2 and ErbB3, unrelated RTKs, and multiple intracellular adaptor proteins and signaling kinases. Thus, the complexes formed on the cytoplasmic tail of active receptors and the downstream signaling kinases they control are highly dynamic and antagonized by potent phosphatases. We develop a kinetic scheme for binding of anti-ErbB1 drugs to receptors and show that rapid phospho-turnover significantly impacts their mechanisms of action. Copyright © 2011 Elsevier Inc. All rights reserved.

  9. Ph1 chromosomes and bcr gene rearrangements in chronic myelocytic leukemia patients developed from atomic bomb survivors

    International Nuclear Information System (INIS)

    Tanaka, Kimio; Takechi, Miho; Shigeta, Chiharu; Sakatani, Keiko; Oguma, Nobuo; Kamada, Nanao; Takimoto, Yasuo; Kuramoto, Atsushi

    1989-01-01

    This study compared findings of chronic myelocytic leukemia (CML) in A-bomb survivors (n=8) developing CML within 10 years after the bombing and in non-exposed CML patients (n=14). Both Ph 1 chromosomes and bcr rearrangement were observed in all patients in both exposed and non-exposed groups. There was no significant difference in distribution sites of bcr rearrangement between the groups. These results suggest that bcr-abl chimera mRNA and chimera protein associated with Ph 1 chromosomes have an important role in the development of CML among A-bomb survivors, as well as among non-exposed patients. (N.K.)

  10. Refined localization of the Escherichia coli F4ab/F4ac receptor locus on pig chromosome 13

    DEFF Research Database (Denmark)

    Joller, D.; Jørgensen, Claus Bøttcher; Bertschinger, H.U.

    2009-01-01

    Diarrhoea in newborn and weaned pigs caused by enterotoxigenic Escherichia coli (ETEC) expressing F4 fimbriae leads to considerable losses in pig production. In this study, we refined the mapping of the receptor locus for ETEC F4ab/F4ac adhesion (F4bcR) by joint analysis of Nordic and Swiss data...... (MUC4-8227) were used to create the linkage map. The region for F4bcR was refined to the interval SW207-S0075 on pig chromosome 13. The most probable position of F4bcR was in the SW207-MUC4 region. The order of six markers was supported by physical mapping on the BAC fingerprint contig from...

  11. Differences in signal activation by LH and hCG are mediated by the LH/CG receptor`s extracellular hinge region

    Directory of Open Access Journals (Sweden)

    Paul eGrzesik

    2015-09-01

    Full Text Available The human lutropin/choriogonadotropin receptor (LHCGR can be activated by binding two slightly different gonadotropic glycoprotein hormones, choriogonadotropin (CG - secreted by the placenta, and lutropin (LH - produced by the pituitary. They induce different signaling profiles at the LHCGR. This cannot be explained by binding to the receptor's leucine-rich repeat domain (LRRD, as this binding is similar for the two hormones. We therefore speculate that there are previously unknown differences in the hormone/receptor interaction at the extracellular hinge region, which might help to understand functional differences between the two hormones. We have therefore performed a detailed study of the binding and action of LH and CG at the LHCGR hinge region. We focused on a primate-specific additional exon in the hinge region, which is located between LRRD and the serpentine domain. The segment of the hinge region encoded by exon10 was previously reported to be only relevant to hLH signaling, as the exon10-deletion receptor exhibits decreased hLH signaling, but unchanged hCG signaling. We designed an advanced homology model of the hormone/LHCGR complex, followed by experimental characterization of relevant fragments in the hinge region. In addition, we examined predictions of a helical exon10-encoded conformation by block-wise polyalanine (helix supporting mutations. These helix preserving modifications showed no effect on hormone induced signaling. However, introduction of a structure-disturbing double-proline mutant LHCGR-Q303P/E305P within the exon10-helix has, in contrast to exon10 deletion, no impact on hLH, but only on hCG signaling. This opposite effect on signaling by hLH and hCG can be explained by distinct sites of hormone interaction in the hinge region s. In conclusion, our analysis provides details of the differences between hLH- and hCG-induced signaling that are mainly determined in the L2-beta loop of the hormones and in the hinge region

  12. Plant immune and growth receptors share common signalling components but localise to distinct plasma membrane nanodomains.

    Science.gov (United States)

    Bücherl, Christoph A; Jarsch, Iris K; Schudoma, Christian; Segonzac, Cécile; Mbengue, Malick; Robatzek, Silke; MacLean, Daniel; Ott, Thomas; Zipfel, Cyril

    2017-03-06

    Cell surface receptors govern a multitude of signalling pathways in multicellular organisms. In plants, prominent examples are the receptor kinases FLS2 and BRI1, which activate immunity and steroid-mediated growth, respectively. Intriguingly, despite inducing distinct signalling outputs, both receptors employ common downstream signalling components, which exist in plasma membrane (PM)-localised protein complexes. An important question is thus how these receptor complexes maintain signalling specificity. Live-cell imaging revealed that FLS2 and BRI1 form PM nanoclusters. Using single-particle tracking we could discriminate both cluster populations and we observed spatiotemporal separation between immune and growth signalling platforms. This finding was confirmed by visualising FLS2 and BRI1 within distinct PM nanodomains marked by specific remorin proteins and differential co-localisation with the cytoskeleton. Our results thus suggest that signalling specificity between these pathways may be explained by the spatial separation of FLS2 and BRI1 with their associated signalling components within dedicated PM nanodomains.

  13. Somatic hypermutation of T cell receptor α chain contributes to selection in nurse shark thymus.

    Science.gov (United States)

    Ott, Jeannine A; Castro, Caitlin D; Deiss, Thaddeus C; Ohta, Yuko; Flajnik, Martin F; Criscitiello, Michael F

    2018-04-17

    Since the discovery of the T cell receptor (TcR), immunologists have assigned somatic hypermutation (SHM) as a mechanism employed solely by B cells to diversify their antigen receptors. Remarkably, we found SHM acting in the thymus on α chain locus of shark TcR. SHM in developing shark T cells likely is catalyzed by activation-induced cytidine deaminase (AID) and results in both point and tandem mutations that accumulate non-conservative amino acid replacements within complementarity-determining regions (CDRs). Mutation frequency at TcRα was as high as that seen at B cell receptor loci (BcR) in sharks and mammals, and the mechanism of SHM shares unique characteristics first detected at shark BcR loci. Additionally, fluorescence in situ hybridization showed the strongest AID expression in thymic corticomedullary junction and medulla. We suggest that TcRα utilizes SHM to broaden diversification of the primary αβ T cell repertoire in sharks, the first reported use in vertebrates. © 2018, Ott et al.

  14. Signal transduction by VEGF receptors in regulation of angiogenesis and lymphangiogenesis

    International Nuclear Information System (INIS)

    Shibuya, Masabumi; Claesson-Welsh, Lena

    2006-01-01

    The VEGF/VPF (vascular endothelial growth factor/vascular permeability factor) ligands and receptors are crucial regulators of vasculogenesis, angiogenesis, lymphangiogenesis and vascular permeability in vertebrates. VEGF-A, the prototype VEGF ligand, binds and activates two tyrosine kinase receptors: VEGFR1 (Flt-1) and VEGFR2 (KDR/Flk-1). VEGFR1, which occurs in transmembrane and soluble forms, negatively regulates vasculogenesis and angiogenesis during early embryogenesis, but it also acts as a positive regulator of angiogenesis and inflammatory responses, playing a role in several human diseases such as rheumatoid arthritis and cancer. The soluble VEGFR1 is overexpressed in placenta in preeclampsia patients. VEGFR2 has critical functions in physiological and pathological angiogenesis through distinct signal transduction pathways regulating proliferation and migration of endothelial cells. VEGFR3, a receptor for the lymphatic growth factors VEGF-C and VEGF-D, but not for VEGF-A, regulates vascular and lymphatic endothelial cell function during embryogenesis. Loss-of-function variants of VEGFR3 have been identified in lymphedema. Formation of tumor lymphatics may be stimulated by tumor-produced VEGF-C, allowing increased spread of tumor metastases through the lymphatics. Mapping the signaling system of these important receptors may provide the knowledge necessary to suppress specific signaling pathways in major human diseases

  15. Modulating Neurotrophin Receptor Signaling as a Therapeutic Strategy for Huntington’s Disease

    Science.gov (United States)

    Simmons, Danielle A.

    2017-01-01

    Huntington’s disease (HD) is an autosomal dominant neurodegenerative disorder caused by CAG repeat expansions in the IT15 gene which encodes the huntingtin (HTT) protein. Currently, no treatments capable of preventing or slowing disease progression exist. Disease modifying therapeutics for HD would be expected to target a comprehensive set of degenerative processes given the diverse mechanisms contributing to HD pathogenesis including neuroinflammation, excitotoxicity, and transcription dysregulation. A major contributor to HD-related degeneration is mutant HTT-induced loss of neurotrophic support. Thus, neurotrophin (NT) receptors have emerged as therapeutic targets in HD. The considerable overlap between NT signaling networks and those dysregulated by mutant HTT provides strong theoretical support for this approach. This review will focus on the contributions of disrupted NT signaling in HD-related neurodegeneration and how targeting NT receptors to augment pro-survival signaling and/or to inhibit degenerative signaling may combat HD pathologies. Therapeutic strategies involving NT delivery, peptidomimetics, and the targeting of specific NT receptors (e.g., Trks or p75NTR), particularly with small molecule ligands, are discussed. PMID:29254102

  16. Bicaudal-D1 regulates the intracellular sorting and signalling of neurotrophin receptors.

    Science.gov (United States)

    Terenzio, Marco; Golding, Matthew; Russell, Matthew R G; Wicher, Krzysztof B; Rosewell, Ian; Spencer-Dene, Bradley; Ish-Horowicz, David; Schiavo, Giampietro

    2014-07-17

    We have identified a new function for the dynein adaptor Bicaudal D homolog 1 (BICD1) by screening a siRNA library for genes affecting the dynamics of neurotrophin receptor-containing endosomes in motor neurons (MNs). Depleting BICD1 increased the intracellular accumulation of brain-derived neurotrophic factor (BDNF)-activated TrkB and p75 neurotrophin receptor (p75(NTR)) by disrupting the endosomal sorting, reducing lysosomal degradation and increasing the co-localisation of these neurotrophin receptors with retromer-associated sorting nexin 1. The resulting re-routing of active receptors increased their recycling to the plasma membrane and altered the repertoire of signalling-competent TrkB isoforms and p75(NTR) available for ligand binding on the neuronal surface. This resulted in attenuated, but more sustained, AKT activation in response to BDNF stimulation. These data, together with our observation that Bicd1 expression is restricted to the developing nervous system when neurotrophin receptor expression peaks, indicate that BICD1 regulates neurotrophin signalling by modulating the endosomal sorting of internalised ligand-activated receptors. © 2014 The Authors.

  17. Ibrutinib: a paradigm shift in management of CLL.

    Science.gov (United States)

    Badar, Talha; Burger, Jan A; Wierda, William G; O'Brien, Susan

    2014-12-01

    B-cell receptor (BCR) signaling plays a vital role in B-cell malignancies; Bruton tyrosine kinase is a critical mediator of this signaling. BCR signaling, either constitutively or following antigen binding, leads to activation of several downstream pathways involved in cell survival, proliferation and migration. The efficacy observed in studies of the Bruton tyrosine kinase inhibitor, ibrutinib, confirms that BCR signaling is critical for the growth of B-cell malignancies. Ibrutinib characteristically induces redistribution of malignant B cells from tissues into the peripheral blood and rapid resolution of adenopathy. Furthermore, ibrutinib therapy results in normalization of lymphocyte counts and improvement in cytopenias. Ibrutinib has been shown to have an excellent safety profile and does not cause myelosuppression. Early data from combination studies of ibrutinib with anti-CD20 monoclonal antibodies have shown more rapid responses compared to those seen with ibrutinib monotherapy. Current data strongly support continued clinical evaluation of ibrutinib in B-cell malignancies.

  18. Signaling by myeloid C-type lectin receptors in immunity and homeostasis.

    Science.gov (United States)

    Sancho, David; Reis e Sousa, Caetano

    2012-01-01

    Myeloid cells are key drivers of physiological responses to pathogen invasion or tissue damage. Members of the C-type lectin receptor (CLR) family stand out among the specialized receptors utilized by myeloid cells to orchestrate these responses. CLR ligands include carbohydrate, protein, and lipid components of both pathogens and self, which variably trigger endocytic, phagocytic, proinflammatory, or anti-inflammatory reactions. These varied outcomes rely on a versatile system for CLR signaling that includes tyrosine-based motifs that recruit kinases, phosphatases, or endocytic adaptors as well as nontyrosine-based signals that modulate the activation of other pathways or couple to the uptake machinery. Here, we review the signaling properties of myeloid CLRs and how they impact the role of myeloid cells in innate and adaptive immunity.

  19. Activation of Toll-like receptors nucleates assembly of the MyDDosome signaling hub.

    Science.gov (United States)

    Latty, Sarah Louise; Sakai, Jiro; Hopkins, Lee; Verstak, Brett; Paramo, Teresa; Berglund, Nils A; Cammorota, Eugenia; Cicuta, Pietro; Gay, Nicholas J; Bond, Peter J; Klenerman, David; Bryant, Clare E

    2018-01-24

    Infection and tissue damage induces assembly of supramolecular organizing centres (SMOCs)), such as the Toll-like receptor (TLR) MyDDosome, to co-ordinate inflammatory signaling. SMOC assembly is thought to drive digital all-or-none responses, yet TLR activation by diverse microbes induces anything from mild to severe inflammation. Using single-molecule imaging of TLR4-MyDDosome signaling in living macrophages, we find that MyDDosomes assemble within minutes of TLR4 stimulation. TLR4/MD2 activation leads only to formation of TLR4/MD2 heterotetramers, but not oligomers, suggesting a stoichiometric mismatch between activated receptors and MyDDosomes. The strength of TLR4 signalling depends not only on the number and size of MyDDosomes formed but also how quickly these structures assemble. Activated TLR4, therefore, acts transiently nucleating assembly of MyDDosomes, a process that is uncoupled from receptor activation. These data explain how the oncogenic mutation of MyD88 (L265P) assembles MyDDosomes in the absence of receptor activation to cause constitutive activation of pro-survival NF-κB signalling. © 2018, Latty et al.

  20. Phytomelatonin receptor PMTR1-mediated signaling regulates stomatal closure in Arabidopsis thaliana.

    Science.gov (United States)

    Wei, Jian; Li, Dong-Xu; Zhang, Jia-Rong; Shan, Chi; Rengel, Zed; Song, Zhong-Bang; Chen, Qi

    2018-04-27

    Melatonin has been detected in plants in 1995; however, the function and signaling pathway of this putative phytohormone are largely undetermined due to a lack of knowledge about its receptor. Here, we discovered the first phytomelatonin receptor (CAND2/PMTR1) in Arabidopsis thaliana and found that melatonin governs the receptor-dependent stomatal closure. The application of melatonin induced stomatal closure through the heterotrimeric G protein α subunit-regulated H 2 O 2 and Ca 2+ signals. The Arabidopsis mutant lines lacking AtCand2 that encodes a candidate G protein-coupled receptor were insensitive to melatonin-induced stomatal closure. Accordingly, the melatonin-induced H 2 O 2 production and Ca 2+ influx were completely abolished in cand2. CAND2 is a membrane protein that interacts with GPA1 and the expression of AtCand2 was tightly regulated by melatonin in various organs and guard cells. CAND2 showed saturable and specific 125 I-melatonin binding, with apparent K d (dissociation constant) of 0.73 ± 0.10 nmol/L (r 2  = .99), demonstrating this protein is a phytomelatonin receptor (PMTR1). Our results suggest that the phytomelatonin regulation of stomatal closure is dependent on its receptor CAND2/PMTR1-mediated H 2 O 2 and Ca 2+ signaling transduction cascade. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  1. Peroxisome proliferator-activated receptor gamma signaling in human sperm physiology.

    Science.gov (United States)

    Liu, Li-Li; Xian, Hua; Cao, Jing-Chen; Zhang, Chong; Zhang, Yong-Hui; Chen, Miao-Miao; Qian, Yi; Jiang, Ming

    2015-01-01

    Peroxisome proliferator-activated receptor gamma (PPARγ) is a member of the PPARs, which are transcription factors of the steroid receptor superfamily. PPARγ acts as an important molecule for regulating energy homeostasis, modulates the hypothalamic-pituitary-gonadal (HPG) axis, and is reciprocally regulated by HPG. In the human, PPARγ protein is highly expressed in ejaculated spermatozoa, implying a possible role of PPARγ signaling in regulating sperm energy dissipation. PPARγ protein is also expressed in Sertoli cells and germ cells (spermatocytes). Its activation can be induced during capacitation and the acrosome reaction. This mini-review will focus on how PPARγ signaling may affect fertility and sperm quality and the potential reversibility of these adverse effects.

  2. The mTORC1-Signaling Pathway and Hepatic Polyribosome Profile Are Enhanced after the Recovery of a Protein Restricted Diet by a Combination of Soy or Black Bean with Corn Protein.

    Science.gov (United States)

    Márquez-Mota, Claudia C; Rodriguez-Gaytan, Cinthya; Adjibade, Pauline; Mazroui, Rachid; Gálvez, Amanda; Granados, Omar; Tovar, Armando R; Torres, Nimbe

    2016-09-20

    Between 6% and 11% of the world's population suffers from malnutrition or undernutrition associated with poverty, aging or long-term hospitalization. The present work examined the effect of different types of proteins on the mechanistic target of rapamycin (mTORC1)-signaling pathway in: (1) healthy; and (2) protein restricted rats. (1) In total, 200 rats were divided into eight groups and fed one of the following diets: 20% casein (C), soy (S), black bean (B), B + Corn (BCr), Pea (P), spirulina (Sp), sesame (Se) or Corn (Cr). Rats fed C or BCr had the highest body weight gain; rats fed BCr had the highest pS6K1/S6K1 ratio; rats fed B, BCr or P had the highest eIF4G expression; (2) In total, 84 rats were fed 0.5% C for 21 day and protein rehabilitated with different proteins. The S, soy + Corn (SCr) and BCr groups had the highest body weight gain. Rats fed SCr and BCr had the highest eIF4G expression and liver polysome formation. These findings suggest that the quality of the dietary proteins modulate the mTORC1-signaling pathway. In conclusion, the combination of BCr or SCr are the best proteins for dietary protein rehabilitation due to the significant increase in body weight, activation of the mTORC1-signaling pathway in liver and muscle, and liver polysome formation.

  3. Quantitative phosphoproteomics dissection of seven-transmembrane receptor signaling using full and biased agonists

    DEFF Research Database (Denmark)

    Christensen, Gitte L; Kelstrup, Christian D; Lyngsø, Christina

    2010-01-01

    (q)-dependent and -independent AT(1)R signaling. This study provides substantial novel insight into angiotensin II signal transduction and is the first study dissecting the differences between a full agonist and a biased agonist from a 7TMR on a systems-wide scale. Importantly, it reveals a previously unappreciated diversity......Seven-transmembrane receptors (7TMRs) signal through the well described heterotrimeric G proteins but can also activate G protein-independent signaling pathways of which the impact and complexity are less understood. The angiotensin II type 1 receptor (AT(1)R) is a prototypical 7TMR...... and quantity of Galpha(q) protein-independent signaling and uncovers novel signaling pathways. We foresee that the amount and diversity of G protein-independent signaling may be more pronounced than previously recognized for other 7TMRs as well. Quantitative mass spectrometry is a promising tool for evaluation...

  4. Allogeneic stem cell transplantation for patients harboring T315I BCR-ABL mutated leukemias

    DEFF Research Database (Denmark)

    Nicolini, Franck Emmanuel; Basak, Grzegorz W; Soverini, Simona

    2011-01-01

    T315I(+) Philadelphia chromosome-positive leukemias are inherently resistant to all licensed tyrosine kinase inhibitors, and therapeutic options remain limited. We report the outcome of allogeneic stem cell transplantation in 64 patients with documented BCR-ABL(T315I) mutations. Median follow......) as unfavorable factors. We conclude that allogeneic stem cell transplantation represents a valuable therapeutic tool for eligible patients with BCR-ABL(T315I) mutation, a tool that may or may not be replaced by third-generation tyrosine kinase inhibitors....

  5. Suppression of bcr-abl synthesis by siRNAs or tyrosine kinase activity by Glivec alters different oncogenes, apoptotic/antiapoptotic genes and cell proliferation factors (microarray study).

    Science.gov (United States)

    Zhelev, Zhivko; Bakalova, Rumiana; Ohba, Hideki; Ewis, Ashraf; Ishikawa, Mitsuru; Shinohara, Yasuo; Baba, Yoshinobu

    2004-07-16

    Short 21-mer double-stranded/small-interfering RNAs (ds/siRNAs) were designed to target bcr-abl mRNA in chronic myelogenous leukemia. The ds/siRNAs were transfected into bcr-abl-positive K-562 (derived from blast crisis chronic myelogenous leukemia), using lipofectamine. Penetrating of ds/siRNAs into the cells was detected by fluorescent confocal microscopy, using fluorescein-labeled ds/siRNAs. The cells were treated with mix of three siRNA sequences (3 x 60 nM) during 6 days with three repetitive transfections. The siRNA-treatment was accompanied with significant reduction of bcr-abl mRNA, p210, protein tyrosine kinase activity and cell proliferation index. Treatment of cells with Glivec (during 8 days with four repetitive doses, 180 nM single dose) resulted in analogous reduction of cell proliferation activity, stronger suppression of protein tyrosine kinase activity, and very low reduction of p210. siRNA-mix and Glivec did not affect significantly the viability of normal lymphocytes. Microarray analysis of siRNA- and Glivec-treated K-562 cells demonstrated that both pathways of bcr-abl suppression were accompanied with overexpression and suppression of many different oncogenes, apoptotic/antiapoptotic and cell proliferation factors. The following genes of interest were found to decrease in relatively equal degree in both siRNA- and Glivec-treated cells: Bcd orf1 and orf2 proto-oncogene, chromatin-specific transcription elongation factor FACT 140-kDa subunit mRNA, gene encoding splicing factor SF1, and mRNA for Tec protein tyrosine kinase. siRNA-mix and Glivec provoked overexpression of the following common genes: c-jun proto-oncogene, protein kinase C-alpha, pvt-1 oncogene homologue (myc activator), interleukin-6, 1-8D gene from interferon-inducible gene family, tumor necrosis factor receptor superfamily (10b), and STAT-induced STAT inhibitor.

  6. Rational Targeting of Cellular Cholesterol in Diffuse Large B-Cell Lymphoma (DLBCL) Enabled by Functional Lipoprotein Nanoparticles: A Therapeutic Strategy Dependent on Cell of Origin.

    Science.gov (United States)

    Rink, Jonathan S; Yang, Shuo; Cen, Osman; Taxter, Tim; McMahon, Kaylin M; Misener, Sol; Behdad, Amir; Longnecker, Richard; Gordon, Leo I; Thaxton, C Shad

    2017-11-06

    Cancer cells have altered metabolism and, in some cases, an increased demand for cholesterol. It is important to identify novel, rational treatments based on biology, and cellular cholesterol metabolism as a potential target for cancer is an innovative approach. Toward this end, we focused on diffuse large B-cell lymphoma (DLBCL) as a model because there is differential cholesterol biosynthesis driven by B-cell receptor (BCR) signaling in germinal center (GC) versus activated B-cell (ABC) DLBCL. To specifically target cellular cholesterol homeostasis, we employed high-density lipoprotein-like nanoparticles (HDL NP) that can generally reduce cellular cholesterol by targeting and blocking cholesterol uptake through the high-affinity HDL receptor, scavenger receptor type B-1 (SCARB1). As we previously reported, GC DLBCL are exquisitely sensitive to HDL NP as monotherapy, while ABC DLBCL are less sensitive. Herein, we report that enhanced BCR signaling and resultant de novo cholesterol synthesis in ABC DLBCL drastically reduces the ability of HDL NPs to reduce cellular cholesterol and induce cell death. Therefore, we combined HDL NP with the BCR signaling inhibitor ibrutinib and the SYK inhibitor R406. By targeting both cellular cholesterol uptake and BCR-associated de novo cholesterol synthesis, we achieved cellular cholesterol reduction and induced apoptosis in otherwise resistant ABC DLBCL cell lines. These results in lymphoma demonstrate that reduction of cellular cholesterol is a powerful mechanism to induce apoptosis. Cells rich in cholesterol require HDL NP therapy to reduce uptake and molecularly targeted agents that inhibit upstream pathways that stimulate de novo cholesterol synthesis, thus, providing a new paradigm for rationally targeting cholesterol metabolism as therapy for cancer.

  7. AP24534, a Pan-BCR-ABL Inhibitor for Chronic Myeloid Leukemia, Potently Inhibits the T315I Mutant and Overcomes Mutation-Based Resistance

    Science.gov (United States)

    O’Hare, Thomas; Shakespeare, William C.; Zhu, Xiaotian; Eide, Christopher A.; Rivera, Victor M.; Wang, Frank; Adrian, Lauren T.; Zhou, Tianjun; Huang, Wei-Sheng; Xu, Qihong; Metcalf, Chester A.; Tyner, Jeffrey W.; Loriaux, Marc M.; Corbin, Amie S.; Wardwell, Scott; Ning, Yaoyu; Keats, Jeffrey A.; Wang, Yihan; Sundaramoorthi, Raji; Thomas, Mathew; Zhou, Dong; Snodgrass, Joseph; Commodore, Lois; Sawyer, Tomi K.; Dalgarno, David C.; Deininger, Michael W.N.; Druker, Brian J.; Clackson, Tim

    2009-01-01

    SUMMARY Inhibition of BCR-ABL by imatinib induces durable responses in many patients with chronic myeloid leukemia (CML), but resistance attributable to kinase domain mutations can lead to relapse and a switch to second-line therapy with nilotinib or dasatinib. Despite three approved therapeutic options, the cross-resistant BCR-ABLT315I mutation and compound mutants selected on sequential inhibitor therapy remain major clinical challenges. We report design and pre-clinical evaluation of AP24534, a potent, orally available multi-targeted kinase inhibitor active against T315I and other BCR-ABL mutants. AP24534 inhibited all tested BCR-ABL mutants in cellular and biochemical assays, suppressed BCR-ABLT315I-driven tumor growth in mice, and completely abrogated resistance in cell-based mutagenesis screens. Our work supports clinical evaluation of AP24534 as a pan-BCR-ABL inhibitor for treatment of CML. PMID:19878872

  8. AP24534, a Pan-BCR-ABL Inhibitor for Chronic Myeloid Leukemia, Potently Inhibits the T315I Mutant and Overcomes Mutation-Based Resistance

    Energy Technology Data Exchange (ETDEWEB)

    O’Hare, Thomas; Shakespeare, William C.; Zhu, Xiaotian; Eide, Christopher A.; Rivera, Victor M.; Wang, Frank; Adrian, Lauren T.; Zhou, Tianjun; Huang, Wei-Sheng; Xu, Qihong; Metcalf, III, Chester A.; Tyner, Jeffrey W.; Loriaux, Marc M.; Corbin, Amie S.; Wardwell, Scott; Ning, Yaoyu; Keats, Jeffrey A.; Wang, Yihan; Sundaramoorthi, Raji; Thomas, Mathew; Zhou, Dong; Snodgrass, Joseph; Commodore, Lois; Sawyer, Tomi K.; Dalgarno, David C.; Deininger, Michael W.N.; Druker, Brian J.; Clackson, Tim; (OHSU- Cancer Instit.); (ARIAD)

    2010-09-07

    Inhibition of BCR-ABL by imatinib induces durable responses in many patients with chronic myeloid leukemia (CML), but resistance attributable to kinase domain mutations can lead to relapse and a switch to second-line therapy with nilotinib or dasatinib. Despite three approved therapeutic options, the cross-resistant BCR-ABL{sup T315I} mutation and compound mutants selected on sequential inhibitor therapy remain major clinical challenges. We report design and preclinical evaluation of AP24534, a potent, orally available multitargeted kinase inhibitor active against T315I and other BCR-ABL mutants. AP24534 inhibited all tested BCR-ABL mutants in cellular and biochemical assays, suppressed BCR-ABL{sup T315I}-driven tumor growth in mice, and completely abrogated resistance in cell-based mutagenesis screens. Our work supports clinical evaluation of AP24534 as a pan-BCR-ABL inhibitor for treatment of CML.

  9. Characterization of the CDR3 structure of the Vβ21 T cell clone in patients with P210(BCR-ABL)-positive chronic myeloid leukemia and B-cell acute lymphoblastic leukemia.

    Science.gov (United States)

    Zha, Xianfeng; Chen, Shaohua; Yang, Lijian; Li, Bo; Chen, Yu; Yan, Xiaojuan; Li, Yangqiu

    2011-10-01

    The clonally expanded T cells identified in most cancer patients that respond to tumor-associated antigen such as P210(BCR-ABL) protein have definite, specific antitumor cytotoxicity. T cell receptor (TCR) Vβ CDR3 repertoire diversity was analyzed in patients with chronic myeloid leukemia (CML) and BCR-ABL(+) B-cell acute lymphoblastic leukemia (B-ALL) by GeneScan. A high frequency of oligoclonal expansion of the TCR Vβ21 subfamily was observed in the peripheral blood of CML and B-ALL patients. These clonally expanded Vβ21 T cells were correlated with the pathophysiologic process of CML. A conserved amino acid motif (SLxxV) was observed within the CDR3 region in only 3 patients with CML. Preferential usage of the Jβ segments was also observed in a minority of patients. The 3-dimensional structures of the CDR3 region containing the same motif or using the same Jβ segment displayed low similarity; on the contrary, the conformation of the CDR3 region containing no conserved motif in some T cell clones was highly similar. In conclusion, our findings indicate a high frequency of TCR Vβ21 subfamily expansion in p210(BCR-ABL)-positive CML and B-ALL patients. The characterization of the CDR3 structure was complex. Regrettably, at this time it was not possible to confirm that the Vβ21 T cell clones were derived from the stimulation of p210(BCR-ABL) protein. Copyright © 2011 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

  10. Neurotransmitter receptors as signaling platforms in anterior pituitary cells

    Czech Academy of Sciences Publication Activity Database

    Zemková, Hana; Stojilkovic, S. S.

    2018-01-01

    Roč. 463, C (2018), s. 49-64 ISSN 0303-7207 R&D Projects: GA ČR(CZ) GA16-12695S; GA ČR(CZ) GBP304/12/G069; GA MŠk(CZ) LQ1604; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:67985823 Keywords : pituitary * ligand-gated receptor channels * G protein -coupled receptors * neurotransmitters * action potentials * calcium signaling * hormone secretion Subject RIV: FH - Neurology OBOR OECD: Neurosciences (including psychophysiology Impact factor: 3.754, year: 2016

  11. Regulation of G protein-coupled receptor signalling: focus on the cardiovascular system and regulator of G protein signalling proteins

    NARCIS (Netherlands)

    Hendriks-Balk, Mariëlle C.; Peters, Stephan L. M.; Michel, Martin C.; Alewijnse, Astrid E.

    2008-01-01

    G protein-coupled receptors (GPCRs) are involved in many biological processes. Therefore, GPCR function is tightly controlled both at receptor level and at the level of signalling components. Well-known mechanisms by which GPCR function can be regulated comprise desensitization/resensitization

  12. G Protein and β-arrestin signaling bias at the ghrelin receptor.

    Science.gov (United States)

    Evron, Tama; Peterson, Sean M; Urs, Nikhil M; Bai, Yushi; Rochelle, Lauren K; Caron, Marc G; Barak, Larry S

    2014-11-28

    The G protein-coupled ghrelin receptor GHSR1a is a potential pharmacological target for treating obesity and addiction because of the critical role ghrelin plays in energy homeostasis and dopamine-dependent reward. GHSR1a enhances growth hormone release, appetite, and dopamine signaling through G(q/11), G(i/o), and G(12/13) as well as β-arrestin-based scaffolds. However, the contribution of individual G protein and β-arrestin pathways to the diverse physiological responses mediated by ghrelin remains unknown. To characterize whether a signaling bias occurs for GHSR1a, we investigated ghrelin signaling in a number of cell-based assays, including Ca(2+) mobilization, serum response factor response element, stress fiber formation, ERK1/2 phosphorylation, and β-arrestin translocation, utilizing intracellular second loop and C-tail mutants of GHSR1a. We observed that GHSR1a and β-arrestin rapidly form metastable plasma membrane complexes following exposure to an agonist, but replacement of the GHSR1a C-tail by the tail of the vasopressin 2 receptor greatly stabilizes them, producing complexes observable on the plasma membrane and also in endocytic vesicles. Mutations of the contiguous conserved amino acids Pro-148 and Leu-149 in the GHSR1a intracellular second loop generate receptors with a strong bias to G protein and β-arrestin, respectively, supporting a role for conformation-dependent signaling bias in the wild-type receptor. Our results demonstrate more balance in GHSR1a-mediated ERK signaling from G proteins and β-arrestin but uncover an important role for β-arrestin in RhoA activation and stress fiber formation. These findings suggest an avenue for modulating drug abuse-associated changes in synaptic plasticity via GHSR1a and indicate the development of GHSR1a-biased ligands as a promising strategy for selectively targeting downstream signaling events. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. Cocaine Disrupts Histamine H3 Receptor Modulation of Dopamine D1 Receptor Signaling: σ1-D1-H3 Receptor Complexes as Key Targets for Reducing Cocaine's Effects

    Science.gov (United States)

    Moreno, Estefanía; Moreno-Delgado, David; Navarro, Gemma; Hoffmann, Hanne M.; Fuentes, Silvia; Rosell-Vilar, Santi; Gasperini, Paola; Rodríguez-Ruiz, Mar; Medrano, Mireia; Mallol, Josefa; Cortés, Antoni; Casadó, Vicent; Lluís, Carme; Ferré, Sergi; Ortiz, Jordi; Canela, Enric

    2014-01-01

    The general effects of cocaine are not well understood at the molecular level. What is known is that the dopamine D1 receptor plays an important role. Here we show that a key mechanism may be cocaine's blockade of the histamine H3 receptor-mediated inhibition of D1 receptor function. This blockade requires the σ1 receptor and occurs upon cocaine binding to σ1-D1-H3 receptor complexes. The cocaine-mediated disruption leaves an uninhibited D1 receptor that activates Gs, freely recruits β-arrestin, increases p-ERK 1/2 levels, and induces cell death when over activated. Using in vitro assays with transfected cells and in ex vivo experiments using both rats acutely treated or self-administered with cocaine along with mice depleted of σ1 receptor, we show that blockade of σ1 receptor by an antagonist restores the protective H3 receptor-mediated brake on D1 receptor signaling and prevents the cell death from elevated D1 receptor signaling. These findings suggest that a combination therapy of σ1R antagonists with H3 receptor agonists could serve to reduce some effects of cocaine. PMID:24599455

  14. ZINC-INDUCED EGF RECEPTOR SIGNALING REQUIRES SRC-MEDIATED PHOSPHORYLATION OF THE EGF RECEPTOR ON TYROSINE 845 (Y845)

    Science.gov (United States)

    ZINC-INDUCED EGF RECEPTOR SIGNALING REQUIRES Src-MEDIATED PHOSPHORYLATION OF THE EGF RECEPTOR ON TYROSINE 845 (Y845)Weidong Wu1, Lee M. Graves2, Gordon N. Gill3 and James M. Samet4 1Center for Environmental Medicine and Lung Biology; 2Department of Pharmacology, University o...

  15. Allosteric Inhibition of Bcr-Abl Kinase by High Affinity Monobody Inhibitors Directed to the Src Homology 2 (SH2)-Kinase Interface*

    Science.gov (United States)

    Wojcik, John; Lamontanara, Allan Joaquim; Grabe, Grzegorz; Koide, Akiko; Akin, Louesa; Gerig, Barbara; Hantschel, Oliver; Koide, Shohei

    2016-01-01

    Bcr-Abl is a constitutively active kinase that causes chronic myelogenous leukemia. We have shown that a tandem fusion of two designed binding proteins, termed monobodies, directed to the interaction interface between the Src homology 2 (SH2) and kinase domains and to the phosphotyrosine-binding site of the SH2 domain, respectively, inhibits the Bcr-Abl kinase activity. Because the latter monobody inhibits processive phosphorylation by Bcr-Abl and the SH2-kinase interface is occluded in the active kinase, it remained undetermined whether targeting the SH2-kinase interface alone was sufficient for Bcr-Abl inhibition. To address this question, we generated new, higher affinity monobodies with single nanomolar KD values targeting the kinase-binding surface of SH2. Structural and mutagenesis studies revealed the molecular underpinnings of the monobody-SH2 interactions. Importantly, the new monobodies inhibited Bcr-Abl kinase activity in vitro and in cells, and they potently induced cell death in chronic myelogenous leukemia cell lines. This work provides strong evidence for the SH2-kinase interface as a pharmacologically tractable site for allosteric inhibition of Bcr-Abl. PMID:26912659

  16. Allosteric Inhibition of Bcr-Abl Kinase by High Affinity Monobody Inhibitors Directed to the Src Homology 2 (SH2)-Kinase Interface.

    Science.gov (United States)

    Wojcik, John; Lamontanara, Allan Joaquim; Grabe, Grzegorz; Koide, Akiko; Akin, Louesa; Gerig, Barbara; Hantschel, Oliver; Koide, Shohei

    2016-04-15

    Bcr-Abl is a constitutively active kinase that causes chronic myelogenous leukemia. We have shown that a tandem fusion of two designed binding proteins, termed monobodies, directed to the interaction interface between the Src homology 2 (SH2) and kinase domains and to the phosphotyrosine-binding site of the SH2 domain, respectively, inhibits the Bcr-Abl kinase activity. Because the latter monobody inhibits processive phosphorylation by Bcr-Abl and the SH2-kinase interface is occluded in the active kinase, it remained undetermined whether targeting the SH2-kinase interface alone was sufficient for Bcr-Abl inhibition. To address this question, we generated new, higher affinity monobodies with single nanomolar KD values targeting the kinase-binding surface of SH2. Structural and mutagenesis studies revealed the molecular underpinnings of the monobody-SH2 interactions. Importantly, the new monobodies inhibited Bcr-Abl kinase activity in vitro and in cells, and they potently induced cell death in chronic myelogenous leukemia cell lines. This work provides strong evidence for the SH2-kinase interface as a pharmacologically tractable site for allosteric inhibition of Bcr-Abl. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. Identification of potential pathway mediation targets in Toll-like receptor signaling.

    Directory of Open Access Journals (Sweden)

    Fan Li

    2009-02-01

    Full Text Available Recent advances in reconstruction and analytical methods for signaling networks have spurred the development of large-scale models that incorporate fully functional and biologically relevant features. An extended reconstruction of the human Toll-like receptor signaling network is presented herein. This reconstruction contains an extensive complement of kinases, phosphatases, and other associated proteins that mediate the signaling cascade along with a delineation of their associated chemical reactions. A computational framework based on the methods of large-scale convex analysis was developed and applied to this network to characterize input-output relationships. The input-output relationships enabled significant modularization of the network into ten pathways. The analysis identified potential candidates for inhibitory mediation of TLR signaling with respect to their specificity and potency. Subsequently, we were able to identify eight novel inhibition targets through constraint-based modeling methods. The results of this study are expected to yield meaningful avenues for further research in the task of mediating the Toll-like receptor signaling network and its effects.

  18. Loss of the xeroderma pigmentosum group B protein binding site impairs p210 BCR/ABL1 leukemogenic activity

    International Nuclear Information System (INIS)

    Pannucci, N L; Li, D; Sahay, S; Thomas, E K; Chen, R; Tala, I; Hu, T; Ciccarelli, B T; Megjugorac, N J; Adams III, H C; Rodriguez, P L; Fitzpatrick, E R; Lagunoff, D; Williams, D A; Whitehead, I P

    2013-01-01

    Previous studies have demonstrated that p210 BCR/ABL1 interacts directly with the xeroderma pigmentosum group B (XPB) protein, and that XPB is phosphorylated on tyrosine in cells that express p210 BCR/ABL1. In the current study, we have constructed a p210 BCR/ABL1 mutant that can no longer bind to XPB. The mutant has normal kinase activity and interacts with GRB2, but can no longer phosphorylate XPB. Loss of XPB binding is associated with reduced expression of c-MYC and reduced transforming potential in ex-vivo clonogenicity assays, but does not affect nucleotide excision repair in lymphoid or myeloid cells. When examined in a bone marrow transplantation (BMT) model for chronic myelogenous leukemia, mice that express the mutant exhibit attenuated myeloproliferation and lymphoproliferation when compared with mice that express unmodified p210 BCR/ABL1. Thus, the mutant-transplanted mice show predominantly neutrophilic expansion and altered progenitor expansion, and have significantly extended lifespans. This was confirmed in a BMT model for B-cell acute lymphoblastic leukemia, wherein the majority of the mutant-transplanted mice remain disease free. These results suggest that the interaction between p210 BCR/ABL1 and XPB can contribute to disease progression by influencing the lineage commitment of lymphoid and myeloid progenitors

  19. Arabidopsis CPR5 regulates ethylene signaling via molecular association with the ETR1 receptor.

    Science.gov (United States)

    Wang, Feifei; Wang, Lijuan; Qiao, Longfei; Chen, Jiacai; Pappa, Maria Belen; Pei, Haixia; Zhang, Tao; Chang, Caren; Dong, Chun-Hai

    2017-11-01

    The plant hormone ethylene plays various functions in plant growth, development and response to environmental stress. Ethylene is perceived by membrane-bound ethylene receptors, and among the homologous receptors in Arabidopsis, the ETR1 ethylene receptor plays a major role. The present study provides evidence demonstrating that Arabidopsis CPR5 functions as a novel ETR1 receptor-interacting protein in regulating ethylene response and signaling. Yeast split ubiquitin assays and bi-fluorescence complementation studies in plant cells indicated that CPR5 directly interacts with the ETR1 receptor. Genetic analyses indicated that mutant alleles of cpr5 can suppress ethylene insensitivity in both etr1-1 and etr1-2, but not in other dominant ethylene receptor mutants. Overexpression of Arabidopsis CPR5 either in transgenic Arabidopsis plants, or ectopically in tobacco, significantly enhanced ethylene sensitivity. These findings indicate that CPR5 plays a critical role in regulating ethylene signaling. CPR5 is localized to endomembrane structures and the nucleus, and is involved in various regulatory pathways, including pathogenesis, leaf senescence, and spontaneous cell death. This study provides evidence for a novel regulatory function played by CPR5 in the ethylene receptor signaling pathway in Arabidopsis. © 2017 Institute of Botany, Chinese Academy of Sciences.

  20. Association of HLA Class I and Class II genes with bcr-abl transcripts in leukemia patients with t(9;22 (q34;q11

    Directory of Open Access Journals (Sweden)

    Cano Pedro

    2004-06-01

    Full Text Available Abstract Background Based on the site of breakpoint in t(9;22 (q34;q11, bcr-abl fusion in leukemia patients is associated with different types of transcript proteins. In this study we have seen the association of HLA genes with different types of bcr-abl transcripts. The association could predict the bcr-abl peptide presentation by particular HLA molecules. Methods The study included a total of 189 patients of mixed ethnicity with chronic myelogenous leukemia and acute lymphocytic leukemia who were being considered for bone marrow transplantation. Typing of bcr-abl transcripts was done by reverse transcriptase PCR method. HLA typing was performed by molecular methods. The bcr-abl and HLA association was studied by calculating the relative risks and chi-square test. Results Significant negative associations (p Conclusions The negative associations of a particular bcr-abl transcript with specific HLA alleles suggests that these alleles play a critical role in presenting peptides derived from the chimeric proteins and eliciting a successful T-cell cytotoxic response. Knowledge of differential associations between HLA phenotypes and bcr-abl fusion transcript types would help in developing better strategies for immunization with the bcr-abl peptides against t(9;22 (q34;q11-positive leukemia.

  1. TSH Receptor Signaling Abrogation by a Novel Small Molecule.

    Science.gov (United States)

    Latif, Rauf; Realubit, Ronald B; Karan, Charles; Mezei, Mihaly; Davies, Terry F

    2016-01-01

    Pathological activation of the thyroid-stimulating hormone receptor (TSHR) is caused by thyroid-stimulating antibodies in patients with Graves' disease (GD) or by somatic and rare genomic mutations that enhance constitutive activation of the receptor influencing both G protein and non-G protein signaling. Potential selective small molecule antagonists represent novel therapeutic compounds for abrogation of such abnormal TSHR signaling. In this study, we describe the identification and in vitro characterization of a novel small molecule antagonist by high-throughput screening (HTS). The identification of the TSHR antagonist was performed using a transcription-based TSH-inhibition bioassay. TSHR-expressing CHO cells, which also expressed a luciferase-tagged CRE response element, were optimized using bovine TSH as the activator, in a 384 well plate format, which had a Z score of 0.3-0.6. Using this HTS assay, we screened a diverse library of ~80,000 compounds at a final concentration of 16.7 μM. The selection criteria for a positive hit were based on a mean signal threshold of ≥50% inhibition of control TSH stimulation. The screening resulted in 450 positive hits giving a hit ratio of 0.56%. A secondary confirmation screen against TSH and forskolin - a post receptor activator of adenylyl cyclase - confirmed one TSHR-specific candidate antagonist molecule (named VA-K-14). This lead molecule had an IC 50 of 12.3 μM and a unique chemical structure. A parallel analysis for cell viability indicated that the lead inhibitor was non-cytotoxic at its effective concentrations. In silico docking studies performed using a TSHR transmembrane model showed the hydrophobic contact locations and the possible mode of inhibition of TSHR signaling. Furthermore, this molecule was capable of inhibiting TSHR stimulation by GD patient sera and monoclonal-stimulating TSHR antibodies. In conclusion, we report the identification of a novel small molecule TSHR inhibitor, which has the

  2. Quantitative properties and receptor reserve of the IP(3) and calcium branch of G(q)-coupled receptor signaling.

    Science.gov (United States)

    Dickson, Eamonn J; Falkenburger, Björn H; Hille, Bertil

    2013-05-01

    Gq-coupled plasma membrane receptors activate phospholipase C (PLC), which hydrolyzes membrane phosphatidylinositol 4,5-bisphosphate (PIP2) into the second messengers inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). This leads to calcium release, protein kinase C (PKC) activation, and sometimes PIP2 depletion. To understand mechanisms governing these diverging signals and to determine which of these signals is responsible for the inhibition of KCNQ2/3 (KV7.2/7.3) potassium channels, we monitored levels of PIP2, IP3, and calcium in single living cells. DAG and PKC are monitored in our companion paper (Falkenburger et al. 2013. J. Gen. Physiol. http://dx.doi.org/10.1085/jgp.201210887). The results extend our previous kinetic model of Gq-coupled receptor signaling to IP3 and calcium. We find that activation of low-abundance endogenous P2Y2 receptors by a saturating concentration of uridine 5'-triphosphate (UTP; 100 µM) leads to calcium release but not to PIP2 depletion. Activation of overexpressed M1 muscarinic receptors by 10 µM Oxo-M leads to a similar calcium release but also depletes PIP2. KCNQ2/3 channels are inhibited by Oxo-M (by 85%), but not by UTP (calcium responses can be elicited even after PIP2 was partially depleted by overexpressed inducible phosphatidylinositol 5-phosphatases, suggesting that very low amounts of IP3 suffice to elicit a full calcium release. Hence, weak PLC activation can elicit robust calcium signals without net PIP2 depletion or KCNQ2/3 channel inhibition.

  3. Quantitative phosphoproteomics dissection of 7TM receptor signaling using full and biased agonists

    DEFF Research Database (Denmark)

    Christensen, Gitte L; Kelstrup, Christian D; Lyngsø, Christina

    2010-01-01

    only activates the Gaq protein-independent signaling.e quantified more than ten thousand phosphorylation sites of which 1183 were regulated by Angiotensin II or its analogue SII Angiotensin II. 36% of the AT1R regulated phosphorylations were regulated by SII Angiotensin II. Analysis of phosphorylation...... into Angiotensin II signal transduction and is the first study dissecting the differences between a full agonist and a biased agonist from a 7TMR on a systems-wide scale. Importantly, it reveals a previously unappreciated diversity and quantity of Gaq protein-independent signaling and uncovers novel signaling......Seven-transmembrane receptors (7TMRs) signal through the well described heterotrimeric G proteins, but can also activate G protein-independent signaling pathways of which the impact and complexity are less understood. The Angiotensin II type 1 receptor (AT1R) is a prototypical 7TMR and an important...

  4. FUSION TRANSCRIPTS OF BCR/ABL GENE IN PATIENTES WITH CHRONIC MYELOID LEUKEMIA

    OpenAIRE

    Artigas, Carmen Gloria; Melo, Angélica; Roa, Juan Carlos; Roa, Iván; Quijada, Ingrid; Vittini, Cecilia; Cabrera, María Elena; Risueño, Concepción

    2003-01-01

    La anormalidad citogenética más común en la leucemia mieloide crónica (LMC) es el cromosoma Philadelphia, producida por la t(9;22), cuya expresión molecular es el gen de fusión BCR-ABL, que codifica proteínas con actividad tirosinquinasa. Según el punto de ruptura de los genes BCR o ABL se produce una proteína de fusión de 210-kD(p210) o 190-kD(p190). La presencia de este gen de fusión en pacientes con LMC tiene implicancia diagnóstica. Con el propósito de detectar transcriptos de fusión del ...

  5. Postoperative ileus involves interleukin-1 receptor signaling in enteric glia.

    Science.gov (United States)

    Stoffels, Burkhard; Hupa, Kristof Johannes; Snoek, Susanne A; van Bree, Sjoerd; Stein, Kathy; Schwandt, Timo; Vilz, Tim O; Lysson, Mariola; Veer, Cornelis Van't; Kummer, Markus P; Hornung, Veit; Kalff, Joerg C; de Jonge, Wouter J; Wehner, Sven

    2014-01-01

    Postoperative ileus (POI) is a common consequence of abdominal surgery that increases the risk of postoperative complications and morbidity. We investigated the cellular mechanisms and immune responses involved in the pathogenesis of POI. We studied a mouse model of POI in which intestinal manipulation leads to inflammation of the muscularis externa and disrupts motility. We used C57BL/6 (control) mice as well as mice deficient in Toll-like receptors (TLRs) and cytokine signaling components (TLR-2(-/-), TLR-4(-/-), TLR-2/4(-/-), MyD88(-/-), MyD88/TLR adaptor molecule 1(-/-), interleukin-1 receptor [IL-1R1](-/-), and interleukin (IL)-18(-/-) mice). Bone marrow transplantation experiments were performed to determine which cytokine receptors and cell types are involved in the pathogenesis of POI. Development of POI did not require TLRs 2, 4, or 9 or MyD88/TLR adaptor molecule 2 but did require MyD88, indicating a role for IL-1R1. IL-1R1(-/-) mice did not develop POI; however, mice deficient in IL-18, which also signals via MyD88, developed POI. Mice given injections of an IL-1 receptor antagonist (anakinra) or antibodies to deplete IL-1α and IL-1β before intestinal manipulation were protected from POI. Induction of POI activated the inflammasome in muscularis externa tissues of C57BL6 mice, and IL-1α and IL-1β were released in ex vivo organ bath cultures. In bone marrow transplantation experiments, the development of POI required activation of IL-1 receptor in nonhematopoietic cells. IL-1R1 was expressed by enteric glial cells in the myenteric plexus layer, and cultured primary enteric glia cells expressed IL-6 and the chemokine monocyte chemotactic protein 1 in response to IL-1β stimulation. Immunohistochemical analysis of human small bowel tissue samples confirmed expression of IL-1R1 in the ganglia of the myenteric plexus. IL-1 signaling, via IL-1R1 and MyD88, is required for development of POI after intestinal manipulation in mice. Agents that interfere with

  6. G Protein-Coupled Receptor Signaling in Stem Cells and Cancer.

    Science.gov (United States)

    Lynch, Jennifer R; Wang, Jenny Yingzi

    2016-05-11

    G protein-coupled receptors (GPCRs) are a large superfamily of cell-surface signaling proteins that bind extracellular ligands and transduce signals into cells via heterotrimeric G proteins. GPCRs are highly tractable drug targets. Aberrant expression of GPCRs and G proteins has been observed in various cancers and their importance in cancer stem cells has begun to be appreciated. We have recently reported essential roles for G protein-coupled receptor 84 (GPR84) and G protein subunit Gαq in the maintenance of cancer stem cells in acute myeloid leukemia. This review will discuss how GPCRs and G proteins regulate stem cells with a focus on cancer stem cells, as well as their implications for the development of novel targeted cancer therapies.

  7. G Protein-Coupled Receptor Signaling in Stem Cells and Cancer

    Directory of Open Access Journals (Sweden)

    Jennifer R. Lynch

    2016-05-01

    Full Text Available G protein-coupled receptors (GPCRs are a large superfamily of cell-surface signaling proteins that bind extracellular ligands and transduce signals into cells via heterotrimeric G proteins. GPCRs are highly tractable drug targets. Aberrant expression of GPCRs and G proteins has been observed in various cancers and their importance in cancer stem cells has begun to be appreciated. We have recently reported essential roles for G protein-coupled receptor 84 (GPR84 and G protein subunit Gαq in the maintenance of cancer stem cells in acute myeloid leukemia. This review will discuss how GPCRs and G proteins regulate stem cells with a focus on cancer stem cells, as well as their implications for the development of novel targeted cancer therapies.

  8. Two signaling molecules share a phosphotyrosine-containing binding site in the platelet-derived growth factor receptor.

    Science.gov (United States)

    Nishimura, R; Li, W; Kashishian, A; Mondino, A; Zhou, M; Cooper, J; Schlessinger, J

    1993-11-01

    Autophosphorylation sites of growth factor receptors with tyrosine kinase activity function as specific binding sites for Src homology 2 (SH2) domains of signaling molecules. This interaction appears to be a crucial step in a mechanism by which receptor tyrosine kinases relay signals to downstream signaling pathways. Nck is a widely expressed protein consisting exclusively of SH2 and SH3 domains, the overexpression of which causes cell transformation. It has been shown that various growth factors stimulate the phosphorylation of Nck and its association with autophosphorylated growth factor receptors. A panel of platelet-derived growth factor (PDGF) receptor mutations at tyrosine residues has been used to identify the Nck binding site. Here we show that mutation at Tyr-751 of the PDGF beta-receptor eliminates Nck binding both in vitro and in living cells. Moreover, the Y751F PDGF receptor mutant failed to mediate PDGF-stimulated phosphorylation of Nck in intact cells. A phosphorylated Tyr-751 is also required for binding of phosphatidylinositol-3 kinase to the PDGF receptor. Hence, the SH2 domains of p85 and Nck share a binding site in the PDGF receptor. Competition experiments with different phosphopeptides derived from the PDGF receptor suggest that binding of Nck and p85 is influenced by different residues around Tyr-751. Thus, a single tyrosine autophosphorylation site is able to link the PDGF receptor to two distinct SH2 domain-containing signaling molecules.

  9. Negative regulation of Toll-like receptor signalling 

    Directory of Open Access Journals (Sweden)

    Halina Antosz

    2013-04-01

    Full Text Available The mechanism of innate immunity is based on the pattern recognition receptors (PRR that recognize molecular patterns associated with pathogens (PAMPs. Among PRR receptors Toll-like receptors (TLR are distinguished. As a result of contact with pathogens, TLRs activate specific intracellular signaling pathways. It happens through proteins such as adaptor molecules, e.g. MyD88, TIRAP, TRIF, TRAM, and IPS-1, which participate in the cascade activation of kinases (IKK, MAP, RIP-1, TBK-1 as well as transcription factors (NF-κB, AP-1 and regulatory factor (IRF3. The result of this activation is the production of active proinflammatory cytokines, chemokines, interferons and enzymes. The PRR pathways are controlled by extra – and intracellular molecules to prevent overexpression of PRR. They include soluble receptors (sTLR, transmembrane proteins (ST2, SIGIRR, RP105, TRAIL-R and intracellular inhibitors (SOCS-1, SOCS-3, sMyD88, TOLLIP, IRAK-M, SARM, A20, β-arrestin, CYLD, SHP. These molecules maintain the balance between activation and inhibition and ensure balancing of the beneficial and adverse effects of antigen recognition.

  10. DMPD: Signaling to NF-kappaB by Toll-like receptors. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available l Med. 2007 Nov;13(11):460-9. Epub 2007 Oct 29. (.png) (.svg) (.html) (.csml) Show Signaling to NF-kappaB by Toll-like receptors. Pub...medID 18029230 Title Signaling to NF-kappaB by Toll-like receptors. Authors Kawai T

  11. Quantification of BCR-ABL transcripts in peripheral blood cells and ...

    African Journals Online (AJOL)

    Purpose: To investigate the feasibility of using peripheral blood plasma samples as surrogates for blood cell sampling for quantification of breakpoint cluster region-Abelson oncogene (BCR-ABL) transcript levels to monitor treatment responses in chronic myeloid leukemia (CML) patients. Methods: Peripheral blood samples ...

  12. Inositol trisphosphate receptor mediated spatiotemporal calcium signalling.

    Science.gov (United States)

    Miyazaki, S

    1995-04-01

    Spatiotemporal Ca2+ signalling in the cytoplasm is currently understood as an excitation phenomenon by analogy with electrical excitation in the plasma membrane. In many cell types, Ca2+ waves and Ca2+ oscillations are mediated by inositol 1,4,5-trisphosphate (IP3) receptor/Ca2+ channels in the endoplasmic reticulum membrane, with positive feedback between cytosolic Ca2+ and IP3-induced Ca2+ release creating a regenerative process. Remarkable advances have been made in the past year in the analysis of subcellular Ca2+ microdomains using confocal microscopy and of Ca2+ influx pathways that are functionally coupled to IP3-induced Ca2+ release. Ca2+ signals can be conveyed into the nucleus and mitochondria. Ca2+ entry from outside the cell allows repetitive Ca2+ release by providing Ca2+ to refill the endoplasmic reticulum stores, thus giving rise to frequency-encoded Ca2+ signals.

  13. Signalling through C-type lectin receptors: shaping immune responses

    NARCIS (Netherlands)

    Geijtenbeek, Teunis B. H.; Gringhuis, Sonja I.

    2009-01-01

    C-type lectin receptors (CLRs) expressed by dendritic cells are crucial for tailoring immune responses to pathogens. Following pathogen binding, CLRs trigger distinct signalling pathways that induce the expression of specific cytokines which determine T cell polarization fates. Some CLRs can induce

  14. Role of Estrogen Receptor Signaling in Breast Cancer Metastasis

    International Nuclear Information System (INIS)

    Roy, S.S.; Vadlamudi, R.K.

    2012-01-01

    Metastatic breast cancer is a life-threatening stage of cancer and is the leading cause of death in advanced breast cancer patients. Estrogen signaling and the estrogen receptor (ER) are implicated in breast cancer progression, and the majority of the human breast cancers start out as estrogen dependent. Accumulating evidence suggests that ER signaling is complex, involving coregulatory proteins and extranuclear actions. ER-coregualtory proteins are tightly regulated under normal conditions with miss expression primarily reported in cancer. Deregulation of ER coregualtors or ER extranuclear signaling has potential to promote metastasis in ER-positive breast cancer cells. This review summarizes the emerging role of ER signaling in promoting metastasis of breast cancer cells, discusses the molecular mechanisms by which ER signaling contributes to metastasis, and explores possible therapeutic targets to block ER-driven metastasis

  15. 14-3-3 Proteins Buffer Intracellular Calcium Sensing Receptors to Constrain Signaling.

    Directory of Open Access Journals (Sweden)

    Michael P Grant

    Full Text Available Calcium sensing receptors (CaSR interact with 14-3-3 binding proteins at a carboxyl terminal arginine-rich motif. Mutations identified in patients with familial hypocalciuric hypercalcemia, autosomal dominant hypocalcemia, pancreatitis or idiopathic epilepsy support the functional importance of this motif. We combined total internal reflection fluorescence microscopy and biochemical approaches to determine the mechanism of 14-3-3 protein regulation of CaSR signaling. Loss of 14-3-3 binding caused increased basal CaSR signaling and plasma membrane levels, and a significantly larger signaling-evoked increase in plasma membrane receptors. Block of core glycosylation with tunicamycin demonstrated that changes in plasma membrane CaSR levels were due to differences in exocytic rate. Western blotting to quantify time-dependent changes in maturation of expressed wt CaSR and a 14-3-3 protein binding-defective mutant demonstrated that signaling increases synthesis to maintain constant levels of the immaturely and maturely glycosylated forms. CaSR thus operates by a feed-forward mechanism, whereby signaling not only induces anterograde trafficking of nascent receptors but also increases biosynthesis to maintain steady state levels of net cellular CaSR. Overall, these studies suggest that 14-3-3 binding at the carboxyl terminus provides an important buffering mechanism to increase the intracellular pool of CaSR available for signaling-evoked trafficking, but attenuates trafficking to control the dynamic range of responses to extracellular calcium.

  16. BCR/ABL downregulates DNA-PK(CS)-dependent and upregulates backup non-homologous end joining in leukemic cells.

    Science.gov (United States)

    Poplawski, Tomasz; Blasiak, Janusz

    2010-06-01

    Non-homologous end joining (NHEJ) and homologous recombination repair (HRR) are the main mechanisms involved in the processing of DNA double strand breaks (DSBs) in humans. We showed previously that the oncogenic tyrosine kinase BCR/ABL stimulated DSBs repair by HRR. To evaluate the role of BCR/ABL in DSBs repair by NHEJ we examined the ability of leukemic BCR/ABL-expressing cell line BV173 to repair DNA damage induced by two DNA topoisomerase II inhibitors: etoposide and sobuzoxane. DNA lesions induced by sobuzoxane are repaired by a NHEJ pathway which is dependent on the catalytic subunit of protein kinase dependent on DNA (DNA-PK(CS); D-NHEJ), whereas damage evoked by etoposide are repaired by two distinct NHEJ pathways, dependent on or independent of DNA-PK(CS) (backup NHEJ, B-NHEJ). Cells incubated with STI571, a highly specific inhibitor of BCR/ABL, displayed resistance to these agents associated with an accelerated kinetics of DSBs repair, as measured by the neutral comet assay and pulsed field gel electrophoresis. However, in a functional NHEJ assay, cells preincubated with STI571 repaired DSBs induced by a restriction enzyme with a lower efficacy than without the preincubation and addition of wortmannin, a specific inhibitor of DNA-PK(CS), did not change efficacy of the NHEJ reaction. We suggest that BCR/ABL switch on B-NHEJ which is more error-prone then D-NHEJ and in such manner contribute to the increase of the genomic instability of leukemic cells.

  17. Elabela-apelin receptor signaling pathway is functional in mammalian systems.

    Science.gov (United States)

    Wang, Zhi; Yu, Daozhan; Wang, Mengqiao; Wang, Qilong; Kouznetsova, Jennifer; Yang, Rongze; Qian, Kun; Wu, Wenjun; Shuldiner, Alan; Sztalryd, Carole; Zou, Minghui; Zheng, Wei; Gong, Da-Wei

    2015-02-02

    Elabela (ELA) or Toddler is a recently discovered hormone which is required for normal development of heart and vasculature through activation of apelin receptor (APJ), a G protein-coupled receptor (GPCR), in zebrafish. The present study explores whether the ELA-APJ signaling pathway is functional in the mammalian system. Using reverse-transcription PCR, we found that ELA is restrictedly expressed in human pluripotent stem cells and adult kidney whereas APJ is more widely expressed. We next studied ELA-APJ signaling pathway in reconstituted mammalian cell systems. Addition of ELA to HEK293 cells over-expressing GFP-AJP fusion protein resulted in rapid internalization of the fusion receptor. In Chinese hamster ovarian (CHO) cells over-expressing human APJ, ELA suppresses cAMP production with EC50 of 11.1 nM, stimulates ERK1/2 phosphorylation with EC50 of 14.3 nM and weakly induces intracellular calcium mobilization. Finally, we tested ELA biological function in human umbilical vascular endothelial cells and showed that ELA induces angiogenesis and relaxes mouse aortic blood vessel in a dose-dependent manner through a mechanism different from apelin. Collectively, we demonstrate that the ELA-AJP signaling pathways are functional in mammalian systems, indicating that ELA likely serves as a hormone regulating the circulation system in adulthood as well as in embryonic development.

  18. CD147 is a signaling receptor for cyclophilin B.

    Science.gov (United States)

    Yurchenko, V; O'Connor, M; Dai, W W; Guo, H; Toole, B; Sherry, B; Bukrinsky, M

    2001-11-09

    Cyclophilins A and B (CyPA and CyPB) are cyclosporin A binding proteins that can be secreted in response to inflammatory stimuli. We recently identified CD147 as a cell-surface receptor for CyPA and demonstrated that CD147 is an essential component in the CyPA-initiated signaling cascade that culminates in ERK activation and chemotaxis. Here we demonstrate that CD147 also serves as a receptor for CyPB. CyPB induced Ca(2+) flux and chemotaxis of CD147-transfected, but not control, CHO cells, and the chemotactic response of primary human neutrophils to CyPB was blocked by antibodies to CD147. These results suggest that CD147 serves as a receptor for extracellular cyclophilins. Copyright 2001 Academic Press.

  19. Signaling Properties of Chemerin Receptors CMKLR1, GPR1 and CCRL2.

    Science.gov (United States)

    De Henau, Olivier; Degroot, Gaetan-Nagim; Imbault, Virginie; Robert, Virginie; De Poorter, Cédric; Mcheik, Saria; Galés, Céline; Parmentier, Marc; Springael, Jean-Yves

    2016-01-01

    Chemerin is a small chemotactic protein originally identified as the natural ligand of CMKLR1. More recently, two other receptors, GPR1 and CCRL2, have been reported to bind chemerin but their functional relevance remains poorly understood. In this study, we compared the binding and signaling properties of the three human chemerin receptors and showed differences in mode of chemerin binding and receptor signaling. Chemerin binds to all three receptors with low nanomolar affinities. However, the contribution of the chemerin C-terminus to binding efficiency varies greatly amongst receptors. By using BRET-based biosensors monitoring the activation of various G proteins, we showed that binding of chemerin and the chemerin 9 nonapeptide (149YFPGQFAFS157) to CMKLR1 activates the three Gαi subtypes (Gαi1, Gαi2 and Gαi3) and the two Gαo isoforms (Gαoa and Gαob) with potencies correlated to binding affinities. In contrast, no significant activation of G proteins was detected upon binding of chemerin to GPR1 or CCRL2. Binding of chemerin and the chemerin 9 peptide also induced the recruitment of β-arrestin1 and 2 to CMKLR1 and GPR1, though to various degree, but not to CCRL2. However, the propensity of chemerin 9 to activate β-arrestins relative to chemerin is higher when bound to GPR1. Finally, we showed that binding of chemerin to CMKLR1 and GPR1 promotes also the internalization of the two receptors and the phosphorylation of ERK1/2 MAP kinases, although with a different efficiency, and that phosphorylation of ERK1/2 requires both Gαi/o and β-arrestin2 activation but not β-arrestin1. Collectively, these data support a model in which each chemerin receptor displays selective signaling properties.

  20. Age-related changes in expression and signaling of TAM receptor inflammatory regulators in monocytes.

    Science.gov (United States)

    Wang, Xiaomei; Malawista, Anna; Qian, Feng; Ramsey, Christine; Allore, Heather G; Montgomery, Ruth R

    2018-02-09

    The multifactorial immune deterioration in aging--termed "inflamm-aging"--is comprised of a state of low-grade, chronic inflammation and complex dysregulation of responses to immune stimulation. The TAM family (Tyro 3, Axl, and Mer) of receptor tyrosine kinases are negative regulators of Toll like receptor-mediated immune responses that broadly inhibit cytokine receptor cascades to inhibit inflammation. Here we demonstrate elevated expression of TAM receptors in monocytes of older adults, and an age-dependent difference in signaling mediator AKT resulting in dysregulated responses to signaling though Mer. Our results may be especially significant in tissue, where levels of Mer are highest, and may present avenues for modulation of chronic tissue inflammation noted in aging.

  1. Cross-regulation of cytokine signalling: pro-inflammatory cytokines restrict IL-6 signalling through receptor internalisation and degradation.

    Science.gov (United States)

    Radtke, Simone; Wüller, Stefan; Yang, Xiang-ping; Lippok, Barbara E; Mütze, Barbara; Mais, Christine; de Leur, Hildegard Schmitz-Van; Bode, Johannes G; Gaestel, Matthias; Heinrich, Peter C; Behrmann, Iris; Schaper, Fred; Hermanns, Heike M

    2010-03-15

    The inflammatory response involves a complex interplay of different cytokines which act in an auto- or paracrine manner to induce the so-called acute phase response. Cytokines are known to crosstalk on multiple levels, for instance by regulating the mRNA stability of targeted cytokines through activation of the p38-MAPK pathway. In our study we discovered a new mechanism that answers the long-standing question how pro-inflammatory cytokines and environmental stress restrict immediate signalling of interleukin (IL)-6-type cytokines. We show that p38, activated by IL-1beta, TNFalpha or environmental stress, impairs IL-6-induced JAK/STAT signalling through phosphorylation of the common cytokine receptor subunit gp130 and its subsequent internalisation and degradation. We identify MK2 as the kinase that phosphorylates serine 782 in the cytoplasmic part of gp130. Consequently, inhibition of p38 or MK2, deletion of MK2 or mutation of crucial amino acids within the MK2 target site or the di-leucine internalisation motif blocks receptor depletion and restores IL-6-dependent STAT activation as well as gene induction. Hence, a novel negative crosstalk mechanism for cytokine signalling is described, where cytokine receptor turnover is regulated in trans by pro-inflammatory cytokines and stress stimuli to coordinate the inflammatory response.

  2. Heterotrimeric G Protein-coupled Receptor Signaling in Yeast Mating Pheromone Response.

    Science.gov (United States)

    Alvaro, Christopher G; Thorner, Jeremy

    2016-04-08

    The DNAs encoding the receptors that respond to the peptide mating pheromones of the budding yeastSaccharomyces cerevisiaewere isolated in 1985, and were the very first genes for agonist-binding heterotrimeric G protein-coupled receptors (GPCRs) to be cloned in any organism. Now, over 30 years later, this yeast and its receptors continue to provide a pathfinding experimental paradigm for investigating GPCR-initiated signaling and its regulation, as described in this retrospective overview. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. Cadherin adhesion, tissue tension, and noncanonical Wnt signaling regulate fibronectin matrix organization.

    Science.gov (United States)

    Dzamba, Bette J; Jakab, Karoly R; Marsden, Mungo; Schwartz, Martin A; DeSimone, Douglas W

    2009-03-01

    In this study we demonstrate that planar cell polarity signaling regulates morphogenesis in Xenopus embryos in part through the assembly of the fibronectin (FN) matrix. We outline a regulatory pathway that includes cadherin adhesion and signaling through Rac and Pak, culminating in actin reorganization, myosin contractility, and tissue tension, which, in turn, directs the correct spatiotemporal localization of FN into a fibrillar matrix. Increased mechanical tension promotes FN fibril assembly in the blastocoel roof (BCR), while reduced BCR tension inhibits matrix assembly. These data support a model for matrix assembly in tissues where cell-cell adhesions play an analogous role to the focal adhesions of cultured cells by transferring to integrins the tension required to direct FN fibril formation at cell surfaces.

  4. Signaling flux redistribution at toll-like receptor pathway junctions.

    Directory of Open Access Journals (Sweden)

    Kumar Selvarajoo

    Full Text Available Various receptors on cell surface recognize specific extracellular molecules and trigger signal transduction altering gene expression in the nucleus. Gain or loss-of-function mutations of one molecule have shown to affect alternative signaling pathways with a poorly understood mechanism. In Toll-like receptor (TLR 4 signaling, which branches into MyD88- and TRAM-dependent pathways upon lipopolysaccharide (LPS stimulation, we investigated the gain or loss-of-function mutations of MyD88. We predict, using a computational model built on the perturbation-response approach and the law of mass conservation, that removal and addition of MyD88 in TLR4 activation, enhances and impairs, respectively, the alternative TRAM-dependent pathway through signaling flux redistribution (SFR at pathway branches. To verify SFR, we treated MyD88-deficient macrophages with LPS and observed enhancement of TRAM-dependent pathway based on increased IRF3 phosphorylation and induction of Cxcl10 and Ifit2. Furthermore, increasing the amount of MyD88 in cultured cells showed decreased TRAM binding to TLR4. Investigating another TLR4 pathway junction, from TRIF to TRAF6, RIP1 and TBK1, the removal of MyD88-dependent TRAF6 increased expression of TRAM-dependent Cxcl10 and Ifit2. Thus, we demonstrate that SFR is a novel mechanism for enhanced activation of alternative pathways when molecules at pathway junctions are removed. Our data suggest that SFR may enlighten hitherto unexplainable intracellular signaling alterations in genetic diseases where gain or loss-of-function mutations are observed.

  5. The role of insulin receptor signaling in the brain.

    Science.gov (United States)

    Plum, Leona; Schubert, Markus; Brüning, Jens C

    2005-03-01

    The insulin receptor (IR) is expressed in various regions of the developing and adult brain, and its functions have become the focus of recent research. Insulin enters the central nervous system (CNS) through the blood-brain barrier by receptor-mediated transport to regulate food intake, sympathetic activity and peripheral insulin action through the inhibition of hepatic gluconeogenesis and reproductive endocrinology. On a molecular level, some of the effects of insulin converge with those of the leptin signaling machinery at the point of activation of phosphatidylinositol 3-kinase (PI3K), resulting in the regulation of ATP-dependent potassium channels. Furthermore, insulin inhibits neuronal apoptosis via activation of protein kinase B in vitro, and it regulates phosphorylation of tau, metabolism of the amyloid precursor protein and clearance of beta-amyloid from the brain in vivo. These findings indicate that neuronal IR signaling has a direct role in the link between energy homeostasis, reproduction and the development of neurodegenerative diseases.

  6. Expression of G(alpha)(s) proteins and TSH receptor signalling in hyperfunctioning thyroid nodules with TSH receptor mutations.

    Science.gov (United States)

    Holzapfel, Hans-Peter; Bergner, Beate; Wonerow, Peter; Paschke, Ralf

    2002-07-01

    Constitutively activating mutations of the thyrotrophin receptor (TSHR) are the main molecular cause of hyperfunctioning thyroid nodules (HTNs). The G protein coupling is an important and critical step in the TSHR signalling which mainly includes G(alpha)(s), G(alpha)(i) and G(alpha)(q)/11 proteins. We investigated the in vitro consequences of overexpressing G(alpha) proteins on signalling of the wild-type (WT) or mutated TSHR. Moreover, we investigated whether changes in G(alpha) protein expression are pathophysiologically relevant in HTNs or cold thyroid nodules (CTNs). Wild-type TSH receptor and mutated TSH receptors were coexpressed with G(alpha)(s), G(alpha)(i) or G(alpha)(q)/11, and cAMP and inositol phosphate (IP) production was measured after stimulation with TSH. The expression of G(alpha)(s), G(alpha)(i) and G(alpha)(q)/11 proteins was examined by Western blotting in 28 HTNs and 14 CTNs. Coexpression of G(alpha)(s) with the WT TSH receptor in COS 7 cells significantly increased the basal and TSH-stimulated cAMP accumulation while coexpression of the G(alpha)(q) or G(alpha)11 protein significantly increased the production of cAMP and inositol triphosphate (IP(3)). The coexpression of the TSH receptor mutants (I486F, DEL613-621), known to couple constitutively to G(alpha)(s) and G(alpha)(q) with G(alpha)(s) and G(alpha)(q)/11, significantly increased the basal and stimulated cAMP and IP(3) accumulation. Coexpression of the TSH receptor mutant V556F with G(alpha)(s) only increased the basal and stimulated cAMP production while its coexpression with G(alpha)(q)/11 increased the basal and stimulated IP(3) signalling. The expression of G(alpha)(s) protein subunits determined by Western blotting was significantly decreased in 14 HTNs with a constitutively activating TSH receptor mutation in comparison with the corresponding surrounding tissue, while in 14 HTNs without TSH receptor or G(alpha)(s) protein mutation and in 14 CTNs the expression of G

  7. Overcoming Bcr-Abl T315I mutation by combination of GNF-2 and ATP competitors in an Abl-independent mechanism

    International Nuclear Information System (INIS)

    Khateb, Mamduh; Ruimi, Nili; Khamisie, Hazem; Najajreh, Yousef; Mian, Afsar; Metodieva, Anna; Ruthardt, Martin; Mahajna, Jamal

    2012-01-01

    Philadelphia positive leukemias are characterized by the presence of Bcr-Abl fusion protein which exhibits an abnormal kinase activity. Selective Abl kinase inhibitors have been successfully established for the treatment of Ph (+) leukemias. Despite high rates of clinical response, Ph (+) patients can develop resistance against these kinase inhibitors mainly due to point mutations within the Abl protein. Of special interest is the ‘gatekeeper’ T315I mutation, which confers complete resistance to Abl kinase inhibitors. Recently, GNF-2, Abl allosteric kinase inhibitor, was demonstrated to possess cellular activity against Bcr-Abl transformed cells. Similarly to Abl kinase inhibitors (AKIs), GNF-2 failed to inhibit activity of mutated Bcr-Abl carrying the T315I mutation. Ba/F3 cells harboring native or T315I mutated Bcr-Abl constructs were treated with GNF-2 and AKIs. We monitored the effect of GNF-2 with AKIs on the proliferation and clonigenicity of the different Ba/F3 cells. In addition, we monitored the auto-phosphorylation activity of Bcr-Abl and JAK2 in cells treated with GNF-2 and AKIs. In this study, we report a cooperation between AKIs and GNF-2 in inhibiting proliferation and clonigenicity of Ba/F3 cells carrying T315I mutated Bcr-Abl. Interestingly, cooperation was most evident between Dasatinib and GNF-2. Furthermore, we showed that GNF-2 was moderately active in inhibiting the activity of JAK2 kinase, and presence of AKIs augmented GNF-2 activity. Our data illustrated the ability of allosteric inhibitors such as GNF-2 to cooperate with AKIs to overcome T315I mutation by Bcr-Abl-independent mechanisms, providing a possibility of enhancing AKIs efficacy and overcoming resistance in Ph+ leukemia cells

  8. Computational dissection of allosteric inhibition of the SH2 domain of Bcr-Abl kinase by the monobody inhibitor AS25.

    Science.gov (United States)

    Ji, Mingfei; Zheng, Guodong; Li, Xiaolong; Zhang, Zhongqin; Jv, Guanqun; Wang, Xiaowei; Wang, Jialin

    2017-06-01

    The deregulated breakpoint cluster region (Bcr)-Abelson tyrosine kinase (Abl) fusion protein represents an attractive pharmacological target for the treatment of chronic myeloid leukemia (CML). The high affinity of monobody AS25 was designed to target the Src homology 2 (SH2) domain of Bcr-Abl, leading to allosteric inhibition of Bcr-Abl through formation of protein-protein interactions. An I164E mutation in the SH2 domain disrupts AS25 binding to the SH2 domain of Bcr-Abl. The detailed mechanisms, however, remain to be unresolved. Here, molecular dynamics (MD) simulations and binding free energy calculations were performed to explore the conformational and energetic differences between the wild-type (WT) complexes of Bcr-Abl SH2 domain and AS25 (SH2 WT -AS25) as well as the mutated complexes (SH2 I164E -AS25). The results revealed that I164E mutation not only caused an increase in the conformational flexibility of SH2-AS25 complexes, but also weakened the binding affinity of AS25 to SH2. The comparative binding modes of SH2-AS25 complexes between WT and the I164E mutant were comprehensively analyzed to unravel the disruption of hydrophobic and hydrogen bonding interactions in the interface of the SH2-AS25 complex triggered by the I164E mutation. The results obtained may help to design the next generation of higher affinity Bcr-Abl SH2-specific peptide inhibitors.

  9. Frequency of p190 and p210 BCR-ABL rearrangements and survival in Brazilian adult patients with acute lymphoblastic leukemia

    Directory of Open Access Journals (Sweden)

    Ilana de França Azevedo

    2014-10-01

    Full Text Available Objective: This study investigated the occurrence of the p190 and p210 break point clusterregion-Abelson (BCR-ABL rearrangements in adults with acute lymphoblastic leukemia and possible associations with clinical and laboratory characteristics and survival. Methods: Forty-one over 18-year-old patients with acute lymphoblastic leukemia of both genders followed-up between January 2008 and May 2012 were included in this study. Clinical and laboratory data were obtained from the medical charts of the patients. Reverse transcription polymerase chain reaction (RT-PCR using specific primers was employed to identify molecular rearrangements. Results: At diagnosis, the median age was 33 years, and there was a predominance of males (61%. The most common immunophenotype was B lineage (76%. BCR-ABL rearrangements was detected in 14 (34% patients with the following distribution: p190 (28%, p210 (50% and double positive (22%. Overall survival of patients with a mean/median of 331/246 days of follow up was 39%, respectively, negative BCR-ABL (44% and positive BCR-ABL (28%. Conclusion: These results confirm the high frequency of BCR-ABL rearrangements and the low survival rate of adult Brazilian patients with acute lymphoblastic leukemia.

  10. Targeting CB2-GPR55 Receptor Heteromers Modulates Cancer Cell Signaling*

    Science.gov (United States)

    Moreno, Estefanía; Andradas, Clara; Medrano, Mireia; Caffarel, María M.; Pérez-Gómez, Eduardo; Blasco-Benito, Sandra; Gómez-Cañas, María; Pazos, M. Ruth; Irving, Andrew J.; Lluís, Carme; Canela, Enric I.; Fernández-Ruiz, Javier; Guzmán, Manuel; McCormick, Peter J.; Sánchez, Cristina

    2014-01-01

    The G protein-coupled receptors CB2 (CB2R) and GPR55 are overexpressed in cancer cells and human tumors. Because a modulation of GPR55 activity by cannabinoids has been suggested, we analyzed whether this receptor participates in cannabinoid effects on cancer cells. Here we show that CB2R and GPR55 form heteromers in cancer cells, that these structures possess unique signaling properties, and that modulation of these heteromers can modify the antitumoral activity of cannabinoids in vivo. These findings unveil the existence of previously unknown signaling platforms that help explain the complex behavior of cannabinoids and may constitute new targets for therapeutic intervention in oncology. PMID:24942731

  11. Dual Drug Targeting of Mutant Bcr-Abl Induces Inactive Conformation: New Strategy for the Treatment of Chronic Myeloid Leukemia and Overcoming Monotherapy Resistance.

    Science.gov (United States)

    El Rashedy, Ahmed A; Olotu, Fisayo A; Soliman, Mahmoud E S

    2018-03-01

    Bcr-Abl is an oncogenic fusion protein which expression enhances tumorigenesis, and has been highly associated with chronic myeloid leukemia (CML). Acquired drug resistance in mutant Bcr-Abl has enhanced pathogenesis with the use of single therapy agents such as nilotinib. Moreover, allosteric targeting has been identified to consequentially inhibit Bcr-Abl activity, which led to the recent development of ABL-001 (asciminib) that selectively binds the myristoyl pocket. Experimental studies have revealed that the combination of nilotinib and ABL-001 induced a 'bent' conformation in the C-terminal helix of Bcr-Abl; a benchmark of inhibition, thereby exhibiting a greater potency in the treatment of CML, surmounting the setbacks of drug resistance, disease regression and relapse. Therefore, we report the first account of the dynamics and conformational analysis of oncogenic T334I Bcr-Abl by dual targeting. Our findings revealed that unlike in the Bcr-Abl-Nilotinib complex, dual targeting by both inhibitors induced the bent conformation in the C-terminal helix that varied with time. This was coupled with significant alteration in Bcr-Abl stability, flexibility, and compactness and an overall structural re-orientation inwards towards the hydrophobic core, which reduced the solvent-exposed residues indicative of protein folding. This study will facilitate allosteric targeting and the design of more potent allosteric inhibitors for resistive target proteins in cancer. © 2018 Wiley-VHCA AG, Zurich, Switzerland.

  12. Analysis of receptor signaling pathways by mass spectrometry: identification of vav-2 as a substrate of the epidermal and platelet-derived growth factor receptors

    DEFF Research Database (Denmark)

    Pandey, A; Podtelejnikov, A V; Blagoev, B

    2000-01-01

    Oligomerization of receptor protein tyrosine kinases such as the epidermal growth factor receptor (EGFR) by their cognate ligands leads to activation of the receptor. Transphosphorylation of the receptor subunits is followed by the recruitment of signaling molecules containing src homology 2 (SH2...

  13. Spatiotemporal intracellular dynamics of neurotrophin and its receptors. Implications for neurotrophin signaling and neuronal function.

    Science.gov (United States)

    Bronfman, F C; Lazo, O M; Flores, C; Escudero, C A

    2014-01-01

    Neurons possess a polarized morphology specialized to contribute to neuronal networks, and this morphology imposes an important challenge for neuronal signaling and communication. The physiology of the network is regulated by neurotrophic factors that are secreted in an activity-dependent manner modulating neuronal connectivity. Neurotrophins are a well-known family of neurotrophic factors that, together with their cognate receptors, the Trks and the p75 neurotrophin receptor, regulate neuronal plasticity and survival and determine the neuronal phenotype in healthy and regenerating neurons. Is it now becoming clear that neurotrophin signaling and vesicular transport are coordinated to modify neuronal function because disturbances of vesicular transport mechanisms lead to disturbed neurotrophin signaling and to diseases of the nervous system. This chapter summarizes our current understanding of how the regulated secretion of neurotrophin, the distribution of neurotrophin receptors in different locations of neurons, and the intracellular transport of neurotrophin-induced signaling in distal processes are achieved to allow coordinated neurotrophin signaling in the cell body and axons.

  14. Diverse FGF receptor signaling controls astrocyte specification and proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Kang, Kyungjun [School of Life Sciences, Gwangju Institute of Science and Technology, Oryong-dong, Buk-gu, Gwangju 500-712 (Korea, Republic of); Song, Mi-Ryoung, E-mail: msong@gist.ac.kr [School of Life Sciences, Gwangju Institute of Science and Technology, Oryong-dong, Buk-gu, Gwangju 500-712 (Korea, Republic of); Bioimaging Research Center and Cell Dynamics Research Center, Gwangju Institute of Science and Technology, Oryong-dong, Buk-gu, Gwangju 500-712 (Korea, Republic of)

    2010-05-07

    During CNS development, pluripotency neuronal progenitor cells give rise in succession to neurons and glia. Fibroblast growth factor-2 (FGF-2), a major signal that maintains neural progenitors in the undifferentiated state, is also thought to influence the transition from neurogenesis to gliogenesis. Here we present evidence that FGF receptors and underlying signaling pathways transmit the FGF-2 signals that regulate astrocyte specification aside from its mitogenic activity. Application of FGF-2 to cortical progenitors suppressed neurogenesis whereas treatment with an FGFR antagonist in vitro promoted neurogenesis. Introduction of chimeric FGFRs with mutated tyrosine residues into cortical progenitors and drug treatments to specifically block individual downstream signaling pathways revealed that the overall activity of FGFR rather than individual autophosphorylation sites is important for delivering signals for glial specification. In contrast, a signal for cell proliferation by FGFR was mainly delivered by MAPK pathway. Together our findings indicate that FGFR activity promotes astrocyte specification in the developing CNS.

  15. Diverse FGF receptor signaling controls astrocyte specification and proliferation

    International Nuclear Information System (INIS)

    Kang, Kyungjun; Song, Mi-Ryoung

    2010-01-01

    During CNS development, pluripotency neuronal progenitor cells give rise in succession to neurons and glia. Fibroblast growth factor-2 (FGF-2), a major signal that maintains neural progenitors in the undifferentiated state, is also thought to influence the transition from neurogenesis to gliogenesis. Here we present evidence that FGF receptors and underlying signaling pathways transmit the FGF-2 signals that regulate astrocyte specification aside from its mitogenic activity. Application of FGF-2 to cortical progenitors suppressed neurogenesis whereas treatment with an FGFR antagonist in vitro promoted neurogenesis. Introduction of chimeric FGFRs with mutated tyrosine residues into cortical progenitors and drug treatments to specifically block individual downstream signaling pathways revealed that the overall activity of FGFR rather than individual autophosphorylation sites is important for delivering signals for glial specification. In contrast, a signal for cell proliferation by FGFR was mainly delivered by MAPK pathway. Together our findings indicate that FGFR activity promotes astrocyte specification in the developing CNS.

  16. Signaling-sensitive amino acids surround the allosteric ligand binding site of the thyrotropin receptor.

    Science.gov (United States)

    Kleinau, Gunnar; Haas, Ann-Karin; Neumann, Susanne; Worth, Catherine L; Hoyer, Inna; Furkert, Jens; Rutz, Claudia; Gershengorn, Marvin C; Schülein, Ralf; Krause, Gerd

    2010-07-01

    The thyrotropin receptor [thyroid-stimulating hormone receptor (TSHR)], a G-protein-coupled receptor (GPCR), is endogenously activated by thyrotropin, which binds to the extracellular region of the receptor. We previously identified a low-molecular-weight (LMW) agonist of the TSHR and predicted its allosteric binding pocket within the receptor's transmembrane domain. Because binding of the LMW agonist probably disrupts interactions or leads to formation of new interactions among amino acid residues surrounding the pocket, we tested whether mutation of residues at these positions would lead to constitutive signaling activity. Guided by molecular modeling, we performed site-directed mutagenesis of 24 amino acids in this spatial region, followed by functional characterization of the mutant receptors in terms of expression and signaling, measured as cAMP accumulation. We found that mutations V421I, Y466A, T501A, L587V, M637C, M637W, S641A, Y643F, L645V, and Y667A located in several helices exhibit constitutive activity. Of note is mutation M637W at position 6.48 in transmembrane helix 6, which has a significant effect on the interaction of the receptor with the LMW agonist. In summary, we found that a high proportion of residues in several helices surrounding the allosteric binding site of LMW ligands in the TSHR when mutated lead to constitutively active receptors. Our findings of signaling-sensitive residues in this region of the transmembrane bundle may be of general importance as this domain appears to be evolutionarily retained among GPCRs.

  17. Physiological Signaling and Structure of the HGF Receptor MET

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    Gianluca Baldanzi

    2014-12-01

    Full Text Available The “hepatocyte growth factor” also known as “scatter factor”, is a multifunctional cytokine with the peculiar ability of simultaneously triggering epithelial cell proliferation, movement and survival. The combination of those proprieties results in the induction of an epithelial to mesenchymal transition in target cells, fundamental for embryogenesis but also exploited by tumor cells during metastatization. The hepatocyte growth factor receptor, MET, is a proto-oncogene and a prototypical transmembrane tyrosine kinase receptor. Inhere we discuss the MET molecular structure and the hepatocyte growth factor driven physiological signaling which coordinates epithelial proliferation, motility and morphogenesis.

  18. Association of cannabis use during adolescence, prefrontal CB1 receptor signaling and schizophrenia

    Directory of Open Access Journals (Sweden)

    Adriana eCaballero

    2012-05-01

    Full Text Available The cannabinoid receptor 1 (CB1R is the G-protein coupled receptor responsible for the majority of the endocannabinoid signaling in the human brain. It is widely distributed in the limbic system, basal ganglia, and cerebellum, which are areas responsible for cognition, memory, and motor control. Because of this widespread distribution, it is not surprising that drugs that co-opt CB1R have expected behavioral outcomes consistent with dysregulated signaling from these areas (e.g. memory loss, cognitive deficits, etc. In the context of this review, we present evidence for the role of CB1R signaling in the prefrontal cortex (PFC, an area involved in executive functions, with emphasis on the developmental regulation of CB1R signaling in the acquisition of mature PFC function. We further hypothesize how alterations of CB1R signaling specifically during adolescent maturation might confer liability to psychiatric disorders.

  19. Collagen Type I as a Ligand for Receptor-Mediated Signaling

    Directory of Open Access Journals (Sweden)

    Iris Boraschi-Diaz

    2017-05-01

    Full Text Available Collagens form the fibrous component of the extracellular matrix in all multi-cellular animals. Collagen type I is the most abundant collagen present in skin, tendons, vasculature, as well as the organic portion of the calcified tissue of bone and teeth. This review focuses on numerous receptors for which collagen acts as a ligand, including integrins, discoidin domain receptors DDR1 and 2, OSCAR, GPVI, G6b-B, and LAIR-1 of the leukocyte receptor complex (LRC and mannose family receptor uPARAP/Endo180. We explore the process of collagen production and self-assembly, as well as its degradation by collagenases and gelatinases in order to predict potential temporal and spatial sites of action of different collagen receptors. While the interactions of the mature collagen matrix with integrins and DDR are well-appreciated, potential signals from immature matrix as well as collagen degradation products are possible but not yet described. The role of multiple collagen receptors in physiological processes and their contribution to pathophysiology of diseases affecting collagen homeostasis require further studies.

  20. Hindbrain ghrelin receptor signaling is sufficient to maintain fasting glucose.

    Directory of Open Access Journals (Sweden)

    Michael M Scott

    Full Text Available The neuronal coordination of metabolic homeostasis requires the integration of hormonal signals with multiple interrelated central neuronal circuits to produce appropriate levels of food intake, energy expenditure and fuel availability. Ghrelin, a peripherally produced peptide hormone, circulates at high concentrations during nutrient scarcity. Ghrelin promotes food intake, an action lost in ghrelin receptor null mice and also helps maintain fasting blood glucose levels, ensuring an adequate supply of nutrients to the central nervous system. To better understand mechanisms of ghrelin action, we have examined the roles of ghrelin receptor (GHSR expression in the mouse hindbrain. Notably, selective hindbrain ghrelin receptor expression was not sufficient to restore ghrelin-stimulated food intake. In contrast, the lowered fasting blood glucose levels observed in ghrelin receptor-deficient mice were returned to wild-type levels by selective re-expression of the ghrelin receptor in the hindbrain. Our results demonstrate the distributed nature of the neurons mediating ghrelin action.

  1. Anxiolytic-Like Effects of Increased Ghrelin Receptor Signaling in the Amygdala

    DEFF Research Database (Denmark)

    Jensen, Morten; Ratner, Cecilia; Rudenko, Olga

    2016-01-01

    BACKGROUND: Besides the well-known effects of ghrelin on adiposity and food intake regulation, the ghrelin system has been shown to regulate aspects of behavior including anxiety and stress. However, the effect of virus-mediated overexpression of the ghrelin receptor in the amygdala has...... not previously been addressed directly. METHOD: First, we examined the acute effect of peripheral ghrelin administration on anxiety- and depression-like behavior using the open field, elevated plus maze, forced swim and tail suspension tests. Next, we examined the effect of peripheral ghrelin administration...... and ghrelin receptor deficiency on stress in a familiar and social environment using the Intellicage system. Importantly, we also used a novel approach to study ghrelin receptor signaling in the brain by overexpressing the ghrelin receptor in the amygdala. We examined the effect of ghrelin receptor...

  2. The role of Ryk and Ror receptor tyrosine kinases in Wnt signal transduction

    NARCIS (Netherlands)

    Green, J.; Nusse, R.; van Amerongen, R.

    2014-01-01

    Receptor tyrosine kinases of the Ryk and Ror families were initially classified as orphan receptors because their ligands were unknown. They are now known to contain functional extracellular Wnt-binding domains and are implicated in Wnt-signal transduction in multiple species. Although their

  3. Receptor Activity-modifying Protein-directed G Protein Signaling Specificity for the Calcitonin Gene-related Peptide Family of Receptors.

    Science.gov (United States)

    Weston, Cathryn; Winfield, Ian; Harris, Matthew; Hodgson, Rose; Shah, Archna; Dowell, Simon J; Mobarec, Juan Carlos; Woodlock, David A; Reynolds, Christopher A; Poyner, David R; Watkins, Harriet A; Ladds, Graham

    2016-10-14

    The calcitonin gene-related peptide (CGRP) family of G protein-coupled receptors (GPCRs) is formed through the association of the calcitonin receptor-like receptor (CLR) and one of three receptor activity-modifying proteins (RAMPs). Binding of one of the three peptide ligands, CGRP, adrenomedullin (AM), and intermedin/adrenomedullin 2 (AM2), is well known to result in a Gα s -mediated increase in cAMP. Here we used modified yeast strains that couple receptor activation to cell growth, via chimeric yeast/Gα subunits, and HEK-293 cells to characterize the effect of different RAMP and ligand combinations on this pathway. We not only demonstrate functional couplings to both Gα s and Gα q but also identify a Gα i component to CLR signaling in both yeast and HEK-293 cells, which is absent in HEK-293S cells. We show that the CGRP family of receptors displays both ligand- and RAMP-dependent signaling bias among the Gα s , Gα i , and Gα q/11 pathways. The results are discussed in the context of RAMP interactions probed through molecular modeling and molecular dynamics simulations of the RAMP-GPCR-G protein complexes. This study further highlights the importance of RAMPs to CLR pharmacology and to bias in general, as well as identifying the importance of choosing an appropriate model system for the study of GPCR pharmacology. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  4. Insulin signaling inhibits the 5-HT2C receptor in choroid plexus via MAP kinase

    Directory of Open Access Journals (Sweden)

    Guan Kunliang

    2003-06-01

    Full Text Available Abstract Background G protein-coupled receptors (GPCRs interact with heterotrimeric GTP-binding proteins (G proteins to modulate acute changes in intracellular messenger levels and ion channel activity. In contrast, long-term changes in cellular growth, proliferation and differentiation are often mediated by tyrosine kinase receptors and certain GPCRs by activation of mitogen-activated protein (MAP kinases. Complex interactions occur between these signaling pathways, but the specific mechanisms of such regulatory events are not well-understood. In particular it is not clear whether GPCRs are modulated by tyrosine kinase receptor-MAP kinase pathways. Results Here we describe tyrosine kinase receptor regulation of a GPCR via MAP kinase. Insulin reduced the activity of the 5-HT2C receptor in choroid plexus cells which was blocked by the MAP kinase kinase (MEK inhibitor, PD 098059. We demonstrate that the inhibitory effect of insulin and insulin-like growth factor type 1 (IGF-1 on the 5-HT2C receptor is dependent on tyrosine kinase, RAS and MAP kinase. The effect may be receptor-specific: insulin had no effect on another GPCR that shares the same G protein signaling pathway as the 5-HT2C receptor. This effect is also direct: activated MAP kinase mimicked the effect of insulin, and removing a putative MAP kinase site from the 5-HT2C receptor abolished the effect of insulin. Conclusion These results show that insulin signaling can inhibit 5-HT2C receptor activity and suggest that MAP kinase may play a direct role in regulating the function of a specific GPCR.

  5. Assembly of Oligomeric Death Domain Complexes during Toll Receptor Signaling*

    Science.gov (United States)

    Moncrieffe, Martin C.; Grossmann, J. Günter; Gay, Nicholas J.

    2008-01-01

    The Drosophila Toll receptor is activated by the endogenous protein ligand Spätzle in response to microbial stimuli in immunity and spatial cues during embryonic development. Downstream signaling is mediated by the adaptor proteins Tube, the kinase Pelle, and the Drosophila homologue of myeloid differentiation primary response protein (dMyD88). Here we have characterized heterodimeric (dMyD88-Tube) and heterotrimeric (dMyD88-Tube-Pelle) death domain complexes. We show that both the heterodimeric and heterotrimeric complexes form kidney-shaped structures and that Tube is bivalent and has separate high affinity binding sites for dMyD88 and Pelle. Additionally we found no interaction between the isolated death domains of Pelle and dMyD88. These results indicate that the mode of assembly of the heterotrimeric dMyD88-Tube-Pelle complex downstream of the activated Toll receptor is unique. The measured dissociation constants for the interaction between the death domains of dMyD88 and Tube and of Pelle and a preformed dMyD88-Tube complex are used to propose a model of the early postreceptor events in Drosophila Toll receptor signaling. PMID:18829464

  6. Assembly of oligomeric death domain complexes during Toll receptor signaling.

    Science.gov (United States)

    Moncrieffe, Martin C; Grossmann, J Günter; Gay, Nicholas J

    2008-11-28

    The Drosophila Toll receptor is activated by the endogenous protein ligand Spätzle in response to microbial stimuli in immunity and spatial cues during embryonic development. Downstream signaling is mediated by the adaptor proteins Tube, the kinase Pelle, and the Drosophila homologue of myeloid differentiation primary response protein (dMyD88). Here we have characterized heterodimeric (dMyD88-Tube) and heterotrimeric (dMyD88-Tube-Pelle) death domain complexes. We show that both the heterodimeric and heterotrimeric complexes form kidney-shaped structures and that Tube is bivalent and has separate high affinity binding sites for dMyD88 and Pelle. Additionally we found no interaction between the isolated death domains of Pelle and dMyD88. These results indicate that the mode of assembly of the heterotrimeric dMyD88-Tube-Pelle complex downstream of the activated Toll receptor is unique. The measured dissociation constants for the interaction between the death domains of dMyD88 and Tube and of Pelle and a preformed dMyD88-Tube complex are used to propose a model of the early postreceptor events in Drosophila Toll receptor signaling.

  7. Purinergic receptors have different effects in rat exocrine pancreas. Calcium signals monitored by fura-2 using confocal microscopy

    DEFF Research Database (Denmark)

    Novak, Ivana; Nitschke, Roland; Amstrup, Jan

    2002-01-01

    Pancreatic ducts have several types of purinergic P2 receptors, however, nothing is known about P2 receptors in acini. The aim was to establish whether acini express functional P2 receptors coupled to intracellular Ca2+ signals and to measure the signals ratiometrically in a confocal laser scanning...

  8. PSM/SH2-B distributes selected mitogenic receptor signals to distinct components in the PI3-kinase and MAP kinase signaling pathways.

    Science.gov (United States)

    Deng, Youping; Xu, Hu; Riedel, Heimo

    2007-02-15

    The Pro-rich, PH, and SH2 domain containing mitogenic signaling adapter PSM/SH2-B has been implicated as a cellular partner of various mitogenic receptor tyrosine kinases and related signaling mechanisms. Here, we report in a direct comparison of three peptide hormones, that PSM participates in the assembly of distinct mitogenic signaling complexes in response to insulin or IGF-I when compared to PDGF in cultured normal fibroblasts. The complex formed in response to insulin or IGF-I involves the respective peptide hormone receptor and presumably the established components leading to MAP kinase activation. However, our data suggest an alternative link from the PDGF receptor via PSM directly to MEK1/2 and consequently also to p44/42 activation, possibly through a scaffold protein. At least two PSM domains participate, the SH2 domain anticipated to link PSM to the respective receptor and the Pro-rich region in an association with an unidentified downstream component resulting in direct MEK1/2 and p44/42 regulation. The PDGF receptor signaling complex formed in response to PDGF involves PI 3-kinase in addition to the same components and interactions as described for insulin or IGF-I. PSM associates with PI 3-kinase via p85 and in addition the PSM PH domain participates in the regulation of PI 3-kinase activity, presumably through membrane interaction. In contrast, the PSM Pro-rich region appears to participate only in the MAP kinase signal. Both pathways contribute to the mitogenic response as shown by cell proliferation, survival, and focus formation. PSM regulates p38 MAP kinase activity in a pathway unrelated to the mitogenic response.

  9. [Development of Ph negative acute myeloid leukemia in a patient with minor-BCR/ABL positive chronic myeloid leukemia achieving a partial cytogenetic response during tyrosine kinase inhibitor treatment].

    Science.gov (United States)

    Fujii, Soichiro; Miura, Ikuo; Tanaka, Hideo

    2015-06-01

    A 78-year-old male, who had CKD and chronic heart failure, was referred to our hospital for evaluation of leukocytosis. His bone marrow contained 12% blast cells and chromosome analysis showed the Ph chromosome as well as other changes. The patient was diagnosed with the accelerated-phase CML because FISH and RT-PCR disclosed BCR/ABL fusion signals and minor BCR/ABL, respectively. Imatinib was administered, but the CML was resistant to this treatment. We gave him nilotinib employing a reduced and intermittent administration protocol because of the progression of anemia and heart failure. The patient achieved PCyR in 8 months, but, 12 months later, his WBC count increased and 83% of the cells were blasts. Because the probable diagnosis was the blast crisis of CML, we switched from nilotinib to dasatinib. However, leukocytosis worsened and he died of pneumonia. It was later revealed that he had a normal karyotype and both FISH and RT-PCR analysis of BCR/ABL were negative. His final diagnosis was Ph negative AML developing from Ph positive CML in PCyR. Since there were no dysplastic changes indicative of MDS, it was assumed that the AML was not secondary leukemia caused by the tyrosine kinase inhibitor but, rather, de novo AML.

  10. PKCζ regulates Notch receptor routing and activity in a Notch signaling-dependent manner

    NARCIS (Netherlands)

    Sjöqvist, M.; Antfolk, D.; Ferraris, S.; Rraklli, V.; Haga, C.; Antila, C.; Mutvei, A.; Imanishi, S.Y.; Holmberg, J.; Jin, S.; Eriksson, J.E.; Lendahl, U.; Sahlgren, C.M.

    Activation of Notch signaling requires intracellular routing of the receptor, but the mechanisms controlling the distinct steps in the routing process is poorly understood. We identify PKCζ as a key regulator of Notch receptor intracellular routing. When PKCζ was inhibited in the developing chick

  11. Nuclear receptor TLX inhibits TGF-β signaling in glioblastoma.

    Science.gov (United States)

    Johansson, Erik; Zhai, Qiwei; Zeng, Zhao-Jun; Yoshida, Takeshi; Funa, Keiko

    2016-05-01

    TLX (also called NR2E1) is an orphan nuclear receptor that maintains stemness of neuronal stem cells. TLX is highly expressed in the most malignant form of glioma, glioblastoma multiforme (GBM), and is important for the proliferation and maintenance of the stem/progenitor cells of the tumor. Transforming Growth Factor-β (TGF-β) is a cytokine regulating many different cellular processes such as differentiation, migration, adhesion, cell death and proliferation. TGF-β has an important function in cancer where it can work as either a tumor suppressor or oncogene, depending on the cancer type and stage of tumor development. Since glioblastoma often have dysfunctional TGF-β signaling we wanted to find out if there is any interaction between TLX and TGF-β in glioblastoma cells. We demonstrate that knockdown of TLX enhances the canonical TGF-β signaling response in glioblastoma cell lines. TLX physically interacts with and stabilizes Smurf1, which can ubiquitinate and target TGF-β receptor II for degradation, whereas knockdown of TLX leads to stabilization of TGF-β receptor II, increased nuclear translocation of Smad2/3 and enhanced expression of TGF-β target genes. The interaction between TLX and TGF-β may play an important role in the regulation of proliferation and tumor-initiating properties of glioblastoma cells. Copyright © 2016. Published by Elsevier Inc.

  12. Calcium-sensing receptor (CaSR): pharmacological properties and signaling pathways.

    Science.gov (United States)

    Conigrave, Arthur D; Ward, Donald T

    2013-06-01

    In this article we consider the mechanisms by which the calcium-sensing receptor (CaSR) induces its cellular responses via the control (activation or inhibition) of signaling pathways. We consider key features of CaSR-mediated signaling including its control of the heterotrimeric G-proteins Gq/11, Gi/o and G12/13 and the downstream consequences recognizing that very few CaSR-mediated cell phenomena have been fully described. We also consider the manner in which the CaSR contributes to the formation of specific signaling scaffolds via peptide recognition sequences in its intracellular C-terminal along with the origins of its high level of cooperativity, particularly for Ca(2+)o, and its remarkable resistance to desensitization. We also consider the nature of the mechanisms by which the CaSR controls oscillatory and sustained Ca(2+)i mobilizing responses and inhibits or elevates cyclic adenosine monophosphate (cAMP) levels dependent on the cellular and signaling context. Finally, we consider the diversity of the receptor's ligands, ligand binding sites and broader compartment-dependent physiological roles leading to the identification of pronounced ligand-biased signaling for agonists including Sr(2+) and modulators including l-amino acids and the clinically effective calcimimetic cinacalcet. We note the implications of these findings for the development of new designer drugs that might target the CaSR in pathophysiological contexts beyond those established for the treatment of disorders of calcium metabolism. Copyright © 2013 Elsevier Ltd. All rights reserved.

  13. B cell receptor inhibition as a target for CLL therapy.

    Science.gov (United States)

    Jeyakumar, Deepa; O'Brien, Susan

    2016-03-01

    Inhibitors of the B cell receptor (BCR) represent an attractive therapeutic option for patients with chronic lymphocytic leukemia. Recently approved inhibitors of Bruton's tyrosine kinase (ibrutinib) and phosphatidylinositol 3-kinase (idelalisib), are promising agents because they are generally well tolerated and highly effective. These agents may be particularly important in the treatment of older patients who are less able to tolerate the myelosuppression (and infections) associated with chemoimmunotherapy. As a class of medications, BCR inhibitors have some unique side effects including redistribution lymphocytosis. Ibrutinib has specific toxicities including increased risk for bleeding and atrial fibrillation. Idelalisib also has some unique toxicities consisting of transaminitis, diarrhea and pneumonitis. Ongoing clinical trials are evaluating these agents in combination with antibodies, chemotherapy and other small molecules. Copyright © 2016 Elsevier Ltd. All rights reserved.

  14. New twist on neuronal insulin receptor signaling in health, disease, and therapeutics.

    Science.gov (United States)

    Wada, Akihiko; Yokoo, Hiroki; Yanagita, Toshihiko; Kobayashi, Hideyuki

    2005-10-01

    Long after the pioneering studies documenting the existence of insulin (year 1967) and insulin receptor (year 1978) in brain, the last decade has witnessed extraordinary progress in the understanding of brain region-specific multiple roles of insulin receptor signalings in health and disease. In the hypothalamus, insulin regulates food intake, body weight, peripheral fat deposition, hepatic gluconeogenesis, reproductive endocrine axis, and compensatory secretion of counter-regulatory hormones to hypoglycemia. In the hippocampus, insulin promotes learning and memory, independent of the glucoregulatory effect of insulin. Defective insulin receptor signalings are associated with the dementia in normal aging and patients with age-related neurodegenerative diseases (e.g., Alzheimer's disease); the cognitive impairment can be reversed with systemic administration of insulin in the euglycemic condition. Intranasal administration of insulin enhances memory and mood and decreases body weight in healthy humans, without causing hypoglycemia. In the hypothalamus, insulin-induced activation of the phosphoinositide 3-kinase pathway followed by opening of ATP-sensitive K+ channel has been shown to be related to multiple effects of insulin. However, the precise molecular mechanisms of insulin's pleiotropic effects still remain obscure. More importantly, much remains unknown about the quality control mechanisms ensuring correct conformational maturation of the insulin receptor, and the cellular mechanisms regulating density of cell surface functional insulin receptors.

  15. Recruitment of SHP-1 protein tyrosine phosphatase and signalling by a chimeric T-cell receptor-killer inhibitory receptor

    DEFF Research Database (Denmark)

    Christensen, M D; Geisler, C

    2000-01-01

    Receptors expressing the immunoreceptor tyrosine-based inhibitory motif (ITIM) in their cytoplasmic tail play an important role in the negative regulation of natural killer and B-cell activation. A subpopulation of T cells expresses the ITIM containing killer cell inhibitory receptor (KIR), which...... recognize MHC class I molecules. Following coligation of KIR with an activating receptor, the tyrosine in the ITIM is phosphorylated and the cytoplasmic protein tyrosine phosphatase SHP-1 is recruited to the ITIM via its SH2 domains. It is still not clear how SHP-1 affects T-cell receptor (TCR) signalling...... regarding total protein tyrosine phosphorylation, TCR down-regulation, mobilization of intracellular free calcium, or induction of the activation markers CD69 and CD25....

  16. Interaction of the B cell-specific transcriptional coactivator OCA-B and galectin-1 and a possible role in regulating BCR-mediated B cell proliferation.

    Science.gov (United States)

    Yu, Xin; Siegel, Rachael; Roeder, Robert G

    2006-06-02

    OCA-B is a B cell-specific transcriptional coactivator for OCT factors during the activation of immunoglobulin genes. In addition, OCA-B is crucial for B cell activation and germinal center formation. However, the molecular mechanisms for OCA-B function in these processes are not clear. Our previous studies documented two OCA-B isoforms and suggested a novel mechanism for the function of the myristoylated, membrane-bound form of OCA-B/p35 as a signaling molecule. Here, we report the identification of galectin-1, and related galectins, as a novel OCA-B-interacting protein. The interaction of OCA-B and galectin-1 can be detected both in vivo and in vitro. The galectin-1 binding domain in OCA-B has been localized to the N terminus of OCA-B. In B cells lacking OCA-B expression, increased galectin-1 expression, secretion, and cell surface association are observed. Consistent with these observations, and a reported inhibitory interaction of galectin-1 with CD45, the phosphatase activity of CD45 is reduced modestly, but significantly, in OCA-B-deficient B cells. Finally, galectin-1 is shown to negatively regulate B cell proliferation and tyrosine phosphorylation upon BCR stimulation. Together, these results raise the possibility that OCA-B may regulate BCR signaling through an association with galectin-1.

  17. Crammed signaling motifs in the T-cell receptor.

    Science.gov (United States)

    Borroto, Aldo; Abia, David; Alarcón, Balbino

    2014-09-01

    Although the T cell antigen receptor (TCR) is long known to contain multiple signaling subunits (CD3γ, CD3δ, CD3ɛ and CD3ζ), their role in signal transduction is still not well understood. The presence of at least one immunoreceptor tyrosine-based activation motif (ITAM) in each CD3 subunit has led to the idea that the multiplication of such elements essentially serves to amplify signals. However, the evolutionary conservation of non-ITAM sequences suggests that each CD3 subunit is likely to have specific non-redundant roles at some stage of development or in mature T cell function. The CD3ɛ subunit is paradigmatic because in a relatively short cytoplasmic sequence (∼55 amino acids) it contains several docking sites for proteins involved in intracellular trafficking and signaling, proteins whose relevance in T cell activation is slowly starting to be revealed. In this review we will summarize our current knowledge on the signaling effectors that bind directly to the TCR and we will propose a hierarchy in their response to TCR triggering. Copyright © 2014 Elsevier B.V. All rights reserved.

  18. Phosphorylation site dynamics of early T-cell receptor signaling

    DEFF Research Database (Denmark)

    Chylek, Lily A; Akimov, Vyacheslav; Dengjel, Jörn

    2014-01-01

    In adaptive immune responses, T-cell receptor (TCR) signaling impacts multiple cellular processes and results in T-cell differentiation, proliferation, and cytokine production. Although individual protein-protein interactions and phosphorylation events have been studied extensively, we lack...... that diverse dynamic patterns emerge within seconds. We detected phosphorylation dynamics as early as 5 s and observed widespread regulation of key TCR signaling proteins by 30 s. Development of a computational model pointed to the presence of novel regulatory mechanisms controlling phosphorylation of sites...... a systems-level understanding of how these components cooperate to control signaling dynamics, especially during the crucial first seconds of stimulation. Here, we used quantitative proteomics to characterize reshaping of the T-cell phosphoproteome in response to TCR/CD28 co-stimulation, and found...

  19. Metazoan-like signaling in a unicellular receptor tyrosine kinase

    Directory of Open Access Journals (Sweden)

    Schultheiss Kira P

    2013-02-01

    Full Text Available Abstract Background Receptor tyrosine kinases (RTKs are crucial components of signal transduction systems in multicellular animals. Surprisingly, numerous RTKs have been identified in the genomes of unicellular choanoflagellates and other protists. Here, we report the first biochemical study of a unicellular RTK, namely RTKB2 from Monosiga brevicollis. Results We cloned, expressed, and purified the RTKB2 kinase, and showed that it is enzymatically active. The activity of RTKB2 is controlled by autophosphorylation, as in metazoan RTKs. RTKB2 possesses six copies of a unique domain (designated RM2 in its C-terminal tail. An isolated RM2 domain (or a synthetic peptide derived from the RM2 sequence served as a substrate for RTKB2 kinase. When phosphorylated, the RM2 domain bound to the Src homology 2 domain of MbSrc1 from M. brevicollis. NMR structural studies of the RM2 domain indicated that it is disordered in solution. Conclusions Our results are consistent with a model in which RTKB2 activation stimulates receptor autophosphorylation within the RM2 domains. This leads to recruitment of Src-like kinases (and potentially other M. brevicollis proteins and further phosphorylation, which may serve to increase or dampen downstream signals. Thus, crucial features of signal transduction circuitry were established prior to the evolution of metazoans from their unicellular ancestors.

  20. Two signaling molecules share a phosphotyrosine-containing binding site in the platelet-derived growth factor receptor.

    OpenAIRE

    Nishimura, R; Li, W; Kashishian, A; Mondino, A; Zhou, M; Cooper, J; Schlessinger, J

    1993-01-01

    Autophosphorylation sites of growth factor receptors with tyrosine kinase activity function as specific binding sites for Src homology 2 (SH2) domains of signaling molecules. This interaction appears to be a crucial step in a mechanism by which receptor tyrosine kinases relay signals to downstream signaling pathways. Nck is a widely expressed protein consisting exclusively of SH2 and SH3 domains, the overexpression of which causes cell transformation. It has been shown that various growth fac...

  1. Normalization of TAM post-receptor signaling reveals a cell invasive signature for Axl tyrosine kinase.

    Science.gov (United States)

    Kimani, Stanley G; Kumar, Sushil; Davra, Viralkumar; Chang, Yun-Juan; Kasikara, Canan; Geng, Ke; Tsou, Wen-I; Wang, Shenyan; Hoque, Mainul; Boháč, Andrej; Lewis-Antes, Anita; De Lorenzo, Mariana S; Kotenko, Sergei V; Birge, Raymond B

    2016-09-06

    Tyro3, Axl, and Mertk (TAMs) are a family of three conserved receptor tyrosine kinases that have pleiotropic roles in innate immunity and homeostasis and when overexpressed in cancer cells can drive tumorigenesis. In the present study, we engineered EGFR/TAM chimeric receptors (EGFR/Tyro3, EGFR/Axl, and EGF/Mertk) with the goals to interrogate post-receptor functions of TAMs, and query whether TAMs have unique or overlapping post-receptor activation profiles. Stable expression of EGFR/TAMs in EGFR-deficient CHO cells afforded robust EGF inducible TAM receptor phosphorylation and activation of downstream signaling. Using a series of unbiased screening approaches, that include kinome-view analysis, phosphor-arrays, RNAseq/GSEA analysis, as well as cell biological and in vivo readouts, we provide evidence that each TAM has unique post-receptor signaling platforms and identify an intrinsic role for Axl that impinges on cell motility and invasion compared to Tyro3 and Mertk. These studies demonstrate that TAM show unique post-receptor signatures that impinge on distinct gene expression profiles and tumorigenic outcomes.

  2. Intercellular calcium signaling occurs between human osteoblasts and osteoclasts and requires activation of osteoclast P2X7 receptors

    DEFF Research Database (Denmark)

    Jørgensen, Niklas R; Henriksen, Zanne; Sørensen, Ole

    2002-01-01

    that human osteoclasts expressed functional P2Y1 receptors, but, unexpectedly, desensitization of P2Y1 did not block calcium signaling to osteoclasts. We also found that osteoclasts expressed functional P2X7 receptors and showed that pharmacological inhibition of these receptors blocked calcium signaling...

  3. E3 Ubiquitin Ligase RNF125 Activates Interleukin-36 Receptor Signaling and Contributes to Its Turnover.

    Science.gov (United States)

    Saha, Siddhartha S; Caviness, Gary; Yi, Guanghui; Raymond, Ernest L; Mbow, M Lamine; Kao, C Cheng

    2018-01-01

    Signaling by the interleukin-36 receptor (IL-36R) is linked to inflammatory diseases such as psoriasis. However, the regulation of IL-36R signaling is poorly understood. Activation of IL-36R signaling in cultured cells results in an increased polyubiquitination of the receptor subunit, IL-1Rrp2. Treatment with deubiquitinases shows that the receptor subunit of IL-36R, IL-1Rrp2, is primarily polyubiquitinated at the K63 position, which is associated with endocytic trafficking and signal transduction. A minor amount of ubiquitination is at the K48 position that is associated with protein degradation. A focused siRNA screen identified RNF125, an E3 ubiquitin ligase, to ubiquitinate IL-1Rrp2 upon activation of IL-36R signaling while not affecting the activated IL-1 receptor. Knockdown of RNF125 decreases signal transduction by the IL-36R. Overexpression of RNF125 in HEK293T cells activates IL-36R signaling and increases the ubiquitination of IL-1Rrp2 and its subsequent turnover. RNF125 can coimmunoprecipitate with the IL-36R, and it traffics with IL-1Rrp2 from the cell surface to lysosomes. Mutations of Lys568 and Lys569 in the C-terminal tail of IL-1Rrp2 decrease ubiquitination by RNF125 and increase the steady-state levels of IL-1Rrp2. These results demonstrate that RNF125 has multiple regulatory roles in the signaling, trafficking, and turnover of the IL-36R. © 2017 S. Karger AG, Basel.

  4. Effect of spatial inhomogeneities on the membrane surface on receptor dimerization and signal initiation

    Directory of Open Access Journals (Sweden)

    Romica Kerketta

    2016-08-01

    Full Text Available Important signal transduction pathways originate on the plasma membrane, where microdomains may transiently entrap diffusing receptors. This results in a non-random distribution of receptors even in the resting state, which can be visualized as clusters by high resolution imaging methods. Here, we explore how spatial in-homogeneities in the plasma membrane might influence the dimerization and phosphorylation status of ErbB2 and ErbB3, two receptor tyrosine kinases that preferentially heterodimerize and are often co-expressed in cancer. This theoretical study is based upon spatial stochastic simulations of the two-dimensional membrane landscape, where variables include differential distributions and overlap of transient confinement zones (domains for the two receptor species. The in silico model is parameterized and validated using data from single particle tracking experiments. We report key differences in signaling output based on the degree of overlap between domains and the relative retention of receptors in such domains, expressed as escape probability. Results predict that a high overlap of domains, which favors transient co-confinement of both receptor species, will enhance the rate of hetero-interactions. Where domains do not overlap, simulations confirm expectations that homo-interactions are favored. Since ErbB3 is uniquely dependent on ErbB2 interactions for activation of its catalytic activity, variations in domain overlap or escape probability markedly alter the predicted patterns and time course of ErbB3 and ErbB2 phosphorylation. Taken together, these results implicate membrane domain organization as an important modulator of signal initiation, motivating the design of novel experimental approaches to measure these important parameters across a wider range of receptor systems.

  5. Association of HLA Class I and Class II genes with bcr-abl transcripts in leukemia patients with t(9;22) (q34;q11)

    International Nuclear Information System (INIS)

    Mundhada, Shailendra; Luthra, Rajyalakshmi; Cano, Pedro

    2004-01-01

    Based on the site of breakpoint in t(9;22) (q34;q11), bcr-abl fusion in leukemia patients is associated with different types of transcript proteins. In this study we have seen the association of HLA genes with different types of bcr-abl transcripts. The association could predict the bcr-abl peptide presentation by particular HLA molecules. The study included a total of 189 patients of mixed ethnicity with chronic myelogenous leukemia and acute lymphocytic leukemia who were being considered for bone marrow transplantation. Typing of bcr-abl transcripts was done by reverse transcriptase PCR method. HLA typing was performed by molecular methods. The bcr-abl and HLA association was studied by calculating the relative risks and chi-square test. Significant negative associations (p < 0.05) were observed with HLA-A*02 (b2a2, e1a2), -A*68 (b2a2, b3a2, e1a2), -B*14 (b2a2, b3a2, e1a2), -B*15 (b2a2, b3a2), -B*40 (b2a2), -DQB1*0303 (b2a2, b3a2), -DQB1*0603 (b2a2), -DRB1*0401 (e1a2), -DRB1*0701 (b3a2), and -DRB1*1101 (b2a2). The negative associations of a particular bcr-abl transcript with specific HLA alleles suggests that these alleles play a critical role in presenting peptides derived from the chimeric proteins and eliciting a successful T-cell cytotoxic response. Knowledge of differential associations between HLA phenotypes and bcr-abl fusion transcript types would help in developing better strategies for immunization with the bcr-abl peptides against t(9;22) (q34;q11)-positive leukemia

  6. Flow Cytometric Immunobead Assay for Detection of BCR-ABL1 Fusion Proteins in Chronic Myleoid Leukemia: Comparison with FISH and PCR Techniques

    Science.gov (United States)

    Recchia, Anna Grazia; Caruso, Nadia; Bossio, Sabrina; Pellicanò, Mariavaleria; De Stefano, Laura; Franzese, Stefania; Palummo, Angela; Abbadessa, Vincenzo; Lucia, Eugenio; Gentile, Massimo; Vigna, Ernesto; Caracciolo, Clementina; Agostino, Antolino; Galimberti, Sara; Levato, Luciano; Stagno, Fabio; Molica, Stefano; Martino, Bruno; Vigneri, Paolo; Di Raimondo, Francesco; Morabito, Fortunato

    2015-01-01

    Chronic Myeloid Leukemia (CML) is characterized by a balanced translocation juxtaposing the Abelson (ABL) and breakpoint cluster region (BCR) genes. The resulting BCR-ABL1 oncogene leads to increased proliferation and survival of leukemic cells. Successful treatment of CML has been accompanied by steady improvements in our capacity to accurately and sensitively monitor therapy response. Currently, measurement of BCR-ABL1 mRNA transcript levels by real-time quantitative PCR (RQ-PCR) defines critical response endpoints. An antibody-based technique for BCR-ABL1 protein recognition could be an attractive alternative to RQ-PCR. To date, there have been no studies evaluating whether flow-cytometry based assays could be of clinical utility in evaluating residual disease in CML patients. Here we describe a flow-cytometry assay that detects the presence of BCR-ABL1 fusion proteins in CML lysates to determine the applicability, reliability, and specificity of this method for both diagnosis and monitoring of CML patients for initial response to therapy. We show that: i) CML can be properly diagnosed at onset, (ii) follow-up assessments show detectable fusion protein (i.e. relative mean fluorescent intensity, rMFI%>1) when BCR-ABL1IS transcripts are between 1–10%, and (iii) rMFI% levels predict CCyR as defined by FISH analysis. Overall, the FCBA assay is a rapid technique, fully translatable to the routine management of CML patients. PMID:26111048

  7. Flow Cytometric Immunobead Assay for Detection of BCR-ABL1 Fusion Proteins in Chronic Myleoid Leukemia: Comparison with FISH and PCR Techniques.

    Directory of Open Access Journals (Sweden)

    Anna Grazia Recchia

    Full Text Available Chronic Myeloid Leukemia (CML is characterized by a balanced translocation juxtaposing the Abelson (ABL and breakpoint cluster region (BCR genes. The resulting BCR-ABL1 oncogene leads to increased proliferation and survival of leukemic cells. Successful treatment of CML has been accompanied by steady improvements in our capacity to accurately and sensitively monitor therapy response. Currently, measurement of BCR-ABL1 mRNA transcript levels by real-time quantitative PCR (RQ-PCR defines critical response endpoints. An antibody-based technique for BCR-ABL1 protein recognition could be an attractive alternative to RQ-PCR. To date, there have been no studies evaluating whether flow-cytometry based assays could be of clinical utility in evaluating residual disease in CML patients. Here we describe a flow-cytometry assay that detects the presence of BCR-ABL1 fusion proteins in CML lysates to determine the applicability, reliability, and specificity of this method for both diagnosis and monitoring of CML patients for initial response to therapy. We show that: i CML can be properly diagnosed at onset, (ii follow-up assessments show detectable fusion protein (i.e. relative mean fluorescent intensity, rMFI%>1 when BCR-ABL1IS transcripts are between 1-10%, and (iii rMFI% levels predict CCyR as defined by FISH analysis. Overall, the FCBA assay is a rapid technique, fully translatable to the routine management of CML patients.

  8. A Public Database of Memory and Naive B-Cell Receptor Sequences.

    Directory of Open Access Journals (Sweden)

    William S DeWitt

    Full Text Available The vast diversity of B-cell receptors (BCR and secreted antibodies enables the recognition of, and response to, a wide range of epitopes, but this diversity has also limited our understanding of humoral immunity. We present a public database of more than 37 million unique BCR sequences from three healthy adult donors that is many fold deeper than any existing resource, together with a set of online tools designed to facilitate the visualization and analysis of the annotated data. We estimate the clonal diversity of the naive and memory B-cell repertoires of healthy individuals, and provide a set of examples that illustrate the utility of the database, including several views of the basic properties of immunoglobulin heavy chain sequences, such as rearrangement length, subunit usage, and somatic hypermutation positions and dynamics.

  9. A Public Database of Memory and Naive B-Cell Receptor Sequences.

    Science.gov (United States)

    DeWitt, William S; Lindau, Paul; Snyder, Thomas M; Sherwood, Anna M; Vignali, Marissa; Carlson, Christopher S; Greenberg, Philip D; Duerkopp, Natalie; Emerson, Ryan O; Robins, Harlan S

    2016-01-01

    The vast diversity of B-cell receptors (BCR) and secreted antibodies enables the recognition of, and response to, a wide range of epitopes, but this diversity has also limited our understanding of humoral immunity. We present a public database of more than 37 million unique BCR sequences from three healthy adult donors that is many fold deeper than any existing resource, together with a set of online tools designed to facilitate the visualization and analysis of the annotated data. We estimate the clonal diversity of the naive and memory B-cell repertoires of healthy individuals, and provide a set of examples that illustrate the utility of the database, including several views of the basic properties of immunoglobulin heavy chain sequences, such as rearrangement length, subunit usage, and somatic hypermutation positions and dynamics.

  10. Functional requirements for inhibitory signal transmission by the immunomodulatory receptor CD300a.

    Science.gov (United States)

    DeBell, Karen E; Simhadri, Venkateswara R; Mariano, John L; Borrego, Francisco

    2012-04-26

    Activation signals can be negatively regulated by cell surface receptors bearing immunoreceptor tyrosine-based inhibitory motifs (ITIMs). CD300a, an ITIM bearing type I transmembrane protein, is expressed on many hematopoietic cells, including subsets of lymphocytes. We have taken two approaches to further define the mechanism by which CD300a acts as an inhibitor of immune cell receptor signaling. First, we have expressed in Jurkat T cells a chimeric receptor consisting of the extracellular domains of killer-cell immunoglobulin-like receptor (KIR)2DL2 fused to the transmembrane and cytoplasmic segments of CD300a (KIR-CD300a) to explore surrogate ligand-stimulated inhibition of superantigen stimulated T cell receptor (TCR) mediated cell signaling. We found that intact CD300a ITIMs were essential for inhibition and that the tyrosine phosphorylation of these ITIMs required the src tyrosine kinase Lck. Tyrosine phosphorylation of the CD300a ITIMs created docking sites for both src homology 2 domain containing protein tyrosine phosphatase (SHP)-1 and SHP-2. Suppression of SHP-1 and SHP-2 expression in KIR-CD300a Jurkat T cells with siRNA and the use of DT40 chicken B cell lines expressing CD300a and deficient in several phosphatases revealed that SHP-1, but not SHP-2 or the src homology 2 domain containing inositol 5' phosphatase SHIP, was utilized by CD300a for its inhibitory activity. These studies provide new insights into the function of CD300a in tuning T and B cell responses.

  11. Vitamin D receptor–retinoid X receptor heterodimer signaling regulates oligodendrocyte progenitor cell differentiation

    Science.gov (United States)

    de la Fuente, Alerie Guzman; Errea, Oihana; van Wijngaarden, Peter; Gonzalez, Ginez A.; Kerninon, Christophe; Jarjour, Andrew A.; Lewis, Hilary J.; Jones, Clare A.; Nait-Oumesmar, Brahim; Zhao, Chao; Huang, Jeffrey K.; ffrench-Constant, Charles

    2015-01-01

    The mechanisms regulating differentiation of oligodendrocyte (OLG) progenitor cells (OPCs) into mature OLGs are key to understanding myelination and remyelination. Signaling via the retinoid X receptor γ (RXR-γ) has been shown to be a positive regulator of OPC differentiation. However, the nuclear receptor (NR) binding partner of RXR-γ has not been established. In this study we show that RXR-γ binds to several NRs in OPCs and OLGs, one of which is vitamin D receptor (VDR). Using pharmacological and knockdown approaches we show that RXR–VDR signaling induces OPC differentiation and that VDR agonist vitamin D enhances OPC differentiation. We also show expression of VDR in OLG lineage cells in multiple sclerosis. Our data reveal a role for vitamin D in the regenerative component of demyelinating disease and identify a new target for remyelination medicines. PMID:26644513

  12. Bruton tyrosine kinase represents a promising therapeutic target for treatment of chronic lymphocytic leukemia and is effectively targeted by PCI-32765

    Science.gov (United States)

    Herman, Sarah E. M.; Gordon, Amber L.; Hertlein, Erin; Ramanunni, Asha; Zhang, Xiaoli; Jaglowski, Samantha; Flynn, Joseph; Jones, Jeffrey; Blum, Kristie A.; Buggy, Joseph J.; Hamdy, Ahmed

    2011-01-01

    B-cell receptor (BCR) signaling is aberrantly activated in chronic lymphocytic leukemia (CLL). Bruton tyrosine kinase (BTK) is essential to BCR signaling and in knockout mouse models its mutation has a relatively B cell–specific phenotype. Herein, we demonstrate that BTK protein and mRNA are significantly over expressed in CLL compared with normal B cells. Although BTK is not always constitutively active in CLL cells, BCR or CD40 signaling is accompanied by effective activation of this pathway. Using the irreversible BTK inhibitor PCI-32765, we demonstrate modest apoptosis in CLL cells that is greater than that observed in normal B cells. No influence of PCI-32765 on T-cell survival is observed. Treatment of CD40 or BCR activated CLL cells with PCI-32765 results in inhibition of BTK tyrosine phosphorylation and also effectively abrogates downstream survival pathways activated by this kinase including ERK1/2, PI3K, and NF-κB. In addition, PCI-32765 inhibits activation-induced proliferation of CLL cells in vitro, and effectively blocks survival signals provided externally to CLL cells from the microenvironment including soluble factors (CD40L, BAFF, IL-6, IL-4, and TNF-α), fibronectin engagement, and stromal cell contact. Based on these collective data, future efforts targeting BTK with the irreversible inhibitor PCI-32765 in clinical trials of CLL patients is warranted. PMID:21422473

  13. Bach2 regulates aberrant activation of B cell in systemic lupus erythematosus and can be negatively regulated by BCR-ABL/PI3K.

    Science.gov (United States)

    Zhu, Zhengwei; Yang, Chao; Wen, Leilei; Liu, Lu; Zuo, Xianbo; Zhou, Fusheng; Gao, Jinping; Zheng, Xiaodong; Shi, Yinjuan; Zhu, Caihong; Liang, Bo; Yin, Xianyong; Wang, Wenjun; Cheng, Hui; Shen, Songke; Tang, Xianfa; Tang, Huayang; Sun, Liangdan; Zhang, Anping; Yang, Sen; Cui, Yong; Zhang, Xuejun; Sheng, Yujun

    2018-04-01

    This study was aimed to explore the effect of Bach2 on B cells in systemic lupus erythematosus (SLE), as well as the underlying mechanisms. Expression of Bach2, phosphorylated-Bach2 (p-Bach2), Akt, p-Akt and BCR-ABL (p210) in B cells isolated from SLE patients and the healthy persons were assessed by Western blot. Immunofluorescence staining was performed to assess the localization of Bach2 in B cells. Enzyme-linked immunosorbent assay (ELISA) was employed to detect IgG produced by B cells. Cell counting kit-8 (CCK-8) and Annexin-V FITC/PI double staining assay were adopted to evaluate cell proliferation and apoptosis in B cells, respectively. Compared to the healthy controls, Bach2, p-Akt and p210 were significantly decreased, while nuclear translocation of Bach2, IgG, CD40 and CD86 obviously up-regulated in B cells from SLE patients. Bach2 significantly inhibited the proliferation, promoted apoptosis of B cells from SLE patients, whereas BCR-ABL dramatically reversed cell changes induced by Bach2. Besides, BCR-ABL also inhibited nuclear translocation of Bach2 in B cells from SLE patients. Further, LY294002 treatment had no effect on decreased expression of Bach2 induced by BCR-ABL, but significantly eliminated BCR-ABL-induced phosphorylation of Bach2 and restored reduced nuclear translocation of Bach2 induced by BCR-ABL in B cells from SLE. Bach2 may play a suppressive role in B cells from SLE, and BCR-ABL may inhibit the nuclear translocation of Bach2 via serine phosphorylation through the PI3K pathway. Copyright © 2018 Elsevier Inc. All rights reserved.

  14. Isolation and killing of candidate chronic myeloid leukemia stem cells by antibody targeting of IL-1 receptor accessory protein

    DEFF Research Database (Denmark)

    Järås, Marcus; Johnels, Petra; Hansen, Nils Gunder

    2010-01-01

    Chronic myeloid leukemia (CML) is genetically characterized by the Philadelphia (Ph) chromosome, formed through a reciprocal translocation between chromosomes 9 and 22 and giving rise to the constitutively active tyrosine kinase P210 BCR/ABL1. Therapeutic strategies aiming for a cure of CML...... will require full eradication of Ph chromosome-positive (Ph(+)) CML stem cells. Here we used gene-expression profiling to identify IL-1 receptor accessory protein (IL1RAP) as up-regulated in CML CD34(+) cells and also in cord blood CD34(+) cells as a consequence of retroviral BCR/ABL1 expression. To test...

  15. Agmatine produces antidepressant-like effects by activating AMPA receptors and mTOR signaling.

    Science.gov (United States)

    Neis, Vivian Binder; Moretti, Morgana; Bettio, Luis Eduardo B; Ribeiro, Camille M; Rosa, Priscila Batista; Gonçalves, Filipe Marques; Lopes, Mark William; Leal, Rodrigo Bainy; Rodrigues, Ana Lúcia S

    2016-06-01

    The activation of AMPA receptors and mTOR signaling has been reported as mechanisms underlying the antidepressant effects of fast-acting agents, specially the NMDA receptor antagonist ketamine. In the present study, oral administration of agmatine (0.1mg/kg), a neuromodulator that has been reported to modulate NMDA receptors, caused a significant reduction in the immobility time of mice submitted to the tail suspension test (TST), an effect prevented by the administration of DNQX (AMPA receptor antagonist, 2.5μg/site, i.c.v.), BDNF antibody (1μg/site, i.c.v.), K-252a (TrkB receptor antagonist, 1μg/site, i.c.v.), LY294002 (PI3K inhibitor, 10nmol/site, i.c.v.) or rapamycin (selective mTOR inhibitor, 0.2nmol/site, i.c.v.). Moreover, the administration of lithium chloride (non-selective GSK-3β inhibitor, 10mg/kg, p.o.) or AR-A014418 (selective GSK-3β inhibitor, 0.01μg/site, i.c.v.) in combination with a sub-effective dose of agmatine (0.0001mg/kg, p.o.) reduced the immobility time in the TST when compared with either drug alone. Furthermore, increased immunocontents of BDNF, PSD-95 and GluA1 were found in the prefrontal cortex of mice just 1h after agmatine administration. These results indicate that the antidepressant-like effect of agmatine in the TST may be dependent on the activation of AMPA and TrkB receptors, PI3K and mTOR signaling as well as inhibition of GSK-3β, and increase in synaptic proteins. The results contribute to elucidate the complex signaling pathways involved in the antidepressant effect of agmatine and reinforce the pivotal role of these molecular targets for antidepressant responses. Copyright © 2016 Elsevier B.V. and ECNP. All rights reserved.

  16. Function of Bruton's tyrosine kinase during B cell development is partially independent of its catalytic activity

    NARCIS (Netherlands)

    S. Middendorp; G.M. Dingjan (Gemma); A. Maas (Alex); K. Dahlenborg; R.W. Hendriks (Rudi)

    2003-01-01

    textabstractThe Tec family member Bruton's tyrosine kinase (Btk) is a cytoplasmic protein tyrosine kinase that transduces signals from the pre-B and B cell receptor (BCR). Btk is involved in pre-B cell maturation by regulating IL-7 responsiveness, cell surface phenotype changes,

  17. Inter-domain tagging implicates caveolin-1 in insulin receptor trafficking and Erk signaling bias in pancreatic beta-cells

    Directory of Open Access Journals (Sweden)

    Tobias Boothe

    2016-05-01

    Full Text Available Objective: The role and mechanisms of insulin receptor internalization remain incompletely understood. Previous trafficking studies of insulin receptors involved fluorescent protein tagging at their termini, manipulations that may be expected to result in dysfunctional receptors. Our objective was to determine the trafficking route and molecular mechanisms of functional tagged insulin receptors and endogenous insulin receptors in pancreatic beta-cells. Methods: We generated functional insulin receptors tagged with pH-resistant fluorescent proteins between domains. Confocal, TIRF and STED imaging revealed a trafficking pattern of inter-domain tagged insulin receptors and endogenous insulin receptors detected with antibodies. Results: Surprisingly, interdomain-tagged and endogenous insulin receptors in beta-cells bypassed classical Rab5a- or Rab7-mediated endocytic routes. Instead, we found that removal of insulin receptors from the plasma membrane involved tyrosine-phosphorylated caveolin-1, prior to trafficking within flotillin-1-positive structures to lysosomes. Multiple methods of inhibiting caveolin-1 significantly reduced Erk activation in vitro or in vivo, while leaving Akt signaling mostly intact. Conclusions: We conclude that phosphorylated caveolin-1 plays a role in insulin receptor internalization towards lysosomes through flotillin-1-positive structures and that caveolin-1 helps bias physiological beta-cell insulin signaling towards Erk activation. Author Video: Author Video Watch what authors say about their articles Keywords: Insulin receptor internalization, Insulin resistance, Pancreatic islet beta-cells, Autocrine insulin signaling

  18. Signaling by Kit protein-tyrosine kinase--the stem cell factor receptor.

    Science.gov (United States)

    Roskoski, Robert

    2005-11-11

    Signaling by stem cell factor and Kit, its receptor, plays important roles in gametogenesis, hematopoiesis, mast cell development and function, and melanogenesis. Moreover, human and mouse embryonic stem cells express Kit transcripts. Stem cell factor exists as both a soluble and a membrane-bound glycoprotein while Kit is a receptor protein-tyrosine kinase. The complete absence of stem cell factor or Kit is lethal. Deficiencies of either produce defects in red and white blood cell production, hypopigmentation, and sterility. Gain-of-function mutations of Kit are associated with several human neoplasms including acute myelogenous leukemia, gastrointestinal stromal tumors, and mastocytomas. Kit consists of an extracellular domain, a transmembrane segment, a juxtamembrane segment, and a protein kinase domain that contains an insert of about 80 amino acid residues. Binding of stem cell factor to Kit results in receptor dimerization and activation of protein kinase activity. The activated receptor becomes autophosphorylated at tyrosine residues that serve as docking sites for signal transduction molecules containing SH2 domains. The adaptor protein APS, Src family kinases, and Shp2 tyrosyl phosphatase bind to phosphotyrosine 568. Shp1 tyrosyl phosphatase and the adaptor protein Shc bind to phosphotyrosine 570. C-terminal Src kinase homologous kinase and the adaptor Shc bind to both phosphotyrosines 568 and 570. These residues occur in the juxtamembrane segment of Kit. Three residues in the kinase insert domain are phosphorylated and attract the adaptor protein Grb2 (Tyr703), phosphatidylinositol 3-kinase (Tyr721), and phospholipase Cgamma (Tyr730). Phosphotyrosine 900 in the distal kinase domain binds phosphatidylinositol 3-kinase which in turn binds the adaptor protein Crk. Phosphotyrosine 936, also in the distal kinase domain, binds the adaptor proteins APS, Grb2, and Grb7. Kit has the potential to participate in multiple signal transduction pathways as a result of

  19. Structural Mechanism of the Pan-BCR-ABL Inhibitor Ponatinib (AP24534): Lessons for Overcoming Kinase Inhibitor Resistance

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Tianjun; Commodore, Lois; Huang, Wei-Sheng; Wang, Yihan; Thomas, Mathew; Keats, Jeff; Xu, Qihong; Rivera, Victor M.; Shakespeare, William C.; Clackson, Tim; Dalgarno, David C.; Zhu, Xiaotian (ARIAD)

    2012-01-20

    The BCR-ABL inhibitor imatinib has revolutionized the treatment of chronic myeloid leukemia. However, drug resistance caused by kinase domain mutations has necessitated the development of new mutation-resistant inhibitors, most recently against the T315I gatekeeper residue mutation. Ponatinib (AP24534) inhibits both native and mutant BCR-ABL, including T315I, acting as a pan-BCR-ABL inhibitor. Here, we undertook a combined crystallographic and structure-activity relationship analysis on ponatinib to understand this unique profile. While the ethynyl linker is a key inhibitor functionality that interacts with the gatekeeper, virtually all other components of ponatinib play an essential role in its T315I inhibitory activity. The extensive network of optimized molecular contacts found in the DFG-out binding mode leads to high potency and renders binding less susceptible to disruption by single point mutations. The inhibitory mechanism exemplified by ponatinib may have broad relevance to designing inhibitors against other kinases with mutated gatekeeper residues.

  20. Endothelin B Receptors on Primary Chicken Müller Cells and the Human MIO-M1 Müller Cell Line Activate ERK Signaling via Transactivation of Epidermal Growth Factor Receptors.

    Directory of Open Access Journals (Sweden)

    Mohammad Harun-Or-Rashid

    Full Text Available Injury to the eye or retina triggers Müller cells, the major glia cell of the retina, to dedifferentiate and proliferate. In some species they attain retinal progenitor properties and have the capacity to generate new neurons. The epidermal growth factor receptor (EGFR system and extracellular signal-regulated kinase (ERK signaling are key regulators of these processes in Müller cells. The extracellular signals that modulate and control these processes are not fully understood. In this work we studied whether endothelin receptor signaling can activate EGFR and ERK signaling in Müller cells. Endothelin expression is robustly upregulated at retinal injury and endothelin receptors have been shown to transactivate EGFRs in other cell types. We analyzed the endothelin signaling system in chicken retina and cultured primary chicken Müller cells as well as the human Müller cell line MIO-M1. The Müller cells were stimulated with receptor agonists and treated with specific blockers to key enzymes in the signaling pathway or with siRNAs. We focused on endothelin receptor mediated transactivation of EGFRs by using western blot analysis, quantitative reverse transcriptase PCR and immunocytochemistry. The results showed that chicken Müller cells and the human Müller cell line MIO-M1 express endothelin receptor B. Stimulation by the endothelin receptor B agonist IRL1620 triggered phosphorylation of ERK1/2 and autophosphorylation of (Y1173 EGFR. The effects could be blocked by Src-kinase inhibitors (PP1, PP2, EGFR-inhibitor (AG1478, EGFR-siRNA and by inhibitors to extracellular matrix metalloproteinases (GM6001, consistent with a Src-kinase mediated endothelin receptor response that engage ligand-dependent and ligand-independent EGFR activation. Our data suggest a mechanism for how injury-induced endothelins, produced in the retina, may modulate the Müller cell responses by Src-mediated transactivation of EGFRs. The data give support to a view in

  1. Lenalidomide induces lipid raft assembly to enhance erythropoietin receptor signaling in myelodysplastic syndrome progenitors.

    Science.gov (United States)

    McGraw, Kathy L; Basiorka, Ashley A; Johnson, Joseph O; Clark, Justine; Caceres, Gisela; Padron, Eric; Heaton, Ruth; Ozawa, Yukiyasu; Wei, Sheng; Sokol, Lubomir; List, Alan F

    2014-01-01

    Anemia remains the principal management challenge for patients with lower risk Myelodysplastic Syndromes (MDS). Despite appropriate cytokine production and cellular receptor display, erythropoietin receptor (EpoR) signaling is impaired. We reported that EpoR signaling is dependent upon receptor localization within lipid raft microdomains, and that disruption of raft integrity abolishes signaling capacity. Here, we show that MDS erythroid progenitors display markedly diminished raft assembly and smaller raft aggregates compared to normal controls (p = 0.005, raft number; p = 0.023, raft size). Because lenalidomide triggers raft coalescence in T-lymphocytes promoting immune synapse formation, we assessed effects of lenalidomide on raft assembly in MDS erythroid precursors and UT7 cells. Lenalidomide treatment rapidly induced lipid raft formation accompanied by EpoR recruitment into raft fractions together with STAT5, JAK2, and Lyn kinase. The JAK2 phosphatase, CD45, a key negative regulator of EpoR signaling, was displaced from raft fractions. Lenalidomide treatment prior to Epo stimulation enhanced both JAK2 and STAT5 phosphorylation in UT7 and primary MDS erythroid progenitors, accompanied by increased STAT5 DNA binding in UT7 cells, and increased erythroid colony forming capacity in both UT7 and primary cells. Raft induction was associated with F-actin polymerization, which was blocked by Rho kinase inhibition. These data indicate that deficient raft integrity impairs EpoR signaling, and provides a novel strategy to enhance EpoR signal fidelity in non-del(5q) MDS.

  2. Vitamin D controls T cell antigen receptor signaling and activation of human T cells

    DEFF Research Database (Denmark)

    von Essen, Marina Rode; Kongsbak-Wismann, Martin; Schjerling, Peter

    2010-01-01

    Phospholipase C (PLC) isozymes are key signaling proteins downstream of many extracellular stimuli. Here we show that naive human T cells had very low expression of PLC-gamma1 and that this correlated with low T cell antigen receptor (TCR) responsiveness in naive T cells. However, TCR triggering...... led to an upregulation of approximately 75-fold in PLC-gamma1 expression, which correlated with greater TCR responsiveness. Induction of PLC-gamma1 was dependent on vitamin D and expression of the vitamin D receptor (VDR). Naive T cells did not express VDR, but VDR expression was induced by TCR...... signaling via the alternative mitogen-activated protein kinase p38 pathway. Thus, initial TCR signaling via p38 leads to successive induction of VDR and PLC-gamma1, which are required for subsequent classical TCR signaling and T cell activation....

  3. MHC class I is functionally associated with antigen receptors in human T and B lymphomas

    DEFF Research Database (Denmark)

    Pedersen, Anders Elm; Jacoby, B F; Skov, S

    1996-01-01

    lines the increase in [Ca2+]i after MHC-I cross-linking caused upregulation of CD69, an early marker of activation. When studying the effect of MHC-I cross-linking on the TCR- and B cell antigen receptor (BCR)- mediated increase in [Ca2+]i, respectively, we observed that MHC-I had a costimulatory effect...... on the TCR-mediated increase in [Ca2+]i in Jurkat cells but not on the anti-IgM-mediated activity of Solubo cells. Studies of subpopulations of Jurkat and Solubo cells expressing different levels of MHC-I on their cell surfaces revealed that the TCR- and BCR-mediated increases in [Ca2+]i, respectively, were...

  4. c-MPL provides tumor-targeted T-cell receptor-transgenic T cells with costimulation and cytokine signals.

    Science.gov (United States)

    Nishimura, Christopher D; Brenner, Daniel A; Mukherjee, Malini; Hirsch, Rachel A; Ott, Leah; Wu, Meng-Fen; Liu, Hao; Dakhova, Olga; Orange, Jordan S; Brenner, Malcolm K; Lin, Charles Y; Arber, Caroline

    2017-12-21

    Adoptively transferred T-cell receptor (TCR)-engineered T cells depend on host-derived costimulation and cytokine signals for their full and sustained activation. However, in patients with cancer, both signals are frequently impaired. Hence, we developed a novel strategy that combines both essential signals in 1 transgene by expressing the nonlymphoid hematopoietic growth factor receptor c-MPL (myeloproliferative leukemia), the receptor for thrombopoietin (TPO), in T cells. c-MPL signaling activates pathways shared with conventional costimulatory and cytokine receptor signaling. Thus, we hypothesized that host-derived TPO, present in the tumor microenvironment, or pharmacological c-MPL agonists approved by the US Food and Drug Administration could deliver both signals to c-MPL-engineered TCR-transgenic T cells. We found that c-MPL + polyclonal T cells expand and proliferate in response to TPO, and persist longer after adoptive transfer in immunodeficient human TPO-transgenic mice. In TCR-transgenic T cells, c-MPL activation enhances antitumor function, T-cell expansion, and cytokine production and preserves a central memory phenotype. c-MPL signaling also enables sequential tumor cell killing, enhances the formation of effective immune synapses, and improves antileukemic activity in vivo in a leukemia xenograft model. We identify the type 1 interferon pathway as a molecular mechanism by which c-MPL mediates immune stimulation in T cells. In conclusion, we present a novel immunotherapeutic strategy using c-MPL-enhanced transgenic T cells responding to either endogenously produced TPO (a microenvironment factor in hematologic malignancies) or c-MPL-targeted pharmacological agents. © 2017 by The American Society of Hematology.

  5. SGIP1 alters internalization and modulates signaling of activated cannabinoid receptor 1 in a biased manner

    Czech Academy of Sciences Publication Activity Database

    Hájková, Alena; Techlovská, Šárka; Dvořáková, Michaela; Chambers, Jayne Nicole; Kumpošt, Jiří; Hubálková, Pavla; Prezeau, L.; Blahoš, Jaroslav

    2016-01-01

    Roč. 107, léto (2016), s. 201-214 ISSN 0028-3908 R&D Projects: GA ČR GAP303/12/2408 Institutional support: RVO:68378050 Keywords : Seven transmembrane receptors * G-protein coupled receptors * Cannabinoid receptor 1 * Protein-protein interactions * Bias signaling * Receptor endocytosis Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.012, year: 2016

  6. A single amino acid residue controls Ca2+ signaling by an octopamine receptor from Drosophila melanogaster.

    Science.gov (United States)

    Hoff, Max; Balfanz, Sabine; Ehling, Petra; Gensch, Thomas; Baumann, Arnd

    2011-07-01

    Rhythmic activity of cells and cellular networks plays an important role in physiology. In the nervous system oscillations of electrical activity and/or second messenger concentrations are important to synchronize neuronal activity. At the molecular level, rhythmic activity can be initiated by different routes. We have recently shown that an octopamine-activated G-protein-coupled receptor (GPCR; DmOctα1Rb, CG3856) from Drosophila initiates Ca(2+) oscillations. Here, we have unraveled the molecular basis of cellular Ca(2+) signaling controlled by the DmOctα1Rb receptor using a combination of pharmacological intervention, site-directed mutagenesis, and functional cellular Ca(2+) imaging on heterologously expressed receptors. Phosphorylation of a single amino acid residue in the third intracellular loop of the GPCR by PKC is necessary and sufficient to desensitize the receptor. From its desensitized state, DmOctα1Rb is resensitized by dephosphorylation, and a new Ca(2+) signal occurs on octopamine stimulation. Our findings show that transient changes of the receptor's surface profile have a strong effect on its physiological signaling properties. We expect that the detailed knowledge of DmOctα1Rb-dependent signal transduction fosters the identification of specific drugs that can be used for GPCR-mediated pest control, since octopamine serves important physiological and behavioral functions in arthropods.

  7. Elevated insulin-like growth factor 1 receptor signaling induces antiestrogen resistance through the MAPK/ERK and PI3K/Akt signaling routes

    NARCIS (Netherlands)

    Zhang, Y.; Moerkens, M.; Ramaiahgari, S.; Bont, de H.J.G.M.; Price, L.; Meerman, J.H.N.; Water, van de B.

    2011-01-01

    INTRODUCTION: Insulin-like growth factor 1 (IGF-1) receptor (IGF-1R) is phosphorylated in all breast cancer subtypes. Past findings have shown that IGF-1R mediates antiestrogen resistance through cross-talk with estrogen receptor (ER) signaling and via its action upstream of the epidermal growth

  8. Tyrosine 769 of the keratinocyte growth factor receptor is required for receptor signaling but not endocytosis

    International Nuclear Information System (INIS)

    Ceridono, Mara; Belleudi, Francesca; Ceccarelli, Simona; Torrisi, Maria Rosaria

    2005-01-01

    Keratinocyte growth factor receptor (KGFR) is a receptor tyrosine kinase expressed on epithelial cells which belongs to the family of fibroblast growth factor receptors (FGFRs). Following ligand binding, KGFR is rapidly autophosphorylated on specific tyrosine residues in the intracellular domain, recruits substrate proteins, and is rapidly internalized by clathrin-mediated endocytosis. The role of different autophosphorylation sites in FGFRs, and in particular the role of the tyrosine 766 in FGFR1, first identified as PLCγ binding site, has been extensively studied. We analyzed here the possible role of the tyrosine 769 in KGFR, corresponding to tyrosine 766 in FGFR1, in the regulation of KGFR signal transduction and MAPK activation as well as in the control of the endocytic process of KGFR. A mutant KGFR in which tyrosine 769 was substituted by phenylalanine was generated and transfected in NIH3T3 and HeLa cells. Our results indicate that tyrosine 769 is required for the binding to KGFR and tyrosine phosphorylation of PLCγ as well as for the full activation of MAPKs and for cell proliferation through the regulation of FRS2 tyrosine phosphorylation, suggesting that this residue represents a key regulator of KGFR signal transduction. Our data also show that tyrosine 769 is not involved in the regulation of the endocytic process of KGFR

  9. UDP/P2Y6 receptor signaling regulates IgE-dependent degranulation in human basophils

    Directory of Open Access Journals (Sweden)

    Manabu Nakano

    2017-10-01

    Conclusions: This study showed that UDP/P2Y6 receptor signaling is involved in the regulation of IgE-dependent degranulation in basophils, which might stimulate the P2Y6 receptor via the autocrine secretion of UTP. Thus, this receptor represents a potential target to regulate IgE-dependent degranulation in basophils during allergic diseases.

  10. Signaling properties and pharmacological analysis of two sulfakinin receptors from the red flour beetle, Tribolium castaneum.

    Science.gov (United States)

    Zels, Sven; Verlinden, Heleen; Dillen, Senne; Vleugels, Rut; Nachman, Ronald J; Vanden Broeck, Jozef

    2014-01-01

    Sulfakinin is an insect neuropeptide that constitutes an important component of the complex network of hormonal and neural factors that regulate feeding and digestion. The key modulating functions of sulfakinin are mediated by binding and signaling via G-protein coupled receptors. Although a substantial amount of functional data have already been reported on sulfakinins in different insect species, only little information is known regarding the properties of their respective receptors. In this study, we report on the molecular cloning, functional expression and characterization of two sulfakinin receptors in the red flour beetle, Tribolium castaneum. Both receptor open reading frames show extensive sequence similarity with annotated sulfakinin receptors from other insects. Comparison of the sulfakinin receptor sequences with homologous vertebrate cholecystokinin receptors reveals crucial conserved regions for ligand binding and receptor activation. Quantitative reverse transcriptase PCR shows that transcripts of both receptors are primarily expressed in the central nervous system of the beetle. Pharmacological characterization using 29 different peptide ligands clarified the essential requirements for efficient activation of these sulfakinin receptors. Analysis of the signaling pathway in multiple cell lines disclosed that the sulfakinin receptors of T. castaneum can stimulate both the Ca²⁺ and cyclic AMP second messenger pathways. This in depth characterization of two insect sulfakinin receptors may provide useful leads for the further development of receptor ligands with a potential applicability in pest control and crop protection.

  11. Dissection of pathways leading to antigen receptor-induced and Fas/CD95-induced apoptosis in human B cells

    NARCIS (Netherlands)

    Lens, S. M.; den Drijver, B. F.; Pötgens, A. J.; Tesselaar, K.; van Oers, M. H.; van Lier, R. A.

    1998-01-01

    To dissect intracellular pathways involved in B cell Ag receptor (BCR)-mediated and Fas-induced human B cell death, we isolated clones of the Burkitt lymphoma cell line Ramos with different apoptosis sensitivities. Selection for sensitivity to Fas-induced apoptosis also selected for clones with

  12. High constitutive signaling of the ghrelin receptor--identification of a potent inverse agonist

    DEFF Research Database (Denmark)

    Holst, Birgitte; Cygankiewicz, Adam; Jensen, Tine Halkjaer

    2003-01-01

    Ghrelin is a GH-releasing peptide that also has an important role as an orexigenic hormone-stimulating food intake. By measuring inositol phosphate turnover or by using a reporter assay for transcriptional activity controlled by cAMP-responsive elements, the ghrelin receptor showed strong, ligand......-independent signaling in transfected COS-7 or human embryonic kidney 293 cells. Ghrelin and a number of the known nonpeptide GH secretagogues acted as agonists stimulating inositol phosphate turnover further. In contrast, the low potency ghrelin antagonist, [D-Arg1,D-Phe5,D-Trp7,9,Leu11]-substance P was surprisingly...... found to be a high potency (EC50 = 5.2 nm) full inverse agonist as it decreased the constitutive signaling of the ghrelin receptor down to that observed in untransfected cells. The homologous motilin receptor functioned as a negative control as it did not display any sign of constitutive activity...

  13. Spatio-temporal dependence of the signaling response in immune-receptor trafficking networks regulated by cell density: a theoretical model.

    Directory of Open Access Journals (Sweden)

    Pilar García-Peñarrubia

    Full Text Available Cell signaling processes involve receptor trafficking through highly connected networks of interacting components. The binding of surface receptors to their specific ligands is a key factor for the control and triggering of signaling pathways. In most experimental systems, ligand concentration and cell density vary within a wide range of values. Dependence of the signal response on cell density is related with the extracellular volume available per cell. This dependence has previously been studied using non-spatial models which assume that signaling components are well mixed and uniformly distributed in a single compartment. In this paper, a mathematical model that shows the influence exerted by cell density on the spatio-temporal evolution of ligands, cell surface receptors, and intracellular signaling molecules is developed. To this end, partial differential equations were used to model ligand and receptor trafficking dynamics through the different domains of the whole system. This enabled us to analyze several interesting features involved with these systems, namely: a how the perturbation caused by the signaling response propagates through the system; b receptor internalization dynamics and how cell density affects the robustness of dose-response curves upon variation of the binding affinity; and c that enhanced correlations between ligand input and system response are obtained under conditions that result in larger perturbations of the equilibrium ligand + surface receptor [Please see text] ligand - receptor complex. Finally, the results are compared with those obtained by considering that the above components are well mixed in a single compartment.

  14. Optimisation of beryllium-7 gamma analysis following BCR sequential extraction

    International Nuclear Information System (INIS)

    Taylor, A.; Blake, W.H.; Keith-Roach, M.J.

    2012-01-01

    Graphical abstract: Showing decrease in analytical uncertainty using the optimal (combined preconcentrated sample extract) method. nv (no value) where extract activities were 7 Be geochemical behaviour is required to support tracer studies. ► Sequential extraction with natural 7 Be returns high analytical uncertainties. ► Preconcentrating extracts from a large sample mass improved analytical uncertainty. ► This optimised method can be readily employed in studies using low activity samples. - Abstract: The application of cosmogenic 7 Be as a sediment tracer at the catchment-scale requires an understanding of its geochemical associations in soil to underpin the assumption of irreversible adsorption. Sequential extractions offer a readily accessible means of determining the associations of 7 Be with operationally defined soil phases. However, the subdivision of the low activity concentrations of fallout 7 Be in soils into geochemical fractions can introduce high gamma counting uncertainties. Extending analysis time significantly is not always an option for batches of samples, owing to the on-going decay of 7 Be (t 1/2 = 53.3 days). Here, three different methods of preparing and quantifying 7 Be extracted using the optimised BCR three-step scheme have been evaluated and compared with a focus on reducing analytical uncertainties. The optimal method involved carrying out the BCR extraction in triplicate, sub-sampling each set of triplicates for stable Be analysis before combining each set and coprecipitating the 7 Be with metal oxyhydroxides to produce a thin source for gamma analysis. This method was applied to BCR extractions of natural 7 Be in four agricultural soils. The approach gave good counting statistics from a 24 h analysis period (∼10% (2σ) where extract activity >40% of total activity) and generated statistically useful sequential extraction profiles. Total recoveries of 7 Be fell between 84 and 112%. The stable Be data demonstrated that the

  15. The nanoscale spatial organization of B-cell receptors on immunoglobulin M- and G-expressing human B-cells.

    Science.gov (United States)

    Lee, Jinmin; Sengupta, Prabuddha; Brzostowski, Joseph; Lippincott-Schwartz, Jennifer; Pierce, Susan K

    2017-02-15

    B-cell activation is initiated by the binding of antigen to the B-cell receptor (BCR). Here we used dSTORM superresolution imaging to characterize the nanoscale spatial organization of immunoglobulin M (IgM) and IgG BCRs on the surfaces of resting and antigen--activated human peripheral blood B-cells. We provide insights into both the fundamental process of antigen-driven BCR clustering and differences in the spatial organization of IgM and IgG BCRs that may contribute to the characteristic differences in the responses of naive and memory B-cells to antigen. We provide evidence that although both IgM and IgG BCRs reside in highly heterogeneous protein islands that vary in size and number of BCR single-molecule localizations, both resting and activated B-cells intrinsically maintain a high -frequency of single isolated BCR localizations, which likely represent BCR monomers. IgG BCRs are more clustered than IgM BCRs on resting cells and form larger protein islands after antigen activation. Small, dense BCR clusters likely formed via protein-protein interactions are present on the surface of resting cells, and antigen activation induces these to come together to form less dense, larger islands, a process likely governed, at least in part, by protein-lipid interactions. © 2017 Lee, Sengupta, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  16. Delineation of the GPRC6A Receptor Signaling Pathways Using a Mammalian Cell Line Stably Expressing the Receptor

    DEFF Research Database (Denmark)

    Jacobsen, Stine Engesgaard; Nørskov-Lauritsen, Lenea; Thomsen, Alex Rojas Bie

    2013-01-01

    receptor has been suggested to couple to multiple G protein classes albeit via indirect methods. Thus, the exact ligand preferences and signaling pathways are yet to be elucidated. In the present study, we generated a Chinese hamster ovary (CHO) cell line that stably expresses mouse GPRC6A. In an effort...... and divalent cations, and for the first time, we conclusively show that these responses are mediated through the Gq pathway. We were not able to confirm previously published data demonstrating Gi- and Gs-mediated signaling; neither could we detect agonistic activity of testosterone and osteocalcin. Generation...... of the stable CHO cell line with robust receptor responsiveness and optimization of the highly sensitive homogeneous time resolved fluorescence technology allow fast assessment of Gq activation without previous manipulations like cotransfection of mutated G proteins. This cell-based assay system for GPRC6A...

  17. Estrogen receptor signaling in prostate cancer: Implications for carcinogenesis and tumor progression.

    Science.gov (United States)

    Bonkhoff, Helmut

    2018-01-01

    The androgen receptor (AR) is the classical target for prostate cancer prevention and treatment, but more recently estrogens and their receptors have also been implicated in prostate cancer development and tumor progression. Recent experimental and clinical data were reviewed to elucidate pathogenetic mechanisms how estrogens and their receptors may affect prostate carcinogenesis and tumor progression. The estrogen receptor beta (ERβ) is the most prevalent ER in the human prostate, while the estrogen receptor alpha (ERα) is restricted to basal cells of the prostatic epithelium and stromal cells. In high grade prostatic intraepithelial neoplasia (HGPIN), the ERα is up-regulated and most likely mediates carcinogenic effects of estradiol as demonstrated in animal models. The partial loss of the ERβ in HGPIN indicates that the ERβ acts as a tumor suppressor. The tumor promoting function of the TMPRSS2-ERG fusion, a major driver of prostate carcinogenesis, is triggered by the ERα and repressed by the ERβ. The ERβ is generally retained in hormone naïve and metastatic prostate cancer, but is partially lost in castration resistant disease. The progressive emergence of the ERα and ERα-regulated genes (eg, progesterone receptor (PR), PS2, TMPRSS2-ERG fusion, and NEAT1) during prostate cancer progression and hormone refractory disease suggests that these tumors can bypass the AR by using estrogens and progestins for their growth. In addition, nongenomic estrogen signaling pathways mediated by orphan receptors (eg, GPR30 and ERRα) has also been implicated in prostate cancer progression. Increasing evidences demonstrate that local estrogen signaling mechanisms are required for prostate carcinogenesis and tumor progression. Despite the recent progress in this research topic, the translation of the current information into potential therapeutic applications remains highly challenging and clearly warrants further investigation. © 2017 Wiley Periodicals, Inc.

  18. Role of Cbl-associated protein/ponsin in receptor tyrosine kinase signaling and cell adhesion

    Directory of Open Access Journals (Sweden)

    Ritva Tikkanen

    2012-10-01

    Full Text Available The Cbl-associated protein/ponsin (CAP is an adaptor protein that contains a so-called Sorbin homology (SoHo domain and three Src homology 3 (SH3 domains which are engaged in diverse protein-protein interactions. CAP has been shown to function in the regulation of the actin cytoskeleton and cell adhesion and to be involved in the differentiation of muscle cells and adipocytes. In addition, it participates in signaling pathways through several receptor tyrosine kinases such as insulin and neurotrophin receptors. In the last couple of years, several studies have shed light on the details of these processes and identified novel interaction partners of CAP. In this review, we summarize these recent findings and provide an overview on the function of CAP especially in cell adhesion and membrane receptor signaling.

  19. Combinatory annotation of cell membrane receptors and signalling pathways of Bombyx mori prothoracic glands

    Science.gov (United States)

    Moulos, Panagiotis; Samiotaki, Martina; Panayotou, George; Dedos, Skarlatos G.

    2016-01-01

    The cells of prothoracic glands (PG) are the main site of synthesis and secretion of ecdysteroids, the biochemical products of cholesterol conversion to steroids that shape the morphogenic development of insects. Despite the availability of genome sequences from several insect species and the extensive knowledge of certain signalling pathways that underpin ecdysteroidogenesis, the spectrum of signalling molecules and ecdysteroidogenic cascades is still not fully comprehensive. To fill this gap and obtain the complete list of cell membrane receptors expressed in PG cells, we used combinatory bioinformatic, proteomic and transcriptomic analysis and quantitative PCR to annotate and determine the expression profiles of genes identified as putative cell membrane receptors of the model insect species, Bombyx mori, and subsequently enrich the repertoire of signalling pathways that are present in its PG cells. The genome annotation dataset we report here highlights modules and pathways that may be directly involved in ecdysteroidogenesis and aims to disseminate data and assist other researchers in the discovery of the role of such receptors and their ligands. PMID:27576083

  20. Rapid Phospho-Turnover by Receptor Tyrosine Kinases Impacts Downstream Signaling and Drug Binding

    OpenAIRE

    Kleiman, Laura B.; Maiwald, Thomas; Conzelmann, Holger; Lauffenburger, Douglas A.; Sorger, Peter K.

    2011-01-01

    Epidermal growth factor receptors (ErbB1–4) are oncogenic receptor tyrosine kinases (RTKs) that regulate diverse cellular processes. In this study, we combine measurement and mathematical modeling to quantify phospho-turnover at ErbB receptors in human cells and to determine the consequences for signaling and drug binding. We find that phosphotyrosine residues on ErbB1 have half-lives of a few seconds and therefore turn over 100–1000 times in the course of a typical immediate-early response t...

  1. Non-Ligand-Induced Dimerization is Sufficient to Initiate the Signalling and Endocytosis of EGF Receptor

    Directory of Open Access Journals (Sweden)

    George Kourouniotis

    2016-07-01

    Full Text Available The binding of epidermal growth factor (EGF to EGF receptor (EGFR stimulates cell mitogenesis and survival through various signalling cascades. EGF also stimulates rapid EGFR endocytosis and its eventual degradation in lysosomes. The immediate events induced by ligand binding include receptor dimerization, activation of intrinsic tyrosine kinase and autophosphorylation. However, in spite of intensified efforts, the results regarding the roles of these events in EGFR signalling and internalization is still very controversial. In this study, we constructed a chimeric EGFR by replacing its extracellular domain with leucine zipper (LZ and tagged a green fluorescent protein (GFP at its C-terminus. We showed that the chimeric LZ-EGFR-GFP was constitutively dimerized. The LZ-EGFR-GFP dimer autophosphorylated each of its five well-defined C-terminal tyrosine residues as the ligand-induced EGFR dimer does. Phosphorylated LZ-EGFR-GFP was localized to both the plasma membrane and endosomes, suggesting it is capable of endocytosis. We also showed that LZ-EGFR-GFP activated major signalling proteins including Src homology collagen-like (Shc, extracellular signal-regulated kinase (ERK and Akt. Moreover, LZ-EGFR-GFP was able to stimulate cell proliferation. These results indicate that non-ligand induced dimerization is sufficient to activate EGFR and initiate cell signalling and EGFR endocytosis. We conclude that receptor dimerization is a critical event in EGF-induced cell signalling and EGFR endocytosis.

  2. ß-Adrenergic Receptor Signaling and Modulation of Long-Term Potentiation in the Mammalian Hippocampus

    Science.gov (United States)

    O'Dell, Thomas J.; Connor, Steven A.; Guglietta, Ryan; Nguyen, Peter V.

    2015-01-01

    Encoding new information in the brain requires changes in synaptic strength. Neuromodulatory transmitters can facilitate synaptic plasticity by modifying the actions and expression of specific signaling cascades, transmitter receptors and their associated signaling complexes, genes, and effector proteins. One critical neuromodulator in the…

  3. bcr-abl oncogene activation in Philadelphia chromosome-positive acute lymphoblastic leukemia

    NARCIS (Netherlands)

    Hermans, A.; Gow, J.; Selleri, L.; von Lindern, M.; Hagemeijer, A.; Wiedemann, L. M.; Grosveld, G.

    1988-01-01

    Tumor-specific alterations in oncogenes are thought to play a central role in the development of cancer. An example is the consistent fusion of the bcr gene to the c-abl oncogene on the Ph chromosome in CML. The Ph chromosome can also be observed in ALL. About 50% of Ph+ ALL cases, in contrast to

  4. Regulation of G-protein coupled receptor traffic by an evolutionary conserved hydrophobic signal.

    Science.gov (United States)

    Angelotti, Tim; Daunt, David; Shcherbakova, Olga G; Kobilka, Brian; Hurt, Carl M

    2010-04-01

    Plasma membrane (PM) expression of G-protein coupled receptors (GPCRs) is required for activation by extracellular ligands; however, mechanisms that regulate PM expression of GPCRs are poorly understood. For some GPCRs, such as alpha2c-adrenergic receptors (alpha(2c)-ARs), heterologous expression in non-native cells results in limited PM expression and extensive endoplasmic reticulum (ER) retention. Recently, ER export/retentions signals have been proposed to regulate cellular trafficking of several GPCRs. By utilizing a chimeric alpha(2a)/alpha(2c)-AR strategy, we identified an evolutionary conserved hydrophobic sequence (ALAAALAAAAA) in the extracellular amino terminal region that is responsible in part for alpha(2c)-AR subtype-specific trafficking. To our knowledge, this is the first luminal ER retention signal reported for a GPCR. Removal or disruption of the ER retention signal dramatically increased PM expression and decreased ER retention. Conversely, transplantation of this hydrophobic sequence into alpha(2a)-ARs reduced their PM expression and increased ER retention. This evolutionary conserved hydrophobic trafficking signal within alpha(2c)-ARs serves as a regulator of GPCR trafficking.

  5. Lipoprotein receptor LRP1 regulates leptin signaling and energy homeostasis in the adult central nervous system.

    Science.gov (United States)

    Liu, Qiang; Zhang, Juan; Zerbinatti, Celina; Zhan, Yan; Kolber, Benedict J; Herz, Joachim; Muglia, Louis J; Bu, Guojun

    2011-01-11

    Obesity is a growing epidemic characterized by excess fat storage in adipocytes. Although lipoprotein receptors play important roles in lipid uptake, their role in controlling food intake and obesity is not known. Here we show that the lipoprotein receptor LRP1 regulates leptin signaling and energy homeostasis. Conditional deletion of the Lrp1 gene in the brain resulted in an obese phenotype characterized by increased food intake, decreased energy consumption, and decreased leptin signaling. LRP1 directly binds to leptin and the leptin receptor complex and is required for leptin receptor phosphorylation and Stat3 activation. We further showed that deletion of the Lrp1 gene specifically in the hypothalamus by Cre lentivirus injection is sufficient to trigger accelerated weight gain. Together, our results demonstrate that the lipoprotein receptor LRP1, which is critical in lipid metabolism, also regulates food intake and energy homeostasis in the adult central nervous system.

  6. Receptor-receptor interactions within receptor mosaics. Impact on neuropsychopharmacology.

    Science.gov (United States)

    Fuxe, K; Marcellino, D; Rivera, A; Diaz-Cabiale, Z; Filip, M; Gago, B; Roberts, D C S; Langel, U; Genedani, S; Ferraro, L; de la Calle, A; Narvaez, J; Tanganelli, S; Woods, A; Agnati, L F

    2008-08-01

    Future therapies for diseases associated with altered dopaminergic signaling, including Parkinson's disease, schizophrenia and drug addiction or drug dependence may substantially build on the existence of intramembrane receptor-receptor interactions within dopamine receptor containing receptor mosaics (RM; dimeric or high-order receptor oligomers) where it is believed that the dopamine D(2) receptor may operate as the 'hub receptor' within these complexes. The constitutive adenosine A(2A)/dopamine D(2) RM, located in the dorsal striato-pallidal GABA neurons, are of particular interest in view of the demonstrated antagonistic A(2A)/D(2) interaction within these heteromers; an interaction that led to the suggestion and later demonstration that A(2A) antagonists could be used as novel anti-Parkinsonian drugs. Based on the likely existence of A(2A)/D(2)/mGluR5 RM located both extrasynaptically on striato-pallidal GABA neurons and on cortico-striatal glutamate terminals, multiple receptor-receptor interactions within this RM involving synergism between A(2A)/mGluR5 to counteract D(2) signaling, has led to the proposal of using combined mGluR5 and A(2A) antagonists as a future anti-Parkinsonian treatment. Based on the same RM in the ventral striato-pallidal GABA pathways, novel strategies for the treatment of schizophrenia, building on the idea that A(2A) agonists and/or mGluR5 agonists will help reduce the increased dopaminergic signaling associated with this disease, have been suggested. Such treatment may ensure the proper glutamatergic drive from the mediodorsal thalamic nucleus to the prefrontal cortex, one which is believed to be reduced in schizophrenia due to a dominance of D(2)-like signaling in the ventral striatum. Recently, A(2A) receptors also have been shown to counteract the locomotor and sensitizing actions of cocaine and increases in A(2A) receptors have also been observed in the nucleus accumbens after extended cocaine self-administration, probably

  7. Signaling properties and pharmacological analysis of two sulfakinin receptors from the red flour beetle, Tribolium castaneum.

    Directory of Open Access Journals (Sweden)

    Sven Zels

    Full Text Available Sulfakinin is an insect neuropeptide that constitutes an important component of the complex network of hormonal and neural factors that regulate feeding and digestion. The key modulating functions of sulfakinin are mediated by binding and signaling via G-protein coupled receptors. Although a substantial amount of functional data have already been reported on sulfakinins in different insect species, only little information is known regarding the properties of their respective receptors. In this study, we report on the molecular cloning, functional expression and characterization of two sulfakinin receptors in the red flour beetle, Tribolium castaneum. Both receptor open reading frames show extensive sequence similarity with annotated sulfakinin receptors from other insects. Comparison of the sulfakinin receptor sequences with homologous vertebrate cholecystokinin receptors reveals crucial conserved regions for ligand binding and receptor activation. Quantitative reverse transcriptase PCR shows that transcripts of both receptors are primarily expressed in the central nervous system of the beetle. Pharmacological characterization using 29 different peptide ligands clarified the essential requirements for efficient activation of these sulfakinin receptors. Analysis of the signaling pathway in multiple cell lines disclosed that the sulfakinin receptors of T. castaneum can stimulate both the Ca²⁺ and cyclic AMP second messenger pathways. This in depth characterization of two insect sulfakinin receptors may provide useful leads for the further development of receptor ligands with a potential applicability in pest control and crop protection.

  8. Novel method for the study of receptor Ca2+ signalling exemplified by the NK1 receptor

    DEFF Research Database (Denmark)

    Heding, A; Elling, C E; Schwartz, T W

    2002-01-01

    We have used a novel technology (NovoStar from BMG Labtechnologies) for the study of the Ca2+ signalling of the human tackykinin NK1 (hNK-I receptor). The NovoStar is a microplate reader based on fluorescence and luminescence. The instrument implements a robotic pipettor arm and two microplate...... carriers, typically one for samples and one for cells. The robotic pipettor arm can transfer sample (agonist or antagonist) from the sample plate or other liquid containers to the cell plate, facilitating the study of Ca2+ signalling to such a degree that the instrument can be used for Medium Throughput...

  9. Prolonged treatment with imatinib mesylate in patients with advanced chronic myeloid leukemia causes a reduction of bcr/abl mRNA levels independent of cytogenetic response.

    Science.gov (United States)

    Cariani, E; Capucci, M; Micheletti, M; Spalenza, F; Zanella, I; Albertini, A; Rossi, G

    2003-06-01

    Bcr/abl mRNA levels were monitored in 13 patients with chronic myeloid leukemia receiving imatinib mesylate over a period of 78 weeks. During treatment median bcr/abl mRNA levels progressively declined from 77.2 normalized dose (nD) at baseline to 11.28 nD after 13 weeks ( P<0.05) and to 1.28 nD after 78 weeks ( P<0.05). After 13 weeks, bcr/abl mRNA levels were significantly lower in cytogenetic responders compared to nonresponders ( P<0.05), but subsequent decrease in the transcript levels caused the loss of any correlation to the cytogenetic status. These results suggest that bcr/abl mRNA levels may reflect cytogenetic response only during the early phases of imatinib therapy.

  10. Innate immune receptor Toll-like receptor 4 signalling in neuropsychiatric diseases.

    Science.gov (United States)

    García Bueno, B; Caso, J R; Madrigal, J L M; Leza, J C

    2016-05-01

    The innate immunity is a stereotyped first line of defense against pathogens and unspecified damage signals. One of main actors of innate immunity are the Toll-like receptors (TLRs), and one of the better characterized members of this family is TLR-4, that it is mainly activated by Gram-negative bacteria lipopolysaccharide. In brain, TLR-4 organizes innate immune responses against infections or cellular damage, but also possesses other physiological functions. In the last years, some evidences suggest a role of TLR-4 in stress and stress-related neuropsychiatric diseases. Peripheral and brain TLR-4 activation triggers sickness behavior, and its expression is a risk factor of depression. Some elements of the TLR-4 signaling pathway are up-regulated in peripheral samples and brain post-mortem tissue from depressed and suicidal patients. The "leaky gut" hypothesis of neuropsychiatric diseases is based on the existence of an increase of the intestinal permeability which results in bacterial translocation able to activate TLR-4. Enhanced peripheral TLR-4 expression/activity has been described in subjects diagnosed with schizophrenia, bipolar disorder and in autistic children. A role for TLR-4 in drugs abuse has been also proposed. The therapeutic potential of pharmacological/genetic modulation of TLRs signaling pathways in neuropsychiatry is promising, but a great preclinical/clinical scientific effort is still needed. Copyright © 2016 Elsevier Ltd. All rights reserved.

  11. Converging evolution leads to near maximal junction diversity through parallel mechanisms in B and T cell receptors

    Science.gov (United States)

    Benichou, Jennifer I. C.; van Heijst, Jeroen W. J.; Glanville, Jacob; Louzoun, Yoram

    2017-08-01

    T and B cell receptor (TCR and BCR) complementarity determining region 3 (CDR3) genetic diversity is produced through multiple diversification and selection stages. Potential holes in the CDR3 repertoire were argued to be linked to immunodeficiencies and diseases. In contrast with BCRs, TCRs have practically no Dβ germline genetic diversity, and the question emerges as to whether they can produce a diverse CDR3 repertoire. In order to address the genetic diversity of the adaptive immune system, appropriate quantitative measures for diversity and large-scale sequencing are required. Such a diversity method should incorporate the complex diversification mechanisms of the adaptive immune response and the BCR and TCR loci structure. We combined large-scale sequencing and diversity measures to show that TCRs have a near maximal CDR3 genetic diversity. Specifically, TCR have a larger junctional and V germline diversity, which starts more 5‧ in Vβ than BCRs. Selection decreases the TCR repertoire diversity, but does not affect BCR repertoire. As a result, TCR is as diverse as BCR repertoire, with a biased CDR3 length toward short TCRs and long BCRs. These differences suggest parallel converging evolutionary tracks to reach the required diversity to avoid holes in the CDR3 repertoire.

  12. Plasticity of Signaling by Spinal Estrogen Receptor α, κ-Opioid Receptor, and Metabotropic Glutamate Receptors over the Rat Reproductive Cycle Regulates Spinal Endomorphin 2 Antinociception: Relevance of Endogenous-Biased Agonism.

    Science.gov (United States)

    Liu, Nai-Jiang; Murugaiyan, Vijaya; Storman, Emiliya M; Schnell, Stephen A; Kumar, Arjun; Wessendorf, Martin W; Gintzler, Alan R

    2017-11-15

    We previously showed that intrathecal application of endomorphin 2 [EM2; the highly specific endogenous μ-opioid receptor (MOR) ligand] induces antinociception that varies with stage of the rat estrous cycle: minimal during diestrus and prominent during proestrus. Earlier studies, however, did not identify proestrus-activated signaling strategies that enable spinal EM2 antinociception. We now report that in female rats, increased spinal dynorphin release and κ-opioid receptor (KOR) signaling, as well as the emergence of glutamate-activated metabotropic glutamate receptor 1 (mGluR 1 ) signaling, are critical to the transition from an EM2 nonresponsive state (during diestrus) to an analgesically responsive state (during proestrus). Differential signaling by mGluR 1 , depending on its activation by membrane estrogen receptor α (mERα; during diestrus) versus glutamate (during proestrus), concomitant with the ebb and flow of spinal dynorphin/KOR signaling, functions as a switch, preventing or promoting, respectively, spinal EM2 antinociception. Importantly, EM2 and glutamate-containing varicosities appose spinal neurons that express MOR along with mGluRs and mERα, suggesting that signaling mechanisms regulating analgesic effectiveness of intrathecally applied EM2 also pertain to endogenous EM2. Regulation of spinal EM2 antinociception by both the nature of the endogenous mGluR 1 activator (i.e., endogenous biased agonism at mGluR 1 ) and changes in spinal dynorphin/KOR signaling represent a novel mechanism for modulating analgesic responsiveness to endogenous EM2 (and perhaps other opioids). This points the way for developing noncanonical pharmacological approaches to pain management by harnessing endogenous opioids for pain relief. SIGNIFICANCE STATEMENT The current prescription opioid abuse epidemic underscores the urgency to develop alternative pharmacotherapies for managing pain. We find that the magnitude of spinal endomorphin 2 (EM2) antinociception not only

  13. Signal interaction of Hedgehog/GLI and epidermal growth factor receptor signaling in cancer development

    International Nuclear Information System (INIS)

    Eberl, M.

    2012-01-01

    The subject of this PhD thesis is based on the cooperation of Hedgehog (HH)/GLI with epidermal growth factor receptor (EGFR) signaling synergistically promoting oncogenic transformation and cancer growth. In previous studies we have demonstrated that the HH/GLI and EGFR signaling pathways interact synergistically resulting not only in selective induction of HH/GLI-EGFR target genes, but also in the onset of oncogenic transformation and tumor formation (Kasper, Schnidar et al. 2006; Schnidar, Eberl et al. 2009). However, the molecular key mediators acting downstream of HH/GLI and EGFR signal cooperation were largely unknown and the in vivo evidence for the therapeutic relevance of HH/GLI and EGFR signal cooperation in HH-associated cancers was lacking. During my PhD thesis I could demonstrate that the integration of EGFR and HH/GLI signaling involves activation of RAS/MEK/ERK and JUN/AP1 signaling in response to EGFR activation. Furthermore I succeeded in identifying genes, including stem cell- (SOX2, SOX9), tumor growth- (JUN, TGFA, FGF19) and metastasis-associated genes (SPP1/osteopontin, CXCR4) that showed synergistic transcriptional activation by HH/GLI-EGFR signal integration. Importantly, I could demonstrate that these genes arrange themselves within a stable interdependent signaling network, which is required for in vivo growth of basal cell carcinoma (BCC) and tumor-initiating pancreatic cancer cells. These data validate EGFR signaling as additional drug target in HH/GLI driven cancers and provide new therapeutic strategies based on combined targeting of cooperative HH/GLI-EGFR signaling and selected downstream target genes (Eberl, Klingler et al. 2012). (author) [de

  14. Cross-talk between an activator of nuclear receptors-mediated transcription and the D1 dopamine receptor signaling pathway.

    Science.gov (United States)

    Schmidt, Azriel; Vogel, Robert; Rutledge, Su Jane; Opas, Evan E; Rodan, Gideon A; Friedman, Eitan

    2005-03-01

    Nuclear receptors are transcription factors that usually interact, in a ligand-dependent manner, with specific DNA sequences located within promoters of target genes. The nuclear receptors can also be controlled in a ligand-independent manner via the action of membrane receptors and cellular signaling pathways. 5-Tetradecyloxy-2-furancarboxylic acid (TOFA) was shown to stimulate transcription from the MMTV promoter via chimeric receptors that consist of the DNA binding domain of GR and the ligand binding regions of the PPARbeta or LXRbeta nuclear receptors (GR/PPARbeta and GR/LXRbeta). TOFA and hydroxycholesterols also modulate transcription from NF-kappaB- and AP-1-controlled reporter genes and induce neurite differentiation in PC12 cells. In CV-1 cells that express D(1) dopamine receptors, D(1) dopamine receptor stimulation was found to inhibit TOFA-stimulated transcription from the MMTV promoter that is under the control of chimeric GR/PPARbeta and GR/LXRbeta receptors. Treatment with the D(1) dopamine receptor antagonist, SCH23390, prevented dopamine-mediated suppression of transcription, and by itself increased transcription controlled by GR/LXRbeta. Furthermore, combined treatment of CV-1 cells with TOFA and SCH23390 increased transcription controlled by the GR/LXRbeta chimeric receptor synergistically. The significance of this in vitro synergy was demonstrated in vivo, by the observation that SCH23390 (but not haloperidol)-mediated catalepsy in rats was potentiated by TOFA, thus showing that an agent that mimics the in vitro activities of compounds that activate members of the LXR and PPAR receptor families can influence D1 dopamine receptor elicited responses.

  15. Heterogeneity of genomic fusion of BCR and ABL in Philadelphia chromosome-positive acute lymphoblastic leukemia

    International Nuclear Information System (INIS)

    Rubin, C.M.; Carrino, J.J.; Dickler, M.N.; Leibowitz, D.; Smith, S.D.; Westbrook, C.A.

    1988-01-01

    Philadelphia chromosome-positive acute lymphoblastic leukemia occurs in two molecular forms, those with and those without rearrangement of the breakpoint cluster region on chromosome 22. The molecular abnormality in the former group is similar to that found in chronic myelogenous leukemia. To characterize the abnormality in the breakpoint cluster region-unrearranged form, the authors have mapped a 9; 22 translocation from the Philadelphia chromosome-positive acute lymphoblastic leukemia cell line SUP-B13 by using pulsed-field gel electrophoresis and have cloned the DNA at the translocation junctions. They demonstrate a BCR-ABL fusion gene on the Philadelphia chromosome. The exons from ABL are the same. Analysis of leukemic cells from four other patients with breakpoint cluster region-unrearranged Philadelphia chromosome-positive acute lymphoblastic leukemia revealed a rearrangement on chromosome 22 close to the breakpoint in SUP-B13 in only one patient. These data indicate that breakpoints do not cluster tightly in this region but are scattered, possibly in a large intron. Given the large size of BCR and the heterogeneity in breakpoint location, detection of BCR rearrangement by standard Southern blot analysis is difficult. Pulsed-field gel electrophoresis should allow detection at the DNA level in every patient and thus will permit clinical correlation of the breakpoint location with prognosis

  16. Signaling cross-talk between peroxisome proliferator-activated receptor/retinoid X receptor and estrogen receptor through estrogen response elements.

    Science.gov (United States)

    Keller, H; Givel, F; Perroud, M; Wahli, W

    1995-07-01

    Peroxisome proliferator-activated receptors (PPARs) and retinoid X receptors (RXRs) are nuclear hormone receptors that are activated by fatty acids and 9-cis-retinoic acid, respectively. PPARs and RXRs form heterodimers that activate transcription by binding to PPAR response elements (PPREs) in the promoter of target genes. The PPREs described thus far consist of a direct tandem repeat of the AGGTCA core element with one intervening nucleotide. We show here that the vitellogenin A2 estrogen response element (ERE) can also function as a PPRE and is bound by a PPAR/RXR heterodimer. Although this heterodimer can bind to several other ERE-related palindromic response elements containing AGGTCA half-sites, only the ERE is able to confer transactivation of test reporter plasmids, when the ERE is placed either close to or at a distance from the transcription initiation site. Examination of natural ERE-containing promoters, including the pS2, very-low-density apolipoprotein II and vitellogenin A2 genes, revealed considerable differences in the binding of PPAR/RXR heterodimers to these EREs. In their natural promoter context, these EREs did not allow transcriptional activation by PPARs/RXRs. Analysis of this lack of stimulation of the vitellogenin A2 promoter demonstrated that PPARs/RXRs bind to the ERE but cannot transactivate due to a nonpermissive promoter structure. As a consequence, PPARs/RXRs inhibit transactivation by the estrogen receptor through competition for ERE binding. This is the first example of signaling cross-talk between PPAR/RXR and estrogen receptor.

  17. Endogenous GAS6 and Mer receptor signaling regulate prostate cancer stem cells in bone marrow.

    Science.gov (United States)

    Jung, Younghun; Decker, Ann M; Wang, Jingcheng; Lee, Eunsohl; Kana, Lulia A; Yumoto, Kenji; Cackowski, Frank C; Rhee, James; Carmeliet, Peter; Buttitta, Laura; Morgan, Todd M; Taichman, Russell S

    2016-05-03

    GAS6 and its receptors (Tryo 3, Axl, Mer or "TAM") are known to play a role in regulating tumor progression in a number of settings. Previously we have demonstrated that GAS6 signaling regulates invasion, proliferation, chemotherapy-induced apoptosis of prostate cancer (PCa) cells. We have also demonstrated that GAS6 secreted from osteoblasts in the bone marrow environment plays a critical role in establishing prostate tumor cell dormancy. Here we investigated the role that endogenous GAS6 and Mer receptor signaling plays in establishing prostate cancer stem cells in the bone marrow microenvironment.We first observed that high levels of endogenous GAS6 are expressed by disseminated tumor cells (DTCs) in the bone marrow, whereas relatively low levels of endogenous GAS6 are expressed in PCa tumors grown in a s.c. Interestingly, elevated levels of endogenous GAS6 were identified in putative cancer stem cells (CSCs, CD133+/CD44+) compared to non-CSCs (CD133-/CD44-) isolated from PCa/osteoblast cocultures in vitro and in DTCs isolated from the bone marrow 24 hours after intracardiac injection. Moreover, we found that endogenous GAS6 expression is associated with Mer receptor expression in growth arrested (G1) PCa cells, which correlates with the increase of the CSC populations. Importantly, we found that overexpression of GAS6 activates phosphorylation of Mer receptor signaling and subsequent induction of the CSC phenotype in vitro and in vivo.Together these data suggest that endogenous GAS6 and Mer receptor signaling contribute to the establishment of PCa CSCs in the bone marrow microenvironment, which may have important implications for targeting metastatic disease.

  18. ITAM-like signalling for efficient phagocytosis : The paradigm of the granulocyte receptor CEACAM3

    OpenAIRE

    Pils, Stefan

    2010-01-01

    Human CEACAM3 is a tailor-made receptor of the innate immune system to fight pathogens exploiting epithelial CEACAM-family members for colonisation and invasion of their host. Previous studies established CEACAM3 as the receptor facilitating rapid phagocytosis and elimination of N. gonorrhoeae by human granulocytes. The studies reported here set out to shed light on the evolution of this highly specialised receptor and the associated signalling machinery.CEACAM3 arose from exon shuffling afte...

  19. Honey bee dopamine and octopamine receptors linked to intracellular calcium signaling have a close phylogenetic and pharmacological relationship.

    Directory of Open Access Journals (Sweden)

    Kyle T Beggs

    Full Text Available BACKGROUND: Three dopamine receptor genes have been identified that are highly conserved among arthropod species. One of these genes, referred to in honey bees as Amdop2, shows a close phylogenetic relationship to the a-adrenergic-like octopamine receptor family. In this study we examined in parallel the functional and pharmacological properties of AmDOP2 and the honey bee octopamine receptor, AmOA1. For comparison, pharmacological properties of the honey bee dopamine receptors AmDOP1 and AmDOP3, and the tyramine receptor AmTYR1, were also examined. METHODOLOGY/PRINCIPAL FINDINGS: Using HEK293 cells heterologously expressing honey bee biogenic amine receptors, we found that activation of AmDOP2 receptors, like AmOA1 receptors, initiates a rapid increase in intracellular calcium levels. We found no evidence of calcium signaling via AmDOP1, AmDOP3 or AmTYR1 receptors. AmDOP2- and AmOA1-mediated increases in intracellular calcium were inhibited by 10 µM edelfosine indicating a requirement for phospholipase C-β activity in this signaling pathway. Edelfosine treatment had no effect on AmDOP2- or AmOA1-mediated increases in intracellular cAMP. The synthetic compounds mianserin and epinastine, like cis-(Z-flupentixol and spiperone, were found to have significant antagonist activity on AmDOP2 receptors. All 4 compounds were effective antagonists also on AmOA1 receptors. Analysis of putative ligand binding sites offers a possible explanation for why epinastine acts as an antagonist at AmDOP2 receptors, but fails to block responses mediated via AmDOP1. CONCLUSIONS/SIGNIFICANCE: Our results indicate that AmDOP2, like AmOA1, is coupled not only to cAMP, but also to calcium-signalling and moreover, that the two signalling pathways are independent upstream of phospholipase C-β activity. The striking similarity between the pharmacological properties of these 2 receptors suggests an underlying conservation of structural properties related to receptor

  20. Localization of preferential sites of rearrangement within the BCR gene in Philadelphia chromosome-positive acute lymphoblastic leukemia

    International Nuclear Information System (INIS)

    Denny, C.T.; Shah, N.P.; Ogden, S.; Willman, C.; McConnell, T.; Crist, W.; Carroll, A.; Witte, O.N.

    1989-01-01

    The Philadelphia chromosome associated with acute lymphoblastic leukemia (ALL) has been linked to a hybrid BCR/ABL protein product that differs from that found in chronic myelogenous leukemia. This implies that the molecular structures of the two chromosomal translocations also differ. Localization of translocation breakpoints in Philadelphia chromosome-positive ALL has been impeded due to the only partial characterization of the BCR locus. The authors have isolated the entire 130-kilobase BCR genomic locus from a human cosmid library. They have demonstrated that these breakpoints are all located at the 3' end of the intron around an unusual restriction fragment length polymorphism caused by deletion of a 1-kilobase fragment containing Alu family reiterated sequences. This clustering is unexpected in light of previous theories of rearrangement in Philadelphia chromosome-positive chronic myelogenous leukemia that would have predicted a random dispersion of breakpoints in the first intron in Philadelphia chromosome-positive ALL. The proximity of the translocation breakpoints to this constitutive deletion may indicate shared mechanisms of rearrangement or that such polymorphisms mark areas of the genome prone to recombination

  1. Early BCR-ABL1 Transcript Decline after 1 Month of Tyrosine Kinase Inhibitor Therapy as an Indicator for Treatment Response in Chronic Myeloid Leukemia.

    Directory of Open Access Journals (Sweden)

    Mohamed El Missiry

    Full Text Available In chronic myeloid leukemia (CML, early treatment prediction is important to identify patients with inferior overall outcomes. We examined the feasibility of using reductions in BCR-ABL1 transcript levels after 1 month of tyrosine kinase inhibitor (TKI treatment to predict therapy response. Fifty-two first-line TKI-treated CML patients were included (imatinib n = 26, dasatinib n = 21, nilotinib n = 5, and BCR-ABL1 transcript levels were measured at diagnosis (dg and 1, 3, 6, 12, 18, 24, and 36 months. The fold change of the BCR-ABL1 transcripts at 1 month compared to initial BCR-ABL1 transcript levels was used to indicate early therapy response. In our cohort, 21% of patients had no decrease in BCR-ABL1 transcript levels after 1 month and were classified as poor responders. Surprisingly, these patients had lower BCR-ABL1 transcript levels at dg compared to responders (31% vs. 48%, p = 0.0083. Poor responders also significantly more often had enlarged spleen (55% vs. 15%; p<0.01 and a higher percentage of Ph+ CD34+CD38- cells in the bone marrow (91% vs. 75%, p<0.05. The major molecular response rates were inferior in the poor responders (at 12m 18% vs. 64%, p<0.01; 18m 27% vs. 75%, p<0.01; 24m 55% vs. 87%, p<0.01. In conclusion, early treatment response analysis defines a biologically distinct patient subgroup with inferior long-term outcomes.

  2. Global analysis of gene expression mediated by OX1 orexin receptor signaling in a hypothalamic cell line.

    Directory of Open Access Journals (Sweden)

    Eric Koesema

    Full Text Available The orexins and their cognate G-protein coupled receptors have been widely studied due to their associations with various behaviors and cellular processes. However, the detailed downstream signaling cascades that mediate these effects are not completely understood. We report the generation of a neuronal model cell line that stably expresses the OX1 orexin receptor (OX1 and an RNA-Seq analysis of changes in gene expression seen upon receptor activation. Upon treatment with orexin, several families of related transcription factors are transcriptionally regulated, including the early growth response genes (Egr, the Kruppel-like factors (Klf, and the Nr4a subgroup of nuclear hormone receptors. Furthermore, some of the transcriptional effects observed have also been seen in data from in vivo sleep deprivation microarray studies, supporting the physiological relevance of the data set. Additionally, inhibition of one of the most highly regulated genes, serum and glucocorticoid-regulated kinase 1 (Sgk1, resulted in the diminished orexin-dependent induction of a subset of genes. These results provide new insight into the molecular signaling events that occur during OX1 signaling and support a role for orexin signaling in the stimulation of wakefulness during sleep deprivation studies.

  3. Differential Role of Insulin/IGF-1 Receptor Signaling in Muscle Growth and Glucose Homeostasis

    Directory of Open Access Journals (Sweden)

    Brian T. O’Neill

    2015-05-01

    Full Text Available Insulin and insulin-like growth factor 1 (IGF-1 are major regulators of muscle protein and glucose homeostasis. To determine how these pathways interact, we generated mice with muscle-specific knockout of IGF-1 receptor (IGF1R and insulin receptor (IR. These MIGIRKO mice showed >60% decrease in muscle mass. Despite a complete lack of insulin/IGF-1 signaling in muscle, MIGIRKO mice displayed normal glucose and insulin tolerance. Indeed, MIGIRKO mice showed fasting hypoglycemia and increased basal glucose uptake. This was secondary to decreased TBC1D1 resulting in increased Glut4 and Glut1 membrane localization. Interestingly, overexpression of a dominant-negative IGF1R in muscle induced glucose intolerance in MIGIRKO animals. Thus, loss of insulin/IGF-1 signaling impairs muscle growth, but not whole-body glucose tolerance due to increased membrane localization of glucose transporters. Nonetheless, presence of a dominant-negative receptor, even in the absence of functional IR/IGF1R, induces glucose intolerance, indicating that interactions between these receptors and other proteins in muscle can impair glucose homeostasis.

  4. What do we really know about 5-HT1A receptor signaling in neuronal cells?

    Directory of Open Access Journals (Sweden)

    JENNY LUCY FIEDLER

    2016-11-01

    Full Text Available Serotonin (5-HT is a neurotransmitter that plays an important role in neuronal plasticity. Variations in the levels of 5-HT at the synaptic cleft, expression or dysfunction of serotonin receptors may alter brain development and predispose to various mental diseases. Here, we review the transduction pathways described in various cell types transfected with recombinant 5-HT1A receptor (5-HT1AR, specially contrasting with those findings obtained in neuronal cells. The 5-HT1AR is detected in early stages of neural development and is located in the soma, dendrites and spines of hippocampal neurons. The 5-HT1AR differs from other serotonin receptors because it is coupled to different pathways, depending on the targeted cell. The signaling pathway associated with this receptor is determined by Gα isoforms and some cascades involve βγ signaling. The activity of 5-HT1AR usually promotes a reduction in neuronal excitability and firing, provokes a variation in cAMP and Ca2+, levels which may be linked to specific types of behavior and cognition. Furthermore, evidence indicates that 5-HT1AR induces neuritogesis and synapse formation, probably by modulation of the neuronal cytoskeleton through MAPK and PI3K-Akt signaling pathways. Advances in understanding the actions of 5-HT1AR and its association with different signaling pathways in the central nervous system will reveal their pivotal role in health and disease.

  5. Transformation of an Unclassified Myeloproliferative Neoplasm with a Rare BCR-JAK2 Fusion Transcript Resulting from the Translocation (9;22)(p24;q11)

    OpenAIRE

    A. N. Chamseddine; P. Etancelin; D. Penther; F. Parmentier; C. Kuadjovi; V. Camus; N. Contentin; P. Lenain; C. Bastard; H. Tilly; F. Jardin

    2015-01-01

    BCR-ABL1 negative myeloproliferative neoplasms (MPNs) are known to contain alterations of the tyrosine kinase JAK2 (located on 9p24) that result in constitutive activation of the encoded protein. JAK2 fusions are reported in acute and chronic leukemias of myeloid and lymphoid phenotypes. Here, we report an unclassified case of MPN (MPN-U) showing a t(9;22)(p24;q11), which generates a BCR-JAK2 fusion gene by fusing the BCR at intron 13 to JAK2 at intron 17 on the derivative chromosome 22. Most...

  6. β2-Adrenergic receptor activation mobilizes intracellular calcium via a non-canonical cAMP-independent signaling pathway.

    Science.gov (United States)

    Galaz-Montoya, Monica; Wright, Sara J; Rodriguez, Gustavo J; Lichtarge, Olivier; Wensel, Theodore G

    2017-06-16

    Beta adrenergic receptors (βARs) are G-protein-coupled receptors essential for physiological responses to the hormones/neurotransmitters epinephrine and norepinephrine which are found in the nervous system and throughout the body. They are the targets of numerous widely used drugs, especially in the case of the most extensively studied βAR, β 2 AR, whose ligands are used for asthma and cardiovascular disease. βARs signal through Gα s G-proteins and via activation of adenylyl cyclase and cAMP-dependent protein kinase, but some alternative downstream pathways have also been proposed that could be important for understanding normal physiological functioning of βAR signaling and its disruption in disease. Using fluorescence-based Ca 2+ flux assays combined with pharmacology and gene knock-out methods, we discovered a previously unrecognized endogenous pathway in HEK-293 cells whereby β 2 AR activation leads to robust Ca 2+ mobilization from intracellular stores via activation of phospholipase C and opening of inositol trisphosphate (InsP 3 ) receptors. This pathway did not involve cAMP, Gα s , or Gα i or the participation of the other members of the canonical β 2 AR signaling cascade and, therefore, constitutes a novel signaling mechanism for this receptor. This newly uncovered mechanism for Ca 2+ mobilization by β 2 AR has broad implications for adrenergic signaling, cross-talk with other signaling pathways, and the effects of βAR-directed drugs. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  7. Somato-dendritic localization and signaling by leptin receptors in hypothalamic POMC and AgRP neurons.

    Directory of Open Access Journals (Sweden)

    Sangdeuk Ha

    Full Text Available Leptin acts via neuronal leptin receptors to control energy balance. Hypothalamic pro-opiomelanocortin (POMC and agouti-related peptide (AgRP/Neuropeptide Y (NPY/GABA neurons produce anorexigenic and orexigenic neuropeptides and neurotransmitters, and express the long signaling form of the leptin receptor (LepRb. Despite progress in the understanding of LepRb signaling and function, the sub-cellular localization of LepRb in target neurons has not been determined, primarily due to lack of sensitive anti-LepRb antibodies. Here we applied light microscopy (LM, confocal-laser scanning microscopy (CLSM, and electron microscopy (EM to investigate LepRb localization and signaling in mice expressing a HA-tagged LepRb selectively in POMC or AgRP/NPY/GABA neurons. We report that LepRb receptors exhibit a somato-dendritic expression pattern. We further show that LepRb activates STAT3 phosphorylation in neuronal fibers within several hypothalamic and hindbrain nuclei of wild-type mice and rats, and specifically in dendrites of arcuate POMC and AgRP/NPY/GABA neurons of Leprb (+/+ mice and in Leprb (db/db mice expressing HA-LepRb in a neuron specific manner. We did not find evidence of LepRb localization or STAT3-signaling in axon-fibers or nerve-terminals of POMC and AgRP/NPY/GABA neurons. Three-dimensional serial EM-reconstruction of dendritic segments from POMC and AgRP/NPY/GABA neurons indicates a high density of shaft synapses. In addition, we found that the leptin activates STAT3 signaling in proximity to synapses on POMC and AgRP/NPY/GABA dendritic shafts. Taken together, these data suggest that the signaling-form of the leptin receptor exhibits a somato-dendritic expression pattern in POMC and AgRP/NPY/GABA neurons. Dendritic LepRb signaling may therefore play an important role in leptin's central effects on energy balance, possibly through modulation of synaptic activity via post-synaptic mechanisms.

  8. Lipoprotein receptor LRP1 regulates leptin signaling and energy homeostasis in the adult central nervous system.

    Directory of Open Access Journals (Sweden)

    Qiang Liu

    2011-01-01

    Full Text Available Obesity is a growing epidemic characterized by excess fat storage in adipocytes. Although lipoprotein receptors play important roles in lipid uptake, their role in controlling food intake and obesity is not known. Here we show that the lipoprotein receptor LRP1 regulates leptin signaling and energy homeostasis. Conditional deletion of the Lrp1 gene in the brain resulted in an obese phenotype characterized by increased food intake, decreased energy consumption, and decreased leptin signaling. LRP1 directly binds to leptin and the leptin receptor complex and is required for leptin receptor phosphorylation and Stat3 activation. We further showed that deletion of the Lrp1 gene specifically in the hypothalamus by Cre lentivirus injection is sufficient to trigger accelerated weight gain. Together, our results demonstrate that the lipoprotein receptor LRP1, which is critical in lipid metabolism, also regulates food intake and energy homeostasis in the adult central nervous system.

  9. The certification of the surface density (kg/m sub 2 ) of BCR CRM 038 ('fly ash from pulverised coal') comprised in methyl cellulose films simulating dust charged filters. BCR 128

    Energy Technology Data Exchange (ETDEWEB)

    Griepink, B; Marchandise, H; Colinet, E; Dams, R

    1988-01-01

    BCR CRM 38 ('Fly Ash') has been embedded in a stable methylcellulose foil of about 10{mu}m thickness. This has been done by making a slurry of fly ash and methylcellulose in water and spiking it with {sup 42}K as a radioactive tracer. The slurry was spread over glass plates and allowed to dry. 'Filters' of uniform size were punched out of the foil. The surface density (e.g. {mu}g/cm{sup 2}) of the fly ash was calculated for each individual 'filter' by comparing the {sup 42}K-activity of the filter with that of the initial suspension. The uncertainties in the so obtained surface density of random and of systematic origin have been estimated. The total relative uncertainty in the surface density of every element certified in BCR 38 is 4-6%. Studies of stability and homogeneity have revealed that this material is well suited for the verification of the results of non-destructive analytical techniques for dust components.

  10. A novel insulinotropic mechanism of whole grain-derived γ-oryzanol via the suppression of local dopamine D2 receptor signalling in mouse islet.

    Science.gov (United States)

    Kozuka, Chisayo; Sunagawa, Sumito; Ueda, Rei; Higa, Moritake; Ohshiro, Yuzuru; Tanaka, Hideaki; Shimizu-Okabe, Chigusa; Takayama, Chitoshi; Matsushita, Masayuki; Tsutsui, Masato; Ishiuchi, Shogo; Nakata, Masanori; Yada, Toshihiko; Miyazaki, Jun-Ichi; Oyadomari, Seiichi; Shimabukuro, Michio; Masuzaki, Hiroaki

    2015-07-03

    γ-Oryzanol, derived from unrefined rice, attenuated the preference for dietary fat in mice, by decreasing hypothalamic endoplasmic reticulum stress. However, no peripheral mechanisms, whereby γ-oryzanol could ameliorate glucose dyshomeostasis were explored. Dopamine D 2 receptor signalling locally attenuates insulin secretion in pancreatic islets, presumably via decreased levels of intracellular cAMP. We therefore hypothesized that γ-oryzanol would improve high-fat diet (HFD)-induced dysfunction of islets through the suppression of local D 2 receptor signalling. Glucose metabolism and regulation of molecules involved in D 2 receptor signalling in pancreatic islets were investigated in male C57BL/6J mice, fed HFD and treated with γ-oryzanol . In isolated murine islets and the beta cell line, MIN6 , the effects of γ-oryzanol on glucose-stimulated insulin secretion (GSIS) was analysed using siRNA for D 2 receptors and a variety of compounds which alter D 2 receptor signalling. In islets, γ-oryzanol enhanced GSIS via the activation of the cAMP/PKA pathway. Expression of molecules involved in D 2 receptor signalling was increased in islets from HFD-fed mice, which were reciprocally decreased by γ-oryzanol. Experiments with siRNA for D 2 receptors and D 2 receptor ligands in vitro suggest that γ-oryzanol suppressed D 2 receptor signalling and augmented GSIS. γ-Oryzanol exhibited unique anti-diabetic properties. The unexpected effects of γ-oryzanol on D 2 receptor signalling in islets may provide a novel; natural food-based, approach to anti-diabetic therapy. © 2015 The British Pharmacological Society.

  11. DMPD: Mechanisms of selection mediated by interleukin-7, the preBCR, and hemokinin-1during B-cell development. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 14962188 Mechanisms of selection mediated by interleukin-7, the preBCR, and hemokin...ng) (.svg) (.html) (.csml) Show Mechanisms of selection mediated by interleukin-7, the preBCR, and hemokinin...-1during B-cell development. PubmedID 14962188 Title Mechanisms of selection medi

  12. Eye on the B-ALL: B-cell receptor repertoires reveal persistence of numerous B-lymphoblastic leukemia subclones from diagnosis to relapse

    Science.gov (United States)

    Bashford-Rogers, R J M; Nicolaou, K A; Bartram, J; Goulden, N J; Loizou, L; Koumas, L; Chi, J; Hubank, M; Kellam, P; Costeas, P A; Vassiliou, G S

    2016-01-01

    The strongest predictor of relapse in B-cell acute lymphoblastic leukemia (B-ALL) is the level of persistence of tumor cells after initial therapy. The high mutation rate of the B-cell receptor (BCR) locus allows high-resolution tracking of the architecture, evolution and clonal dynamics of B-ALL. Using longitudinal BCR repertoire sequencing, we find that the BCR undergoes an unexpectedly high level of clonal diversification in B-ALL cells through both somatic hypermutation and secondary rearrangements, which can be used for tracking the subclonal composition of the disease and detect minimal residual disease with unprecedented sensitivity. We go on to investigate clonal dynamics of B-ALL using BCR phylogenetic analyses of paired diagnosis-relapse samples and find that large numbers of small leukemic subclones present at diagnosis re-emerge at relapse alongside a dominant clone. Our findings suggest that in all informative relapsed patients, the survival of large numbers of clonogenic cells beyond initial chemotherapy is a surrogate for inherent partial chemoresistance or inadequate therapy, providing an increased opportunity for subsequent emergence of fully resistant clones. These results frame early cytoreduction as an important determinant of long-term outcome. PMID:27211266

  13. Exponential signaling gain at the receptor level enhances signal-to-noise ratio in bacterial chemotaxis.

    Directory of Open Access Journals (Sweden)

    Silke Neumann

    Full Text Available Cellular signaling systems show astonishing precision in their response to external stimuli despite strong fluctuations in the molecular components that determine pathway activity. To control the effects of noise on signaling most efficiently, living cells employ compensatory mechanisms that reach from simple negative feedback loops to robustly designed signaling architectures. Here, we report on a novel control mechanism that allows living cells to keep precision in their signaling characteristics - stationary pathway output, response amplitude, and relaxation time - in the presence of strong intracellular perturbations. The concept relies on the surprising fact that for systems showing perfect adaptation an exponential signal amplification at the receptor level suffices to eliminate slowly varying multiplicative noise. To show this mechanism at work in living systems, we quantified the response dynamics of the E. coli chemotaxis network after genetically perturbing the information flux between upstream and downstream signaling components. We give strong evidence that this signaling system results in dynamic invariance of the activated response regulator against multiplicative intracellular noise. We further demonstrate that for environmental conditions, for which precision in chemosensing is crucial, the invariant response behavior results in highest chemotactic efficiency. Our results resolve several puzzling features of the chemotaxis pathway that are widely conserved across prokaryotes but so far could not be attributed any functional role.

  14. Activated factor X signaling via protease-activated receptor 2 suppresses pro-inflammatory cytokine production from LPS-stimulated myeloid cells.

    LENUS (Irish Health Repository)

    Gleeson, Eimear M

    2013-07-19

    Vitamin K-dependent proteases generated in response to vascular injury and infection enable fibrin clot formation, but also trigger distinct immuno-regulatory signaling pathways on myeloid cells. Factor Xa, a protease crucial for blood coagulation, also induces protease-activated receptor-dependent cell signaling. Factor Xa can bind both monocytes and macrophages, but whether factor Xa-dependent signaling stimulates or suppresses myeloid cell cytokine production in response to Toll-like receptor activation is not known. In this study, exposure to factor Xa significantly impaired pro-inflammatory cytokine production from lipopolysaccharide-treated peripheral blood mononuclear cells, THP-1 monocytic cells and murine macrophages. Furthermore, factor Xa inhibited nuclear factor-kappa B activation in THP-1 reporter cells, requiring phosphatidylinositide 3-kinase activity for its anti-inflammatory effect. Active-site blockade, γ-carboxyglutamic acid domain truncation and a peptide mimic of the factor Xa inter-epidermal growth factor-like region prevented factor Xa inhibition of lipopolysaccharide-induced tumour necrosis factor-α release. In addition, factor Xa anti-inflammatory activity was markedly attenuated by the presence of an antagonist of protease-activated receptor 2, but not protease-activated receptor 1. The key role of protease-activated receptor 2 in eliciting factor Xa-dependent anti-inflammatory signaling on macrophages was further underscored by the inability of factor Xa to mediate inhibition of tumour necrosis factor-α and interleukin-6 release from murine bone marrow-derived protease-activated receptor 2-deficient macrophages. We also show for the first time that, in addition to protease-activated receptor 2, factor Xa requires a receptor-associated protein-sensitive low-density lipoprotein receptor to inhibit lipopolysaccharide-induced cytokine production. Collectively, this study supports a novel function for factor Xa as an endogenous, receptor

  15. Disruption of in vivo chronic lymphocytic leukemia tumor-microenvironment interactions by ibrutinib - findings from an investigator initiated phase 2 study

    DEFF Research Database (Denmark)

    Niemann, Carsten U; Herman, Sarah E M; Maric, Irina

    2016-01-01

    PURPOSE: Chronic lymphocytic leukemia (CLL) cells depend on microenvironmental interactions for proliferation and survival that are at least partially mediated through B cell receptor (BCR) signaling. Ibrutinib, a Bruton's tyrosine kinase inhibitor, disrupts BCR signaling and leads to the egress...... of tumor cells from the microenvironment. While the on-target effects on CLL cells are well defined, the impact on the microenvironment is less well studied. We therefore sought to characterize the in vivo effects of ibrutinib on the tumor microenvironment. EXPERIMENTAL DESIGN: Patients received single...... agent ibrutinib on an investigator-initiated phase 2 trial. Serial blood and tissue samples were collected pre-treatment and during treatment. Changes in cytokine levels, cellular subsets and microenvironmental interactions were assessed. RESULTS: Serum levels of key chemokines and inflammatory...

  16. Role of type I interferon receptor signaling on NK cell development and functions.

    Directory of Open Access Journals (Sweden)

    Jean Guan

    Full Text Available Type I interferons (IFN are unique cytokines transcribed from intronless genes. They have been extensively studied because of their anti-viral functions. The anti-viral effects of type I IFN are mediated in part by natural killer (NK cells. However, the exact contribution of type I IFN on NK cell development, maturation and activation has been somewhat difficult to assess. In this study, we used a variety of approaches to define the consequences of the lack of type I interferon receptor (IFNAR signaling on NK cells. Using IFNAR deficient mice, we found that type I IFN affect NK cell development at the pre-pro NK stage. We also found that systemic absence of IFNAR signaling impacts NK cell maturation with a significant increase in the CD27+CD11b+ double positive (DP compartment in all organs. However, there is tissue specificity, and only in liver and bone marrow is the maturation defect strictly dependent on cell intrinsic IFNAR signaling. Finally, using adoptive transfer and mixed bone marrow approaches, we also show that cell intrinsic IFNAR signaling is not required for NK cell IFN-γ production in the context of MCMV infection. Taken together, our studies provide novel insights on how type I IFN receptor signaling regulates NK cell development and functions.

  17. Signal Peptide and Denaturing Temperature are Critical Factors for Efficient Mammalian Expression and Immunoblotting of Cannabinoid Receptors*

    Science.gov (United States)

    WANG, Chenyun; WANG, Yingying; WANG, Miao; CHEN, Jiankui; YU, Nong; SONG, Shiping; KAMINSKI, Norbert E.; ZHANG, Wei

    2013-01-01

    Summary Many researchers employed mammalian expression system to artificially express cannabinoid receptors, but immunoblot data that directly prove efficient protein expression can hardly be seen in related research reports. In present study, we demonstrated cannabinoid receptor protein was not able to be properly expressed with routine mammalian expression system. This inefficient expression was rescued by endowing an exogenous signal peptide ahead of cannabinoid receptor peptide. In addition, the artificially synthesized cannabinoid receptor was found to aggregate under routine sample denaturing temperatures (i.e., ≥95°C), forming a large molecular weight band when analyzed by immunoblotting. Only denaturing temperatures ≤75°C yielded a clear band at the predicted molecular weight. Collectively, we showed that efficient mammalian expression of cannabinoid receptors need a signal peptide sequence, and described the requirement for a low sample denaturing temperature in immunoblot analysis. These findings provide very useful information for efficient mammalian expression and immunoblotting of membrane receptors. PMID:22528237

  18. Transmembrane signalling at the epidermal growth factor receptor. Positive regulation by the C-terminal phosphotyrosine residues

    DEFF Research Database (Denmark)

    Magni, M; Pandiella, A; Helin, K

    1991-01-01

    a positive role in the regulation of transmembrane signalling at the EGF receptor. The stepwise decrease in signal generation observed in single, double and triple point mutants suggest that the role of phosphotyrosine residues is not in the participation in specific amino acid sequences, but rather...... in the double and the triple mutants. In the latter mutant, expression of the EGF-receptor-activated lipolytic enzyme phospholipase C gamma was unchanged, whereas its tyrosine phosphorylation induced by the growth factor was lowered to approx. 25% of that in the controls. In all of the cell clones employed......, the accumulation of inositol phosphates induced by treatment with fetal calf serum varied only slightly, whereas the same effect induced by EGF was consistently lowered in those lines expressing mutated receptors. This decrease was moderate for those receptors missing only the distal tyrosine (point and deletion...

  19. Dopamine D2L receptor-interacting proteins regulate dopaminergic signaling

    Directory of Open Access Journals (Sweden)

    Norifumi Shioda

    2017-10-01

    Full Text Available Dopamine receptor family proteins include seven transmembrane and trimeric GTP-binding protein-coupled receptors (GPCRs. Among them, the dopamine D2 receptor (D2R is most extensively studied. All clinically used antipsychotic drugs serve as D2R antagonists in the mesolimbic dopamine system, and their ability to block D2R signaling is positively correlated with antipsychotic efficiency. Human genetic studies also show a significant association of DRD2 polymorphisms with disorders including schizophrenia and Parkinson's disease. D2R exists as two alternatively spliced isoforms, the long isoform (D2LR and the short isoform (D2SR, which differ in a 29-amino acid (AA insert in the third cytoplasmic loop. Importantly, previous reports demonstrate functional diversity between the two isoforms in humans. In this review, we focus on binding proteins that specifically interact with the D2LR 29AA insert. We discuss how D2R activities are mediated not only by heterotrimeric G proteins but by D2LR-interacting proteins, which in part regulate diverse D2R activities. Keywords: Dopamine D2L receptor, Antipsychotic drugs, DRD2 polymorphisms, Alternatively spliced isoforms, D2LR-interacting proteins

  20. The brassinosteroid receptor BRI1 can generate cGMP enabling cGMP-dependent downstream signaling

    KAUST Repository

    Wheeler, Janet I.

    2017-05-08

    The brassinosteroid receptor BRASSINOSTEROID INSENSITIVE 1 (BRI1) is a member of the leucine rich repeat receptor like kinase family. The intracellular kinase domain of BRI1 is an active kinase and also encapsulates a guanylate cyclase catalytic centre. Using liquid chromatography tandem mass spectrometry, we confirmed that the recombinant cytoplasmic domain of BRI1 generates pmol amounts of cGMP per μg protein with a preference for magnesium over manganese as a co-factor. Importantly, a functional BRI1 kinase is essential for optimal cGMP generation. Therefore, the guanylate cyclase activity of BRI1 is modulated by the kinase while cGMP, the product of the guanylate cyclase, in turn inhibits BRI1 kinase activity. Furthermore, we show using Arabidopsis root cell cultures that cGMP rapidly potentiates phosphorylation of the downstream substrate BRASSINOSTEROID SIGNALING KINASE 1 (BSK1). Taken together, our results suggest that cGMP acts as a modulator that enhances downstream signaling while dampening signal generation from the receptor. This article is protected by copyright. All rights reserved.

  1. The brassinosteroid receptor BRI1 can generate cGMP enabling cGMP-dependent downstream signaling

    KAUST Repository

    Wheeler, Janet I.; Wong, Aloysius Tze; Marondedze, Claudius; Groen, Arnoud J.; Kwezi, Lusisizwe; Freihat, Lubna; Vyas, Jignesh; Raji, Misjudeen; Irving, Helen R.; Gehring, Christoph A

    2017-01-01

    The brassinosteroid receptor BRASSINOSTEROID INSENSITIVE 1 (BRI1) is a member of the leucine rich repeat receptor like kinase family. The intracellular kinase domain of BRI1 is an active kinase and also encapsulates a guanylate cyclase catalytic centre. Using liquid chromatography tandem mass spectrometry, we confirmed that the recombinant cytoplasmic domain of BRI1 generates pmol amounts of cGMP per μg protein with a preference for magnesium over manganese as a co-factor. Importantly, a functional BRI1 kinase is essential for optimal cGMP generation. Therefore, the guanylate cyclase activity of BRI1 is modulated by the kinase while cGMP, the product of the guanylate cyclase, in turn inhibits BRI1 kinase activity. Furthermore, we show using Arabidopsis root cell cultures that cGMP rapidly potentiates phosphorylation of the downstream substrate BRASSINOSTEROID SIGNALING KINASE 1 (BSK1). Taken together, our results suggest that cGMP acts as a modulator that enhances downstream signaling while dampening signal generation from the receptor. This article is protected by copyright. All rights reserved.

  2. Transmembrane signal transduction by peptide hormones via family B G protein-coupled receptors

    Directory of Open Access Journals (Sweden)

    Kelly J Culhane

    2015-11-01

    Full Text Available Although family B G protein-coupled receptors (GPCRs contain only 15 members, they play key roles in transmembrane signal transduction of hormones. Family B GPCRs are drug targets for developing therapeutics for diseases ranging from metabolic to neurological disorders. Despite their importance, the molecular mechanism of activation of family B GPCRs remains largely unexplored due to the challenges in expression and purification of functional receptors to the quantity for biophysical characterization. Currently, there is no crystal structure available of a full-length family B GPCR. However, structures of key domains, including the extracellular ligand binding regions and seven-helical transmembrane regions, have been solved by X-ray crystallography and NMR, providing insights into the mechanisms of ligand recognition and selectivity, and helical arrangements within the cell membrane. Moreover, biophysical and biochemical methods have been used to explore functions, key residues for signaling, and the kinetics and dynamics of signaling processes. This review summarizes the current knowledge of the signal transduction mechanism of family B GPCRs at the molecular level and comments on the challenges and outlook for mechanistic studies of family B GPCRs.

  3. A threshold model for receptor tyrosine kinase signaling specificity and cell fate determination [version 1; referees: 4 approved

    Directory of Open Access Journals (Sweden)

    Allen Zinkle

    2018-06-01

    Full Text Available Upon ligand engagement, the single-pass transmembrane receptor tyrosine kinases (RTKs dimerize to transmit qualitatively and quantitatively different intracellular signals that alter the transcriptional landscape and thereby determine the cellular response. The molecular mechanisms underlying these fundamental events are not well understood. Considering recent insights into the structural biology of fibroblast growth factor signaling, we propose a threshold model for RTK signaling specificity in which quantitative differences in the strength/longevity of ligand-induced receptor dimers on the cell surface lead to quantitative differences in the phosphorylation of activation loop (A-loop tyrosines as well as qualitative differences in the phosphorylation of tyrosines mediating substrate recruitment. In this model, quantitative differences on A-loop tyrosine phosphorylation result in gradations in kinase activation, leading to the generation of intracellular signals of varying amplitude/duration. In contrast, qualitative differences in the pattern of tyrosine phosphorylation on the receptor result in the recruitment/activation of distinct substrates/intracellular pathways. Commensurate with both the dynamics of the intracellular signal and the types of intracellular pathways activated, unique transcriptional signatures are established. Our model provides a framework for engineering clinically useful ligands that can tune receptor dimerization stability so as to bias the cellular transcriptome to achieve a desired cellular output.

  4. Expression of p210 BCR/ABl increases hematopoietic progenitor cell radiosensitivity

    International Nuclear Information System (INIS)

    Santucci, M.A.; Anklesaria, P.; Das, I.J.; Sakakeeny, M.A.; FitzGerald, T.J.; Greenberger, J.S.; Laneuville, P.

    1993-01-01

    The cytogenetic finding of the Ph1+ chromosome and its molecular biologic marker bcr/abl gene rearrangement in cells from patients with chronic myeloid leukemia are associated with a proliferative advantage of the Ph1+ clone in vivo. Although the transition to the acute terminal phase or blastic crisis is often associated with additional cytogenetic abnormalities, the molecular events which correlate the initial cytogenetic lesion with the terminal phase are poorly understood. Defective cellular DNA repair capacity is often associated with chromosomal instability, increased mutation frequency, and biologic alterations. The authors tested whether the protein product of the bcr/abl translocation (p210) could alter DNA repair after gamma-irradiation of murine cell lines expressing the bcr/abl cDNA. The 32D cl 3 parent, 32D cl 3 pYN (containing the control vector plasmid) and each of two sources of 32D cl 3 cells expressing p210 cDNA (32D-PC1 cell line and 32D-LG7 subclone) showed a D 0 of 1.62, 1.57, 1.16, and 1.27 Gy, respectively. Thus, expression of the p210 product induced a significant increase in radiosensitivity at the clinically relevant radiation therapy dose-rate. The increased radiosensitivity of p210-expressing cells persisted if cells were held before plating in a density-inhibited state for 8 hr after gamma-irradiation, indicating little effect on the repair of potentially lethal gamma-irradiation damage. The IL-3 dependent parent 32D cl 3 cells demonstrated programmed cell death in the absence of growth factor or following gamma-irradiation to 200 cGy. Expression of p210 cDNA in the 32D-PC1 and 32D-LG7 subclones abrogated IL-3 requirement of these cell lines and inhibited gamma-irradiation induced programmed cell death. These data suggest a role for p210 in amplifying gamma-irradiation DNA damage or broadly inhibiting DNA repair, conditions that may stimulate further cytogenetic alterations in hematopoietic cells. 43 refs., 3 figs., 1 tab

  5. Regulation of protease-activated receptor 1 signaling by the adaptor protein complex 2 and R4 subfamily of regulator of G protein signaling proteins.

    Science.gov (United States)

    Chen, Buxin; Siderovski, David P; Neubig, Richard R; Lawson, Mark A; Trejo, Joann

    2014-01-17

    The G protein-coupled protease-activated receptor 1 (PAR1) is irreversibly proteolytically activated by thrombin. Hence, the precise regulation of PAR1 signaling is important for proper cellular responses. In addition to desensitization, internalization and lysosomal sorting of activated PAR1 are critical for the termination of signaling. Unlike most G protein-coupled receptors, PAR1 internalization is mediated by the clathrin adaptor protein complex 2 (AP-2) and epsin-1, rather than β-arrestins. However, the function of AP-2 and epsin-1 in the regulation of PAR1 signaling is not known. Here, we report that AP-2, and not epsin-1, regulates activated PAR1-stimulated phosphoinositide hydrolysis via two different mechanisms that involve, in part, a subset of R4 subfamily of "regulator of G protein signaling" (RGS) proteins. A significantly greater increase in activated PAR1 signaling was observed in cells depleted of AP-2 using siRNA or in cells expressing a PAR1 (420)AKKAA(424) mutant with defective AP-2 binding. This effect was attributed to AP-2 modulation of PAR1 surface expression and efficiency of G protein coupling. We further found that ectopic expression of R4 subfamily members RGS2, RGS3, RGS4, and RGS5 reduced activated PAR1 wild-type signaling, whereas signaling by the PAR1 AKKAA mutant was minimally affected. Intriguingly, siRNA-mediated depletion analysis revealed a function for RGS5 in the regulation of signaling by the PAR1 wild type but not the AKKAA mutant. Moreover, activation of the PAR1 wild type, and not the AKKAA mutant, induced Gαq association with RGS3 via an AP-2-dependent mechanism. Thus, AP-2 regulates activated PAR1 signaling by altering receptor surface expression and through recruitment of RGS proteins.

  6. DESENSITIZATION PROPERTIES OF P2X3 RECEPTORS SHAPING PAIN SIGNALLING

    Directory of Open Access Journals (Sweden)

    Rashid eGiniatullin

    2013-12-01

    Full Text Available ATP-gated P2X3 receptors are mostly expressed by nociceptive sensory neurons and participate in transduction of pain signals. P2X3 receptors show a combination of fast desensitization onset and slow recovery. Moreover, even low nanomolar agonist concentrations unable to evoke a response, can induce desensitization via a phenomenon called ‘high affinity desensitization’. We have also observed that recovery from desensitization is agonist-specific and can range from seconds to minutes. The recovery process displays unusually high temperature dependence. Likewise, recycling of P2X3 receptors in peri-membrane regions shows unexpectedly large temperature sensitivity. By applying kinetic modeling, we have previously shown that desensitization characteristics of P2X3 receptor are best explained with a cyclic model of receptor operation involving three agonist molecules binding a single receptor and that desensitization is primarily developing from the open receptor state. Mutagenesis experiments suggested that desensitization depends on a certain conformation of the ATP binding pocket and on the structure of the transmembrane domains forming the ion pore. Further molecular determinants of desensitization have been identified by mutating the intracellular N- and C-termini of P2X3 receptor. Unlike other P2X receptors, the P2X3 subtype is facilitated by extracellular calcium that acts via specific sites in the ectodomain neighboring the ATP binding pocket. Thus, substitution of serine275 in this region (called ‘left flipper’ converts the natural facilitation induced by extracellular calcium to receptor inhibition. Given such their strategic location in nociceptive neurons and unique desensitization properties, P2X3 receptors represent an attractive target for development of new analgesic drugs via promotion of desensitization aimed at suppressing chronic pain.

  7. Small molecule ErbB inhibitors decrease proliferative signaling and promote apoptosis in philadelphia chromosome-positive acute lymphoblastic leukemia.

    Directory of Open Access Journals (Sweden)

    Mary E Irwin

    Full Text Available The presence of the Philadelphia chromosome in patients with acute lymphoblastic leukemia (Ph(+ALL is a negative prognostic indicator. Tyrosine kinase inhibitors (TKI that target BCR/ABL, such as imatinib, have improved treatment of Ph(+ALL and are generally incorporated into induction regimens. This approach has improved clinical responses, but molecular remissions are seen in less than 50% of patients leaving few treatment options in the event of relapse. Thus, identification of additional targets for therapeutic intervention has potential to improve outcomes for Ph+ALL. The human epidermal growth factor receptor 2 (ErbB2 is expressed in ~30% of B-ALLs, and numerous small molecule inhibitors are available to prevent its activation. We analyzed a cohort of 129 ALL patient samples using reverse phase protein array (RPPA with ErbB2 and phospho-ErbB2 antibodies and found that activity of ErbB2 was elevated in 56% of Ph(+ALL as compared to just 4.8% of Ph(-ALL. In two human Ph+ALL cell lines, inhibition of ErbB kinase activity with canertinib resulted in a dose-dependent decrease in the phosphorylation of an ErbB kinase signaling target p70S6-kinase T389 (by 60% in Z119 and 39% in Z181 cells at 3 µM. Downstream, phosphorylation of S6-kinase was also diminished in both cell lines in a dose-dependent manner (by 91% in both cell lines at 3 µM. Canertinib treatment increased expression of the pro-apoptotic protein Bim by as much as 144% in Z119 cells and 49% in Z181 cells, and further produced caspase-3 activation and consequent apoptotic cell death. Both canertinib and the FDA-approved ErbB1/2-directed TKI lapatinib abrogated proliferation and increased sensitivity to BCR/ABL-directed TKIs at clinically relevant doses. Our results suggest that ErbB signaling is an additional molecular target in Ph(+ALL and encourage the development of clinical strategies combining ErbB and BCR/ABL kinase inhibitors for this subset of ALL patients.

  8. Neurotrophin Signaling via TrkB and TrkC Receptors Promotes the Growth of Brain Tumor-initiating Cells*

    Science.gov (United States)

    Lawn, Samuel; Krishna, Niveditha; Pisklakova, Alexandra; Qu, Xiaotao; Fenstermacher, David A.; Fournier, Michelle; Vrionis, Frank D.; Tran, Nam; Chan, Jennifer A.; Kenchappa, Rajappa S.; Forsyth, Peter A.

    2015-01-01

    Neurotrophins and their receptors are frequently expressed in malignant gliomas, yet their functions are largely unknown. Previously, we have shown that p75 neurotrophin receptor is required for glioma invasion and proliferation. However, the role of Trk receptors has not been examined. In this study, we investigated the importance of TrkB and TrkC in survival of brain tumor-initiating cells (BTICs). Here, we show that human malignant glioma tissues and also tumor-initiating cells isolated from fresh human malignant gliomas express the neurotrophin receptors TrkB and TrkC, not TrkA, and they also express neurotrophins NGF, BDNF, and neurotrophin 3 (NT3). Specific activation of TrkB and TrkC receptors by ligands BDNF and NT3 enhances tumor-initiating cell viability through activation of ERK and Akt pathways. Conversely, TrkB and TrkC knockdown or pharmacologic inhibition of Trk signaling decreases neurotrophin-dependent ERK activation and BTIC growth. Further, pharmacological inhibition of both ERK and Akt pathways blocked BDNF, and NT3 stimulated BTIC survival. Importantly, attenuation of BTIC growth by EGFR inhibitors could be overcome by activation of neurotrophin signaling, and neurotrophin signaling is sufficient for long term BTIC growth as spheres in the absence of EGF and FGF. Our results highlight a novel role for neurotrophin signaling in brain tumor and suggest that Trks could be a target for combinatorial treatment of malignant glioma. PMID:25538243

  9. Neurotrophin signaling via TrkB and TrkC receptors promotes the growth of brain tumor-initiating cells.

    Science.gov (United States)

    Lawn, Samuel; Krishna, Niveditha; Pisklakova, Alexandra; Qu, Xiaotao; Fenstermacher, David A; Fournier, Michelle; Vrionis, Frank D; Tran, Nam; Chan, Jennifer A; Kenchappa, Rajappa S; Forsyth, Peter A

    2015-02-06

    Neurotrophins and their receptors are frequently expressed in malignant gliomas, yet their functions are largely unknown. Previously, we have shown that p75 neurotrophin receptor is required for glioma invasion and proliferation. However, the role of Trk receptors has not been examined. In this study, we investigated the importance of TrkB and TrkC in survival of brain tumor-initiating cells (BTICs). Here, we show that human malignant glioma tissues and also tumor-initiating cells isolated from fresh human malignant gliomas express the neurotrophin receptors TrkB and TrkC, not TrkA, and they also express neurotrophins NGF, BDNF, and neurotrophin 3 (NT3). Specific activation of TrkB and TrkC receptors by ligands BDNF and NT3 enhances tumor-initiating cell viability through activation of ERK and Akt pathways. Conversely, TrkB and TrkC knockdown or pharmacologic inhibition of Trk signaling decreases neurotrophin-dependent ERK activation and BTIC growth. Further, pharmacological inhibition of both ERK and Akt pathways blocked BDNF, and NT3 stimulated BTIC survival. Importantly, attenuation of BTIC growth by EGFR inhibitors could be overcome by activation of neurotrophin signaling, and neurotrophin signaling is sufficient for long term BTIC growth as spheres in the absence of EGF and FGF. Our results highlight a novel role for neurotrophin signaling in brain tumor and suggest that Trks could be a target for combinatorial treatment of malignant glioma. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. Ketamine-induced inhibition of glycogen synthase kinase-3 contributes to the augmentation of AMPA receptor signaling

    Science.gov (United States)

    Beurel, Eléonore; Grieco, Steven F; Amadei, Celeste; Downey, Kimberlee; Jope, Richard S

    2016-01-01

    Objectives Sub-anesthetic doses of ketamine have been found to provide rapid antidepressant actions, indicating that the cellular signaling systems targeted by ketamine are potential sites for therapeutic intervention. Ketamine acts as an antagonist of N-methyl-D-aspartate (NMDA) receptors, and animal studies indicate that subsequent augmentation of signaling by α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors is critical for the antidepressant outcome. Methods In this study, we tested if the inhibitory effect of ketamine on glycogen synthase kinase-3 (GSK3) affected hippocampal cell-surface AMPA receptors using immunoblotting of membrane and synaptosomal extracts from wild-type and GSK3 knockin mice. Results Treatment with an antidepressant dose of ketamine increased the hippocampal membrane level of the AMPA glutamate receptor (GluA)1 subunit, but did not alter the localization of GluA2, GluA3, or GluA4. This effect of ketamine was abrogated in GSK3 knockin mice expressing mutant GSK3 that cannot be inhibited by ketamine, demonstrating that ketamine-induced inhibition of GSK3 is necessary for up-regulation of cell surface AMPA GluA1 subunits. AMPA receptor trafficking is regulated by post-synaptic density-95 (PSD-95), a substrate for GSK3. Ketamine treatment decreased the hippocampal membrane level of phosphorylated PSD-95 on Thr-19, the target of GSK3 that promotes AMPA receptor internalization. Conclusions These results demonstrate that ketamine-induced inhibition of GSK3 causes reduced phosphorylation of PSD-95, diminishing the internalization of AMPA GluA1 subunits to allow for augmented signaling through AMPA receptors following ketamine treatment. PMID:27687706

  11. Large-Scale Phosphoproteomics Reveals Shp-2 Phosphatase-Dependent Regulators of Pdgf Receptor Signaling

    DEFF Research Database (Denmark)

    Batth, Tanveer S; Papetti, Moreno; Pfeiffer, Anamarija

    2018-01-01

    Despite its low cellular abundance, phosphotyrosine (pTyr) regulates numerous cell signaling pathways in health and disease. We applied comprehensive phosphoproteomics to unravel differential regulators of receptor tyrosine kinase (RTK)-initiated signaling networks upon activation by Pdgf-ββ, Fgf-2...... of Pdgfr pTyr signaling. Application of a recently introduced allosteric Shp-2 inhibitor revealed global regulation of the Pdgf-dependent tyrosine phosphoproteome, which significantly impaired cell migration. In addition, we present a list of hundreds of Shp-2-dependent targets and putative substrates...

  12. Neuropeptide Y family receptors traffic via the Bardet-Biedl syndrome pathway to signal in neuronal primary cilia.

    Science.gov (United States)

    Loktev, Alexander V; Jackson, Peter K

    2013-12-12

    Human monogenic obesity syndromes, including Bardet-Biedl syndrome (BBS), implicate neuronal primary cilia in regulation of energy homeostasis. Cilia in hypothalamic neurons have been hypothesized to sense and regulate systemic energy status, but the molecular mechanism of this signaling remains unknown. Here, we report a comprehensive localization screen of 42 G-protein-coupled receptors (GPCR) revealing seven ciliary GPCRs, including the neuropeptide Y (NPY) receptors NPY2R and NPY5R. We show that mice modeling BBS disease or obese tubby mice fail to localize NPY2R to cilia in the hypothalamus and that BBS mutant mice fail to activate c-fos or decrease food intake in response to the NPY2R ligand PYY3-36. We find that cells with ciliary NPY2R show augmented PYY3-36-dependent cAMP signaling. Our data demonstrate that ciliary targeting of NPY receptors is important for controlling energy balance in mammals, revealing a physiologically defined ligand-receptor pathway signaling within neuronal cilia. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

  13. Combined Targeting of BCL-2 and BCR-ABL Tyrosine Kinase Eradicates Chronic Myeloid Leukemia Stem Cells

    Science.gov (United States)

    Mak, Po Yee; Mu, Hong; Zhou, Hongsheng; Mak, Duncan H.; Schober, Wendy; Leverson, Joel D.; Zhang, Bin; Bhatia, Ravi; Huang, Xuelin; Cortes, Jorge; Kantarjian, Hagop; Konopleva, Marina

    2016-01-01

    BCR-ABL tyrosine kinase inhibitors (TKIs) are effective against chronic myeloid leukemia (CML), but they rarely eliminate CML stem cells. Disease relapse is common upon therapy cessation, even in patients with complete molecular responses. Furthermore, once CML progresses to blast crisis (BC), treatment outcomes are dismal. We hypothesized that concomitant targeting of BCL-2 and BCR-ABL tyrosine kinase could overcome these limitations. We demonstrate increased BCL-2 expression at the protein level in bone marrow cells, particularly in Lin−Sca-1+cKit+ cells of inducible CML in mice as determined by CyTOF mass cytometry. Further, selective inhibition of BCL-2, aided by TKI-mediated MCL-1 and BCL-XL inhibition, markedly decreased leukemic Lin−Sca-1+cKit+ cell numbers and long-term stem cell frequency, and prolonged survival in a murine CML model. Additionally, this combination effectively eradicated CD34+CD38−, CD34+CD38+, and quiescent stem/progenitor CD34+ cells from BC CML patient samples. Our results suggest that BCL-2 is a key survival factor for CML stem/progenitor cells and that combined inhibition of BCL-2 and BCR-ABL tyrosine kinase has the potential to significantly improve depth of response and cure rates of chronic phase and BC CML. PMID:27605552

  14. ATAR, a novel tumor necrosis factor receptor family member, signals through TRAF2 and TRAF5.

    Science.gov (United States)

    Hsu, H; Solovyev, I; Colombero, A; Elliott, R; Kelley, M; Boyle, W J

    1997-05-23

    Members of tumor necrosis factor receptor (TNFR) family signal largely through interactions with death domain proteins and TRAF proteins. Here we report the identification of a novel TNFR family member ATAR. Human and mouse ATAR contain 283 and 276 amino acids, respectively, making them the shortest known members of the TNFR superfamily. The receptor is expressed mainly in spleen, thymus, bone marrow, lung, and small intestine. The intracellular domains of human and mouse ATAR share only 25% identity, yet both interact with TRAF5 and TRAF2. This TRAF interaction domain resides at the C-terminal 20 amino acids. Like most other TRAF-interacting receptors, overexpression of ATAR activates the transcription factor NF-kappaB. Co-expression of ATAR with TRAF5, but not TRAF2, results in synergistic activation of NF-kappaB, suggesting potentially different roles for TRAF2 and TRAF5 in post-receptor signaling.

  15. The Role of Stat3 Activation in Androgen Receptor Signaling and Prostate Cancer

    National Research Council Canada - National Science Library

    Gao, Allen C

    2006-01-01

    .... The experiments proposed in this application are based upon the hypothesis that Stat3 activation alters androgen receptor signaling pathways which in turn results in the loss of growth control in prostate cancer cells...

  16. The Role of Stat3 Activation in Androgen Receptor Signaling and Prostate Cancer

    National Research Council Canada - National Science Library

    Gao, Allen

    2004-01-01

    .... The experiments proposed in this application are based upon the hypothesis that stat3 activation alters androgen receptor signaling pathways, that in turn results in the loss of growth control in prostate cancer cells...

  17. The Role of Stat3 Activation in Androgen Receptor Signaling and Prostate Cancer

    National Research Council Canada - National Science Library

    Gao, Allen

    2002-01-01

    .... The experiments proposed in this application are based upon the hypothesis that Stat3 activation alters androgen receptor signaling pathways, that in turn results in the loss of growth control in prostate cancer cells...

  18. The Role of Stat3 Activation in Androgen Receptor Signaling and Prostate Cancer

    National Research Council Canada - National Science Library

    Gao, Allen C

    2005-01-01

    .... The experiments proposed in this application are based upon the hypothesis that Stat3 activation alters androgen receptor signaling pathways, that in turn results in the loss of growth control in prostate cancer cells...

  19. The Role of Stat3 Activation in Androgen Receptor Signaling and Prostate Cancer

    National Research Council Canada - National Science Library

    Gao, Allen

    2003-01-01

    .... The experiments proposed in this application are based upon the hypothesis that Stat3 activation alters androgen receptor signaling pathways, that in turn results in the loss of growth control in prostate cancer cells...

  20. Curcumin blocks interleukin (IL)-2 signaling in T-lymphocytes by inhibiting IL-2 synthesis, CD25 expression, and IL-2 receptor signaling

    International Nuclear Information System (INIS)

    Forward, Nicholas A.; Conrad, David M.; Power Coombs, Melanie R.; Doucette, Carolyn D.; Furlong, Suzanne J.; Lin, Tong-Jun; Hoskin, David W.

    2011-01-01

    Highlights: → Curcumin inhibits CD4 + T-lymphocyte proliferation. → Curcumin inhibits interleukin-2 (IL-2) synthesis and CD25 expression by CD4 + T-lymphocytes. → Curcumin interferes with IL-2 receptor signaling by inhibiting JAK3 and STAT5 phosphorylation. → IL-2-dependent regulatory T-lymphocyte function and Foxp3 expression is downregulated by curcumin. -- Abstract: Curcumin (diferulomethane) is the principal curcuminoid in the spice tumeric and a potent inhibitor of activation-induced T-lymphocyte proliferation; however, the molecular basis of this immunosuppressive effect has not been well studied. Here we show that micromolar concentrations of curcumin inhibited DNA synthesis by mouse CD4 + T-lymphocytes, as well as interleukin-2 (IL-2) and CD25 (α chain of the high affinity IL-2 receptor) expression in response to antibody-mediated cross-linking of CD3 and CD28. Curcumin acted downstream of protein kinase C activation and intracellular Ca 2+ release to inhibit IκB phosphorylation, which is required for nuclear translocation of the transcription factor NFκB. In addition, IL-2-dependent DNA synthesis by mouse CTLL-2 cells, but not constitutive CD25 expression, was impaired in the presence of curcumin, which demonstrated an inhibitory effect on IL-2 receptor (IL-2R) signaling. IL-2-induced phosphorylation of STAT5A and JAK3, but not JAK1, was diminished in the presence of curcumin, indicating inhibition of critical proximal events in IL-2R signaling. In line with the inhibitory action of curcumin on IL-2R signaling, pretreatment of CD4 + CD25 + regulatory T-cells with curcumin downregulated suppressor function, as well as forkhead box p3 (Foxp3) expression. We conclude that curcumin inhibits IL-2 signaling by reducing available IL-2 and high affinity IL-2R, as well as interfering with IL-2R signaling.

  1. A method for the quantification of biased signalling at constitutively active receptors.

    Science.gov (United States)

    Hall, David A; Giraldo, Jesús

    2018-06-01

    Biased agonism, the ability of an agonist to differentially activate one of several signal transduction pathways when acting at a given receptor, is an increasingly recognized phenomenon at many receptors. The Black and Leff operational model lacks a way to describe constitutive receptor activity and hence inverse agonism. Thus, it is impossible to analyse the biased signalling of inverse agonists using this model. In this theoretical work, we develop and illustrate methods for the analysis of biased inverse agonism. Methods were derived for quantifying biased signalling in systems that demonstrate constitutive activity using the modified operational model proposed by Slack and Hall. The methods were illustrated using Monte Carlo simulations. The Monte Carlo simulations demonstrated that, with an appropriate experimental design, the model parameters are 'identifiable'. The method is consistent with methods based on the measurement of intrinsic relative activity (RA i ) (ΔΔlogR or ΔΔlog(τ/K a )) proposed by Ehlert and Kenakin and their co-workers but has some advantages. In particular, it allows the quantification of ligand bias independently of 'system bias' removing the requirement to normalize to a standard ligand. In systems with constitutive activity, the Slack and Hall model provides methods for quantifying the absolute bias of agonists and inverse agonists. This provides an alternative to methods based on RA i and is complementary to the ΔΔlog(τ/K a ) method of Kenakin et al. in systems where use of that method is inappropriate due to the presence of constitutive activity. © 2018 The British Pharmacological Society.

  2. Breast cancer oestrogen independence mediated by BCAR1 or BCAR3 genes is transmitted through mechanisms distinct from the oestrogen receptor signalling pathway or the epidermal growth factor receptor signalling pathway

    International Nuclear Information System (INIS)

    Dorssers, Lambert CJ; Agthoven, Ton van; Brinkman, Arend; Veldscholte, Jos; Smid, Marcel; Dechering, Koen J

    2005-01-01

    Tamoxifen is effective for endocrine treatment of oestrogen receptor-positive breast cancers but ultimately fails due to the development of resistance. A functional screen in human breast cancer cells identified two BCAR genes causing oestrogen-independent proliferation. The BCAR1 and BCAR3 genes both encode components of intracellular signal transduction, but their direct effect on breast cancer cell proliferation is not known. The aim of this study was to investigate the growth control mediated by these BCAR genes by gene expression profiling. We have measured the expression changes induced by overexpression of the BCAR1 or BCAR3 gene in ZR-75-1 cells and have made direct comparisons with the expression changes after cell stimulation with oestrogen or epidermal growth factor (EGF). A comparison with published gene expression data of cell models and breast tumours is made. Relatively few changes in gene expression were detected in the BCAR-transfected cells, in comparison with the extensive and distinct differences in gene expression induced by oestrogen or EGF. Both BCAR1 and BCAR3 regulate discrete sets of genes in these ZR-75-1-derived cells, indicating that the proliferation signalling proceeds along distinct pathways. Oestrogen-regulated genes in our cell model showed general concordance with reported data of cell models and gene expression association with oestrogen receptor status of breast tumours. The direct comparison of the expression profiles of BCAR transfectants and oestrogen or EGF-stimulated cells strongly suggests that anti-oestrogen-resistant cell proliferation is not caused by alternative activation of the oestrogen receptor or by the epidermal growth factor receptor signalling pathway

  3. Kidins220/ARMS as a functional mediator of multiple receptor signalling pathways.

    Science.gov (United States)

    Neubrand, Veronika E; Cesca, Fabrizia; Benfenati, Fabio; Schiavo, Giampietro

    2012-04-15

    An increasing body of evidence suggests that several membrane receptors--in addition to activating distinct signalling cascades--also engage in substantial crosstalk with each other, thereby adjusting their signalling outcome as a function of specific input information. However, little is known about the molecular mechanisms that control their coordination and integration of downstream signalling. A protein that is likely to have a role in this process is kinase-D-interacting substrate of 220 kDa [Kidins220, also known as ankyrin repeat-rich membrane spanning (ARMS), hereafter referred to as Kidins220/ARMS]. Kidins220/ARMS is a conserved membrane protein that is preferentially expressed in the nervous system and interacts with the microtubule and actin cytoskeleton. It interacts with neurotrophin, ephrin, vascular endothelial growth factor (VEGF) and glutamate receptors, and is a common downstream target of several trophic stimuli. Kidins220/ARMS is required for neuronal differentiation and survival, and its expression levels modulate synaptic plasticity. Kidins220/ARMS knockout mice show developmental defects mainly in the nervous and cardiovascular systems, suggesting a crucial role for this protein in modulating the cross talk between different signalling pathways. In this Commentary, we summarise existing knowledge regarding the physiological functions of Kidins220/ARMS, and highlight some interesting directions for future studies on the role of this protein in health and disease.

  4. Correlated cone noise decreases rod signal contributions to the post-receptoral pathways.

    Science.gov (United States)

    Hathibelagal, Amithavikram R; Feigl, Beatrix; Zele, Andrew J

    2018-04-01

    This study investigated how invisible extrinsic temporal white noise that correlates with the activity of one of the three [magnocellular (MC), parvocellular (PC), or koniocellular (KC)] post-receptoral pathways alters mesopic rod signaling. A four-primary photostimulator provided independent control of the rod and three cone photoreceptor excitations. The rod contributions to the three post-receptoral pathways were estimated by perceptually matching a 20% contrast rod pulse by independently varying the LMS (MC pathway), +L-M (PC pathway), and S-cone (KC pathway) excitations. We show that extrinsic cone noise caused a predominant decrease in the overall magnitude and ratio of the rod contributions to each pathway. Thus, the relative cone activity in the post-receptoral pathways determines the relative mesopic rod inputs to each pathway.

  5. Entamoeba histolytica: a beta 1 integrin-like fibronectin receptor assembles a signaling complex similar to those of mammalian cells.

    Science.gov (United States)

    Flores-Robles, Donaciano; Rosales, Carlos; Rosales-Encina, José Luis; Talamás-Rohana, Patricia

    2003-01-01

    During tissue invasion, Entamoeba histolytica trophozoites interact with endothelial cells and extracellular matrix (ECM) proteins such as fibronectin (FN), collagen, and laminin. It has been demonstrated that trophozoites interact with FN through a beta1 integrin-like FN receptor (beta 1EhFNR), activating tyrosine kinases. In order to characterize the signaling process triggered by the amoebic receptor, activation, and association of tyrosine kinases and structural proteins were determined. As a result of FN binding by the beta 1EhFNR, the receptor itself, FAK, and paxillin were phosphorylated in tyrosine. Co-immunoprecipitation experiments showed that a multimolecular signaling complex was formed by the amoebic FN receptor, FAK, paxillin, and vinculin. These results strongly suggest that a signaling pathway, similar to the one used in mammalian cells, is activated when E. histolytica trophozoites adhere to FN.

  6. The receptor locus for escherichia coli F4ab/F4ac in the pig maps distal to the MUC4-LMLN region

    DEFF Research Database (Denmark)

    Rampoldi, Antonio; Jacobsen, Mette Juul; Bertschinger, Hans U.

    2011-01-01

    antigenic variants, F4ab, F4ac, and F4ad, of which F4ac is the most common. Resistance to ETEC F4ab/F4ac adhesion in pigs has been shown to be inherited as an autosomal recessive trait. In previous studies the ETEC F4ab/F4ac receptor locus (F4bcR) was mapped to the q41 region on pig chromosome 13....... A polymorphism within an intron of the mucin 4 (MUC4) gene, which is one of the possible candidate genes located in this region, was shown earlier to cosegregate with the F4bcR alleles. Recently, we discovered a Large White boar from a Swiss experimental herd with a recombination between F4bcR and MUC4. A three...... a newly detected SNP in the leishmanolysin-like gene (LMLN g.15920) and SNP ALGA0072075. In this study the six SNPs ALGA0072075, ALGA0106330, MUC13-226, MUC13-813, DIA0000584, and MARC0006918 were in complete linkage disequilibrium with F4bcR. Based on this finding and earlier investigations, we suggest...

  7. S1P receptor signalling and RGS proteins; expression and function in vascular smooth muscle cells and transfected CHO cells

    NARCIS (Netherlands)

    Hendriks-Balk, Mariëlle C.; van Loenen, Pieter B.; Hajji, Najat; Michel, Martin C.; Peters, Stephan L. M.; Alewijnse, Astrid E.

    2009-01-01

    Sphingosine-1-phosphate (S1P) signalling via G protein-coupled receptors is important for the regulation of cell function and differentiation. Specific Regulators of G protein Signalling (RGS) proteins modulate the function of these receptors in many cell types including vascular smooth muscle cells

  8. The role of complement in the acquired immune response

    DEFF Research Database (Denmark)

    Nielsen, C H; Fischer, E M; Leslie, R G

    2000-01-01

    to specific T cells; the activation of a CD21/CD19 complex-mediated signalling pathway in B cells, which provides a stimulus synergistic to that induced by antigen interaction with the B-cell receptor (BCR); and promotion of the interaction between B cells and FDC, where C3d-bearing immune complexes...

  9. Legume receptors perceive the rhizobial lipochitin oligosaccharide signal molecules by direct binding

    DEFF Research Database (Denmark)

    Broghammer, Angelique; Krusell, Lene; Blaise, Mickael

    2012-01-01

    Lipochitin oligosaccharides called Nod factors function as primary rhizobial signal molecules triggering legumes to develop new plant organs: root nodules that host the bacteria as nitrogen-fixing bacteroids. Here, we show that the Lotus japonicus Nod factor receptor 5 (NFR5) and Nod factor recep...

  10. Growth Hormone Receptor Signaling Pathways and its Negative Regulation by SOCS2

    DEFF Research Database (Denmark)

    Fernández Pérez, Leandro; Flores-Morales, Amilcar; Guerra, Borja

    2016-01-01

    Growth hormone (GH) is a critical regulator of linear body growth during childhood but continues to have important metabolic actions throughout life. The GH receptor (GHR) is ubiquitously expressed, and deficiency of GHR signaling causes a dramatic impact on normal physiology during somatic devel...

  11. Recent Advances on the Role of G Protein-Coupled Receptors in Hypoxia-Mediated Signaling

    OpenAIRE

    Lappano, Rosamaria; Rigiracciolo, Damiano; De Marco, Paola; Avino, Silvia; Cappello, Anna Rita; Rosano, Camillo; Maggiolini, Marcello; De Francesco, Ernestina Marianna

    2016-01-01

    G protein-coupled receptors (GPCRs) are cell surface proteins mainly involved in signal transmission; however, they play a role also in several pathophysiological conditions. Chemically heterogeneous molecules like peptides, hormones, lipids, and neurotransmitters activate second messengers and induce several biological responses by binding to these seven transmembrane receptors, which are coupled to heterotrimeric G proteins. Recently, additional molecular mechanisms have been involved in GP...

  12. Extracellular Ca2+ is a danger signal activating the NLRP3 inflammasome through G protein-coupled calcium sensing receptors

    DEFF Research Database (Denmark)

    Rossol, Manuela; Pierer, Matthias; Raulien, Nora

    2012-01-01

    calcium activates the NLRP3 inflammasome via stimulation of G protein-coupled calcium sensing receptors. Activation is mediated by signalling through the calcium-sensing receptor and GPRC6A via the phosphatidyl inositol/Ca(2+) pathway. The resulting increase in the intracellular calcium concentration......, and this effect was inhibited in GPRC6A(-/-) mice. Our results demonstrate that G-protein-coupled receptors can activate the inflammasome, and indicate that increased extracellular calcium has a role as a danger signal and amplifier of inflammation....

  13. Xingshentongqiao Decoction Mediates Proliferation, Apoptosis, Orexin-A Receptor and Orexin-B Receptor Messenger Ribonucleic Acid Expression and Represses Mitogen-activated Protein Kinase Signaling

    Directory of Open Access Journals (Sweden)

    Yuanli Dong

    2015-01-01

    Full Text Available Background: Hypocretin (HCRT signaling plays an important role in the pathogenesis of narcolepsy and can be significantly influenced by Chinese herbal therapy. Our previous study showed that xingshentongqiao decoction (XSTQ is clinically effective for the treatment of narcolepsy. To determine whether XSTQ improves narcolepsy by modulating HCRT signaling, we investigated its effects on SH-SY5Y cell proliferation, apoptosis, and HCRT receptor 1/2 (orexin receptor 1 [OX1R] and orexin receptor 2 [OX2R] expression. The signaling pathways involved in these processes were also assessed. Methods: The effects of XSTQ on proliferation and apoptosis in SH-SY5Y cells were assessed using cell counting kit-8 and annexin V-fluorescein isothiocyanate assays. OX1R and OX2R expression was assessed by quantitative real-time polymerase chain reaction analysis. Western blotting for mitogen-activated protein kinase (MAPK pathway activation was performed to further assess the signaling mechanism of XSTQ. Results: XSTQ reduced the proliferation and induced apoptosis of SH-SY5Y cells. This effect was accompanied by the upregulation of OX1R and OX2R expression and the reduced phosphorylation of extracellular signal-regulated kinase (Erk 1/2, p38 MAPK and c-Jun N-terminal kinase (JNK. Conclusions: XSTQ inhibits proliferation and induces apoptosis in SH-SY5Y cells. XSTQ also promotes OX1R and OX2R expression. These effects are associated with the repression of the Erk1/2, p38 MAPK, and JNK signaling pathways. These results define a molecular mechanism for XSTQ in regulating HCRT and MAPK activation, which may explain its ability to treat narcolepsy.

  14. Aripiprazole Increases the PKA Signalling and Expression of the GABAA Receptor and CREB1 in the Nucleus Accumbens of Rats.

    Science.gov (United States)

    Pan, Bo; Lian, Jiamei; Huang, Xu-Feng; Deng, Chao

    2016-05-01

    The GABAA receptor is implicated in the pathophysiology of schizophrenia and regulated by PKA signalling. Current antipsychotics bind with D2-like receptors, but not the GABAA receptor. The cAMP-responsive element-binding protein 1 (CREB1) is also associated with PKA signalling and may be related to the positive symptoms of schizophrenia. This study investigated the effects of antipsychotics in modulating D2-mediated PKA signalling and its downstream GABAA receptors and CREB1. Rats were treated orally with aripiprazole (0.75 mg/kg, ter in die (t.i.d.)), bifeprunox (0.8 mg/kg, t.i.d.), haloperidol (0.1 mg/kg, t.i.d.) or vehicle for 1 week. The levels of PKA-Cα and p-PKA in the prefrontal cortex (PFC), nucleus accumbens (NAc) and caudate putamen (CPu) were detected by Western blots. The mRNA levels of Gabrb1, Gabrb2, Gabrb3 and Creb1, and their protein expression were measured by qRT-PCR and Western blots, respectively. Aripiprazole elevated the levels of p-PKA and the ratio of p-PKA/PKA in the NAc, but not the PFC and CPu. Correlated with this elevated PKA signalling, aripiprazole elevated the mRNA and protein expression of the GABAA (β-1) receptor and CREB1 in the NAc. While haloperidol elevated the levels of p-PKA and the ratio of p-PKA/PKA in both NAc and CPu, it only tended to increase the expression of the GABAA (β-1) receptor and CREB1 in the NAc, but not the CPu. Bifeprunox had no effects on PKA signalling in these brain regions. These results suggest that aripiprazole has selective effects on upregulating the GABAA (β-1) receptor and CREB1 in the NAc, probably via activating PKA signalling.

  15. Apo-ghrelin receptor (apo-GHSR1a Regulates Dopamine Signaling in the Brain

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    Andras eKern

    2014-08-01

    Full Text Available The orexigenic peptide hormone ghrelin is synthesized in the stomach and its receptor growth hormone secretagogue receptor (GHSR1a is expressed mainly in the central nervous system (CNS. In this review we confine our discussion to the physiological role of GHSR1a in the brain. Paradoxically, despite broad expression of GHSR1a in the CNS, other than trace amounts in the hypothalamus, ghrelin is undetectable in the brain. In our efforts to elucidate the function of the ligand-free ghrelin receptor (apo-GHSR1a we identified subsets of neurons that co-express GHSR1a and dopamine receptors. In this review we focus on interactions between apo-GHSR1a and dopamine-2 receptor (DRD2 and formation of GHSR1a:DRD2 heteromers in hypothalamic neurons that regulate appetite, and discuss implications for the treatment of Prader-Willi syndrome. GHSR1a antagonists of distinct chemical structures, a quinazolinone and a triazole, respectively enhance and inhibit dopamine signaling through GHSR1a:DRD2 heteromers by an allosteric mechanism. This finding illustrates a potential strategy for designing the next generation of drugs for treating eating disorders as well as psychiatric disorders caused by abnormal dopamine signaling. Treatment with a GHSR1a antagonist that enhances dopamine/DRD2 activity in GHSR1a:DRD2 expressing hypothalamic neurons has the potential to inhibit the uncontrollable hyperphagia associated with Prader-Willi syndrome. DRD2 antagonists are prescribed for treating schizophrenia, but these block dopamine signaling in all DRD2 expressing neurons and are associated with adverse side effects, including enhanced appetite and excessive weight gain. A GHSR1a antagonist of structural class that allosterically blocks dopamine/DRD2 action in GHSR1a:DRD2 expressing neurons would have no effect on neurons expressing DRD2 alone; therefore, the side effects of DRD2 antagonists would potentially be reduced thereby enhancing patient compliance.

  16. Hypocretin/Orexin Regulation of Dopamine Signaling and Cocaine Self-Administration Is Mediated Predominantly by Hypocretin Receptor 1

    OpenAIRE

    Prince, Courtney D.; Rau, Andrew R.; Yorgason, Jordan T.; Espa?a, Rodrigo A.

    2014-01-01

    Extensive evidence suggests that the hypocretins/orexins influence cocaine reinforcement and dopamine signaling via actions at hypocretin receptor 1. By comparison, the involvement of hypocretin receptor 2 in reward and reinforcement processes has received relatively little attention. Thus, although there is some evidence that hypocretin receptor 2 regulates intake of some drugs of abuse, it is currently unclear to what extent hypocretin receptor 2 participates in the regulation of dopamine s...

  17. Plant cell wall signalling and receptor-like kinases.

    Science.gov (United States)

    Wolf, Sebastian

    2017-02-15

    Communication between the extracellular matrix and the cell interior is essential for all organisms as intrinsic and extrinsic cues have to be integrated to co-ordinate development, growth, and behaviour. This applies in particular to plants, the growth and shape of which is governed by deposition and remodelling of the cell wall, a rigid, yet dynamic, extracellular network. It is thus generally assumed that cell wall surveillance pathways exist to monitor the state of the wall and, if needed, elicit compensatory responses such as altered expression of cell wall remodelling and biosynthesis genes. Here, I highlight recent advances in the field of cell wall signalling in plants, with emphasis on the role of plasma membrane receptor-like kinase complexes. In addition, possible roles for cell wall-mediated signalling beyond the maintenance of cell wall integrity are discussed. © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

  18. The LDL Receptor-Related Protein 1: At the Crossroads of Lipoprotein Metabolism and Insulin Signaling

    Directory of Open Access Journals (Sweden)

    Dianaly T. Au

    2017-01-01

    Full Text Available The metabolic syndrome is an escalating worldwide public health concern. Defined by a combination of physiological, metabolic, and biochemical factors, the metabolic syndrome is used as a clinical guideline to identify individuals with a higher risk for type 2 diabetes and cardiovascular disease. Although risk factors for type 2 diabetes and cardiovascular disease have been known for decades, the molecular mechanisms involved in the pathophysiology of these diseases and their interrelationship remain unclear. The LDL receptor-related protein 1 (LRP1 is a large endocytic and signaling receptor that is widely expressed in several tissues. As a member of the LDL receptor family, LRP1 is involved in the clearance of chylomicron remnants from the circulation and has been demonstrated to be atheroprotective. Recently, studies have shown that LRP1 is involved in insulin receptor trafficking and regulation and glucose metabolism. This review summarizes the role of tissue-specific LRP1 in insulin signaling and its potential role as a link between lipoprotein and glucose metabolism in diabetes.

  19. Sensitive detection of pre-existing BCR-ABL kinase domain mutations in CD34+ cells of newly diagnosed chronic-phase chronic myeloid leukemia patients is associated with imatinib resistance: implications in the post-imatinib era.

    Directory of Open Access Journals (Sweden)

    Zafar Iqbal

    Full Text Available BACKGROUND: BCR-ABL kinase domain mutations are infrequently detected in newly diagnosed chronic-phase chronic myeloid leukemia (CML patients. Recent studies indicate the presence of pre-existing BCR-ABL mutations in a higher percentage of CML patients when CD34+ stem/progenitor cells are investigated using sensitive techniques, and these mutations are associated with imatinib resistance and disease progression. However, such studies were limited to smaller number of patients. METHODS: We investigated BCR-ABL kinase domain mutations in CD34+ cells from 100 chronic-phase CML patients by multiplex allele-specific PCR and sequencing at diagnosis. Mutations were re-investigated upon manifestation of imatinib resistance using allele-specific PCR and direct sequencing of BCR-ABL kinase domain. RESULTS: Pre-existing BCR-ABL mutations were detected in 32/100 patients and included F311L, M351T, and T315I. After a median follow-up of 30 months (range 8-48, all patients with pre-existing BCR-ABL mutations exhibited imatinib resistance. Of the 68 patients without pre-existing BCR-ABL mutations, 24 developed imatinib resistance; allele-specific PCR and BCR-ABL kinase domain sequencing detected mutations in 22 of these patients. All 32 patients with pre-existing BCR-ABL mutations had the same mutations after manifestation of imatinib-resistance. In imatinib-resistant patients without pre-existing BCR-ABL mutations, we detected F311L, M351T, Y253F, and T315I mutations. All imatinib-resistant patients except T315I and Y253F mutations responded to imatinib dose escalation. CONCLUSION: Pre-existing BCR-ABL mutations can be detected in a substantial number of chronic-phase CML patients by sensitive allele-specific PCR technique using CD34+ cells. These mutations are associated with imatinib resistance if affecting drug binding directly or indirectly. After the recent approval of nilotinib, dasatinib, bosutinib and ponatinib for treatment of chronic myeloid

  20. The PDZ protein GIPC regulates trafficking of the LPA1 receptor from APPL signaling endosomes and attenuates the cell's response to LPA.

    Directory of Open Access Journals (Sweden)

    Tal Varsano

    Full Text Available Lysophosphatidic acid (LPA mediates diverse cellular responses through the activation of at least six LPA receptors--LPA(1-6, but the interacting proteins and signaling pathways that mediate the specificity of these receptors are largely unknown. We noticed that LPA(1 contains a PDZ binding motif (SVV identical to that present in two other proteins that interact with the PDZ protein GIPC. GIPC is involved in endocytic trafficking of several receptors including TrkA, VEGFR2, lutropin and dopamine D2 receptors. Here we show that GIPC binds directly to the PDZ binding motif of LPA(1 but not that of other LPA receptors. LPA(1 colocalizes and coimmunoprecipitates with GIPC and its binding partner APPL, an activator of Akt signaling found on APPL signaling endosomes. GIPC depletion by siRNA disturbed trafficking of LPA(1 to EEA1 early endosomes and promoted LPA(1 mediated Akt signaling, cell proliferation, and cell motility. We propose that GIPC binds LPA(1 and promotes its trafficking from APPL-containing signaling endosomes to EEA1 early endosomes and thus attenuates LPA-mediated Akt signaling from APPL endosomes.

  1. Insights into Basal Signaling Regulation, Oligomerization, and Structural Organization of the Human G-Protein Coupled Receptor 83.

    Directory of Open Access Journals (Sweden)

    Anne Müller

    Full Text Available The murine G-protein coupled receptor 83 (mGPR83 is expressed in the hypothalamus and was previously suggested to be involved in the regulation of metabolism. The neuropeptide PEN has been recently identified as a potent GPR83 ligand. Moreover, GPR83 constitutes functionally relevant hetero-oligomers with other G-protein coupled receptors (GPCR such as the ghrelin receptor (GHSR or GPR171. Previous deletion studies also revealed that the long N-terminal extracellular receptor domain (eNDo of mGPR83 may act as an intra-molecular ligand, which participates in the regulation of basal signaling activity, which is a key feature of GPCR function. Here, we investigated particular amino acids at the eNDo of human GPR83 (hGPR83 by side-directed mutagenesis to identify determinants of the internal ligand. These studies were accompanied by structure homology modeling to combine functional insights with structural information. The capacity for hetero-oligomer formation of hGPR83 with diverse family A GPCRs such as the melanocortin-4 receptor (MC4R was also investigated, with a specific emphasis on the impact of the eNDo on oligomerization and basal signaling properties. Finally, we demonstrate that hGPR83 exhibits an unusual basal signaling for different effectors, which also supports signaling promiscuity. hGPR83 interacts with a variety of hypothalamic GPCRs such as the MC4R or GHSR. These interactions are not dependent on the ectodomain and most likely occur at interfaces constituted in the transmembrane regions. Moreover, several amino acids at the transition between the eNDo and transmembrane helix 1 were identified, where mutations lead also to biased basal signaling modulation.

  2. Dopamine receptors modulate cytotoxicity of natural killer cells via cAMP-PKA-CREB signaling pathway.

    Directory of Open Access Journals (Sweden)

    Wei Zhao

    Full Text Available Dopamine (DA, a neurotransmitter in the nervous system, has been shown to modulate immune function. We have previously reported that five subtypes of DA receptors, including D1R, D2R, D3R, D4R and D5R, are expressed in T lymphocytes and they are involved in regulation of T cells. However, roles of these DA receptor subtypes and their coupled signal-transduction pathway in modulation of natural killer (NK cells still remain to be clarified. The spleen of mice was harvested and NK cells were isolated and purified by negative selection using magnetic activated cell sorting. After NK cells were incubated with various drugs for 4 h, flow cytometry measured cytotoxicity of NK cells against YAC-1 lymphoma cells. NK cells expressed the five subtypes of DA receptors at mRNA and protein levels. Activation of D1-like receptors (including D1R and D5R with agonist SKF38393 enhanced NK cell cytotoxicity, but activation of D2-like receptors (including D2R, D3R and D4R with agonist quinpirole attenuated NK cells. Simultaneously, SKF38393 elevated D1R and D5R expression, cAMP content, and phosphorylated cAMP-response element-binding (CREB level in NK cells, while quinpirole reduced D3R and D4R expression, cAMP content, and phosphorylated CREB level in NK cells. These effects of SKF38393 were blocked by SCH23390, an antagonist of D1-like receptors, and quinpirole effects were abolished by haloperidol, an antagonist of D2-like receptors. In support these results, H89, an inhibitor of phosphokinase A (PKA, prevented the SKF38393-dependent enhancement of NK cells and forskolin, an activator of adenylyl cyclase (AC, counteracted the quinpirole-dependent suppression of NK cells. These findings show that DA receptor subtypes are involved in modulation of NK cells and suggest that D1-like receptors facilitate NK cells by stimulating D1R/D5R-cAMP-PKA-CREB signaling pathway and D2-like receptors suppress NK cells by inhibiting D3R/D4R-cAMP-PKA-CREB signaling pathway. The

  3. Optogenetic control of chemokine receptor signal and T-cell migration

    Science.gov (United States)

    Xu, Yuexin; Hyun, Young-Min; Lim, Kihong; Lee, Hyunwook; Cummings, Ryan J.; Gerber, Scott A.; Bae, Seyeon; Cho, Thomas Yoonsang; Lord, Edith M.; Kim, Minsoo

    2014-01-01

    Adoptive cell transfer of ex vivo-generated immune-promoting or tolerogenic T cells to either enhance immunity or promote tolerance in patients has been used with some success. However, effective trafficking of the transferred cells to the target tissue sites is the main barrier to achieving successful clinical outcomes. Here we developed a strategy for optically controlling T-cell trafficking using a photoactivatable (PA) chemokine receptor. Photoactivatable-chemokine C-X-C motif receptor 4 (PA-CXCR4) transmitted intracellular CXCR4 signals in response to 505-nm light. Localized activation of PA-CXCR4 induced T-cell polarization and directional migration (phototaxis) both in vitro and in vivo. Directing light onto the melanoma was sufficient to recruit PA-CXCR4–expressing tumor-targeting cytotoxic T cells and improved the efficacy of adoptive T-cell transfer immunotherapy, with a significant reduction in tumor growth in mice. These findings suggest that the use of photoactivatable chemokine receptors allows remotely controlled leukocyte trafficking with outstanding spatial resolution in tissues and may be feasible in other cell transfer therapies. PMID:24733886

  4. Retinoid receptor signaling and autophagy in acute promyelocytic leukemia.

    LENUS (Irish Health Repository)

    Orfali, Nina

    2014-05-15

    Retinoids are a family of signaling molecules derived from vitamin A with well established roles in cellular differentiation. Physiologically active retinoids mediate transcriptional effects on cells through interactions with retinoic acid (RARs) and retinoid-X (RXR) receptors. Chromosomal translocations involving the RARα gene, which lead to impaired retinoid signaling, are implicated in acute promyelocytic leukemia (APL). All-trans-retinoic acid (ATRA), alone and in combination with arsenic trioxide (ATO), restores differentiation in APL cells and promotes degradation of the abnormal oncogenic fusion protein through several proteolytic mechanisms. RARα fusion-protein elimination is emerging as critical to obtaining sustained remission and long-term cure in APL. Autophagy is a degradative cellular pathway involved in protein turnover. Both ATRA and ATO also induce autophagy in APL cells. Enhancing autophagy may therefore be of therapeutic benefit in resistant APL and could broaden the application of differentiation therapy to other cancers. Here we discuss retinoid signaling in hematopoiesis, leukemogenesis, and APL treatment. We highlight autophagy as a potential important regulator in anti-leukemic strategies.

  5. Orphan nuclear receptor TLX regulates astrogenesis by modulating BMP signaling

    Directory of Open Access Journals (Sweden)

    Song eQin

    2014-04-01

    Full Text Available Neural stem cells (NSCs are self-renewing multipotent progenitors that generate both neurons and glia. The precise control of NSC behavior is fundamental to the architecture and function of the central nervous system. We previously demonstrated that the orphan nuclear receptor TLX is required for postnatal NSC activation and neurogenesis in the neurogenic niche. Here, we show that TLX modulates BMP-SMAD signaling to control the timing of postnatal astrogenesis. Genes involved in the BMP signaling pathway, such as Bmp4, Hes1, and Id3, are upregulated in postnatal brains lacking Tlx. Chromatin immunoprecipitation and electrophoretic mobility shift assays reveal that TLX can directly bind the enhancer region of Bmp4. In accordance with elevated BMP signaling, the downstream effectors SMAD1/5/8 are activated by phosphorylation in Tlx mutant mice. Consequently, Tlx mutant brains exhibit an early appearance and increased number of astrocytes with marker expression of glial fibrillary acidic protein (GFAP and S100B. Taken together, these results suggest that TLX tightly controls postnatal astrogenesis through the modulation of BMP-SMAD signaling pathway activity.

  6. Orphan nuclear receptor TLX regulates astrogenesis by modulating BMP signaling.

    Science.gov (United States)

    Qin, Song; Niu, Wenze; Iqbal, Nida; Smith, Derek K; Zhang, Chun-Li

    2014-01-01

    Neural stem cells (NSCs) are self-renewing multipotent progenitors that generate both neurons and glia. The precise control of NSC behavior is fundamental to the architecture and function of the central nervous system. We previously demonstrated that the orphan nuclear receptor TLX is required for postnatal NSC activation and neurogenesis in the neurogenic niche. Here, we show that TLX modulates bone morphogenetic protein (BMP)-SMAD signaling to control the timing of postnatal astrogenesis. Genes involved in the BMP signaling pathway, such as Bmp4, Hes1, and Id3, are upregulated in postnatal brains lacking Tlx. Chromatin immunoprecipitation and electrophoretic mobility shift assays reveal that TLX can directly bind the enhancer region of Bmp4. In accordance with elevated BMP signaling, the downstream effectors SMAD1/5/8 are activated by phosphorylation in Tlx mutant mice. Consequently, Tlx mutant brains exhibit an early appearance and increased number of astrocytes with marker expression of glial fibrillary acidic protein (GFAP) and S100B. Taken together, these results suggest that TLX tightly controls postnatal astrogenesis through the modulation of BMP-SMAD signaling pathway activity.

  7. Intraclonal Cell Expansion and Selection Driven by B Cell Receptor in Chronic Lymphocytic Leukemia

    Science.gov (United States)

    Colombo, Monica; Cutrona, Giovanna; Reverberi, Daniele; Fabris, Sonia; Neri, Antonino; Fabbi, Marina; Quintana, Giovanni; Quarta, Giovanni; Ghiotto, Fabio; Fais, Franco; Ferrarini, Manlio

    2011-01-01

    The mutational status of the immunoglobulin heavy-chain variable region (IGHV) genes utilized by chronic lymphocytic leukemia (CLL) clones defines two disease subgroups. Patients with unmutated IGHV have a more aggressive disease and a worse outcome than patients with cells having somatic IGHV gene mutations. Moreover, up to 30% of the unmutated CLL clones exhibit very similar or identical B cell receptors (BcR), often encoded by the same IG genes. These “stereotyped” BcRs have been classified into defined subsets. The presence of an IGHV gene somatic mutation and the utilization of a skewed gene repertoire compared with normal B cells together with the expression of stereotyped receptors by unmutated CLL clones may indicate stimulation/selection by antigenic epitopes. This antigenic stimulation may occur prior to or during neoplastic transformation, but it is unknown whether this stimulation/selection continues after leukemogenesis has ceased. In this study, we focused on seven CLL cases with stereotyped BcR Subset #8 found among a cohort of 700 patients; in six, the cells expressed IgG and utilized IGHV4-39 and IGKV1-39/IGKV1D-39 genes, as reported for Subset #8 BcR. One case exhibited special features, including expression of IgM or IgG by different subclones consequent to an isotype switch, allelic inclusion at the IGH locus in the IgM-expressing cells and a particular pattern of cytogenetic lesions. Collectively, the data indicate a process of antigenic stimulation/selection of the fully transformed CLL cells leading to the expansion of the Subset #8 IgG-bearing subclone. PMID:21541442

  8. Macroscopic law of conservation revealed in the population dynamics of Toll-like receptor signaling

    Directory of Open Access Journals (Sweden)

    Selvarajoo Kumar

    2011-04-01

    Full Text Available Abstract Stimulating the receptors of a single cell generates stochastic intracellular signaling. The fluctuating response has been attributed to the low abundance of signaling molecules and the spatio-temporal effects of diffusion and crowding. At population level, however, cells are able to execute well-defined deterministic biological processes such as growth, division, differentiation and immune response. These data reflect biology as a system possessing microscopic and macroscopic dynamics. This commentary discusses the average population response of the Toll-like receptor (TLR 3 and 4 signaling. Without requiring detailed experimental data, linear response equations together with the fundamental law of information conservation have been used to decipher novel network features such as unknown intermediates, processes and cross-talk mechanisms. For single cell response, however, such simplicity seems far from reality. Thus, as observed in any other complex systems, biology can be considered to possess order and disorder, inheriting a mixture of predictable population level and unpredictable single cell outcomes.

  9. Disease-associated extracellular loop mutations in the adhesion G protein-coupled receptor G1 (ADGRG1; GPR56) differentially regulate downstream signaling.

    Science.gov (United States)

    Kishore, Ayush; Hall, Randy A

    2017-06-09

    Mutations to the adhesion G protein-coupled receptor ADGRG1 (G1; also known as GPR56) underlie the neurological disorder bilateral frontoparietal polymicrogyria. Disease-associated mutations in G1 studied to date are believed to induce complete loss of receptor function through disruption of either receptor trafficking or signaling activity. Given that N-terminal truncation of G1 and other adhesion G protein-coupled receptors has been shown to significantly increase the receptors' constitutive signaling, we examined two different bilateral frontoparietal polymicrogyria-inducing extracellular loop mutations (R565W and L640R) in the context of both full-length and N-terminally truncated (ΔNT) G1. Interestingly, we found that these mutations reduced surface expression of full-length G1 but not G1-ΔNT in HEK-293 cells. Moreover, the mutations ablated receptor-mediated activation of serum response factor luciferase, a classic measure of Gα 12/13 -mediated signaling, but had no effect on G1-mediated signaling to nuclear factor of activated T cells (NFAT) luciferase. Given these differential signaling results, we sought to further elucidate the pathway by which G1 can activate NFAT luciferase. We found no evidence that ΔNT activation of NFAT is dependent on Gα q/11 -mediated or β-arrestin-mediated signaling but rather involves liberation of Gβγ subunits and activation of calcium channels. These findings reveal that disease-associated mutations to the extracellular loops of G1 differentially alter receptor trafficking, depending on the presence of the N terminus, and differentially alter signaling to distinct downstream pathways. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. Building tolerance by dismantling synapses: inhibitory receptor signaling in natural killer cells.

    Science.gov (United States)

    Huse, Morgan; Catherine Milanoski, S; Abeyweera, Thushara P

    2013-01-01

    Cell surface receptors bearing immunotyrosine-based inhibitory motifs (ITIMs) maintain natural killer (NK) cell tolerance to normal host tissues. These receptors are difficult to analyze mechanistically because they block activating responses in a rapid and comprehensive manner. The advent of high-resolution single cell imaging techniques has enabled investigators to explore the cell biological basis of the inhibitory response. Recent studies using these approaches indicate that ITIM-containing receptors function at least in part by structurally undermining the immunological synapse between the NK cell and its target. In this review, we discuss these new advances and how they might relate to what is known about the biochemistry of inhibitory signaling in NK cells and other cell types. © 2012 John Wiley & Sons A/S. Published by Blackwell Publishing Ltd.

  11. Molecular mechanisms of glucocorticoid receptor signaling

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    Marta Labeur

    2010-10-01

    Full Text Available This review highlights the most recent findings on the molecular mechanisms of the glucocorticoid receptor (GR. Most effects of glucocorticoids are mediated by the intracellular GR which is present in almost every tissue and controls transcriptional activation via direct and indirect mechanisms. Nevertheless the glucocorticoid responses are tissue -and gene- specific. GR associates selectively with corticosteroid ligands produced in the adrenal gland in response to changes of humoral homeostasis. Ligand interaction with GR promotes either GR binding to genomic glucocorticoid response elements, in turn modulating gene transcription, or interaction of GR monomers with other transcription factors activated by other signalling pathways leading to transrepression. The GR regulates a broad spectrum of physiological functions, including cell differentiation, metabolism and inflammatory responses. Thus, disruption or dysregulation of GR function will result in severe impairments in the maintenance of homeostasis and the control of adaptation to stress.

  12. Influence of ER leak on resting cytoplasmic Ca2+ and receptor-mediated Ca2+ signalling in human macrophage.

    Science.gov (United States)

    Layhadi, Janice A; Fountain, Samuel J

    2017-06-03

    Mechanisms controlling endoplasmic reticulum (ER) Ca 2+ homeostasis are important regulators of resting cytoplasmic Ca 2+ concentration ([Ca 2+ ] cyto ) and receptor-mediated Ca 2+ signalling. Here we investigate channels responsible for ER Ca 2+ leak in THP-1 macrophage and human primary macrophage. In the absence of extracellular Ca 2+ we employ ionomycin action at the plasma membrane to stimulate ER Ca 2+ leak. Under these conditions ionomycin elevates [Ca 2+ ] cyto revealing a Ca 2+ leak response which is abolished by thapsigargin. IP 3 receptors (Xestospongin C, 2-APB), ryanodine receptors (dantrolene), and translocon (anisomycin) inhibition facilitated ER Ca 2+ leak in model macrophage, with translocon inhibition also reducing resting [Ca 2+ ] cyto . In primary macrophage, translocon inhibition blocks Ca 2+ leak but does not influence resting [Ca 2+ ] cyto . We identify a role for translocon-mediated ER Ca 2+ leak in receptor-mediated Ca 2+ signalling in both model and primary human macrophage, whereby the Ca 2+ response to ADP (P2Y receptor agonist) is augmented following anisomycin treatment. In conclusion, we demonstrate a role of ER Ca 2+ leak via the translocon in controlling resting cytoplasmic Ca 2+ in model macrophage and receptor-mediated Ca 2+ signalling in model macrophage and primary macrophage. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. Effect of Thai saraphi flower extracts on WT1 and BCR/ABL protein ...

    African Journals Online (AJOL)

    In this study, the cytotoxic effects of crude ethanolic and fractional extracts including hexane, ethyl acetate, and methanol fractions from M. siamensis flowers were investigated in order to determine their effect on WT1 expression in Molt4 and K562 cells and Bcr/Abl expression in K562 cells. Materials and Methods: The ...

  14. Serotonin Signaling in Schistosoma mansoni: A Serotonin–Activated G Protein-Coupled Receptor Controls Parasite Movement

    Science.gov (United States)

    Rashid, Mohammed; Ribeiro, Paula

    2014-01-01

    Serotonin is an important neuroactive substance in all the parasitic helminths. In Schistosoma mansoni, serotonin is strongly myoexcitatory; it potentiates contraction of the body wall muscles and stimulates motor activity. This is considered to be a critical mechanism of motor control in the parasite, but the mode of action of serotonin is poorly understood. Here we provide the first molecular evidence of a functional serotonin receptor (Sm5HTR) in S. mansoni. The schistosome receptor belongs to the G protein-coupled receptor (GPCR) superfamily and is distantly related to serotonergic type 7 (5HT7) receptors from other species. Functional expression studies in transfected HEK 293 cells showed that Sm5HTR is a specific serotonin receptor and it signals through an increase in intracellular cAMP, consistent with a 5HT7 signaling mechanism. Immunolocalization studies with a specific anti-Sm5HTR antibody revealed that the receptor is abundantly distributed in the worm's nervous system, including the cerebral ganglia and main nerve cords of the central nervous system and the peripheral innervation of the body wall muscles and tegument. RNA interference (RNAi) was performed both in schistosomulae and adult worms to test whether the receptor is required for parasite motility. The RNAi-suppressed adults and larvae were markedly hypoactive compared to the corresponding controls and they were also resistant to exogenous serotonin treatment. These results show that Sm5HTR is at least one of the receptors responsible for the motor effects of serotonin in S. mansoni. The fact that Sm5HTR is expressed in nerve tissue further suggests that serotonin stimulates movement via this receptor by modulating neuronal output to the musculature. Together, the evidence identifies Sm5HTR as an important neuronal protein and a key component of the motor control apparatus in S. mansoni. PMID:24453972

  15. Sphingosine Kinases and Sphingosine 1-Phosphate Receptors: Signaling and Actions in the Cardiovascular System

    Directory of Open Access Journals (Sweden)

    Alessandro Cannavo

    2017-08-01

    Full Text Available The sphingosine kinases 1 and 2 (SphK1 and 2 catalyze the phosphorylation of the lipid, sphingosine, generating the signal transmitter, sphingosine 1-phosphate (S1P. The activation of such kinases and the subsequent S1P generation and secretion in the blood serum of mammals represent a major checkpoint in many cellular signaling cascades. In fact, activating the SphK/S1P system is critical for cell motility and proliferation, cytoskeletal organization, cell growth, survival, and response to stress. In the cardiovascular system, the physiological effects of S1P intervene through the binding and activation of a family of five highly selective G protein-coupled receptors, called S1PR1-5. Importantly, SphK/S1P signal is present on both vascular and myocardial cells. S1P is a well-recognized survival factor in many tissues. Therefore, it is not surprising that the last two decades have seen a flourishing of interest and investigative efforts directed to obtain additional mechanistic insights into the signaling, as well as the biological activity of this phospholipid, and of its receptors, especially in the cardiovascular system. Here, we will provide an up-to-date account on the structure and function of sphingosine kinases, discussing the generation, release, and function of S1P. Keeping the bull’s eye on the cardiovascular system, we will review the structure and signaling cascades and biological actions emanating from the stimulation of different S1P receptors. We will end this article with a summary of the most recent, experimental and clinical observations targeting S1PRs and SphKs as possible new therapeutic avenues for cardiovascular disorders, such as heart failure.

  16. BCR translocation to derivative chromosome 2, a new case of chronic myeloid leukemia with complex variant translocation and Philadelphia chromosome

    International Nuclear Information System (INIS)

    Al-Achkar, W.; Wafa, A.; Al-Medani, S.

    2011-01-01

    The well-known typical fusion gene BCR/ABL can be observed in connection with a complex translocation event in only 5-8% of cases with chronic myeloid leukemia (CML). Herein we report an exceptional CML case with complex chromosomal aberrations not observed before, translocated BCR to the derivative chromosome 2 [der(2)], additional to involving a four chromosomes translocation implying chromosomal regions such as 1p32 and 2q21 besides 9q34 and 22q11.2. Which were characterized by molecular cytogenetics. (author)

  17. Regulation of EGF Receptor Signaling by Histone Deacetylase 6 (HDAC6)-Mediated Reversible Acetylation

    National Research Council Canada - National Science Library

    Kovacs, Jeffrey J

    2005-01-01

    One of the hallmarks of cancer is uncontrolled cell growth and proliferation. In cells, a group of proteins called growth factor receptors are responsible for responding to the signals that trigger proliferation...

  18. Clathrin-dependent internalization, signaling, and metabolic processing of guanylyl cyclase/natriuretic peptide receptor-A.

    Science.gov (United States)

    Somanna, Naveen K; Mani, Indra; Tripathi, Satyabha; Pandey, Kailash N

    2018-04-01

    Cardiac hormones, atrial and brain natriuretic peptides (ANP and BNP), have pivotal roles in renal hemodynamics, neuroendocrine signaling, blood pressure regulation, and cardiovascular homeostasis. Binding of ANP and BNP to the guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA) induces rapid internalization and trafficking of the receptor via endolysosomal compartments, with concurrent generation of cGMP. However, the mechanisms of the endocytotic processes of NPRA are not well understood. The present study, using 125 I-ANP binding assay and confocal microscopy, examined the function of dynamin in the internalization of NPRA in stably transfected human embryonic kidney-293 (HEK-293) cells. Treatment of recombinant HEK-293 cells with ANP time-dependently accelerated the internalization of receptor from the cell surface to the cell interior. However, the internalization of ligand-receptor complexes of NPRA was drastically decreased by the specific inhibitors of clathrin- and dynamin-dependent receptor internalization, almost 85% by monodansylcadaverine, 80% by chlorpromazine, and 90% by mutant dynamin, which are specific blockers of endocytic vesicle formation. Visualizing the internalization of NPRA and enhanced GFP-tagged NPRA in HEK-293 cells by confocal microscopy demonstrated the formation of endocytic vesicles after 5 min of ANP treatment; this effect was blocked by the inhibitors of clathrin and by mutant dynamin construct. Our results suggest that NPRA undergoes internalization via clathrin-mediated endocytosis as part of its normal itinerary, including trafficking, signaling, and metabolic degradation.

  19. Determination of cortisol in two BCR reference sera by isotope dilution gas chromatography-mass spectrometry

    NARCIS (Netherlands)

    Derks; H.J.G.M.; Freudenthal; J.; Heiningen; A.van; Gramberg; L.G.; Klaasen; R.

    1984-01-01

    In dit rapport wordt een analytische methode voor de juiste en preciese bepaling van cortisol in humaan serum en zijn toepassing bij de certificatie van twee gevriesdroogde referentiesera van de BCR beschreven. De methode is gebaseerd op isotoopverdunnings gaschromatografie-massaspectrometrie

  20. Cannabinoid Receptors and the Endocannabinoid System: Signaling and Function in the Central Nervous System

    Directory of Open Access Journals (Sweden)

    Shenglong Zou

    2018-03-01

    Full Text Available The biological effects of cannabinoids, the major constituents of the ancient medicinal plant Cannabis sativa (marijuana are mediated by two members of the G-protein coupled receptor family, cannabinoid receptors 1 (CB1R and 2. The CB1R is the prominent subtype in the central nervous system (CNS and has drawn great attention as a potential therapeutic avenue in several pathological conditions, including neuropsychological disorders and neurodegenerative diseases. Furthermore, cannabinoids also modulate signal transduction pathways and exert profound effects at peripheral sites. Although cannabinoids have therapeutic potential, their psychoactive effects have largely limited their use in clinical practice. In this review, we briefly summarized our knowledge of cannabinoids and the endocannabinoid system, focusing on the CB1R and the CNS, with emphasis on recent breakthroughs in the field. We aim to define several potential roles of cannabinoid receptors in the modulation of signaling pathways and in association with several pathophysiological conditions. We believe that the therapeutic significance of cannabinoids is masked by the adverse effects and here alternative strategies are discussed to take therapeutic advantage of cannabinoids.

  1. Differences in Signal Activation by LH and hCG are Mediated by the LH/CG Receptor's Extracellular Hinge Region.

    Science.gov (United States)

    Grzesik, Paul; Kreuchwig, Annika; Rutz, Claudia; Furkert, Jens; Wiesner, Burkhard; Schuelein, Ralf; Kleinau, Gunnar; Gromoll, Joerg; Krause, Gerd

    2015-01-01

    The human lutropin (hLH)/choriogonadotropin (hCG) receptor (LHCGR) can be activated by binding two slightly different gonadotropic glycoprotein hormones, choriogonadotropin (CG) - secreted by the placenta, and lutropin (LH) - produced by the pituitary. They induce different signaling profiles at the LHCGR. This cannot be explained by binding to the receptor's leucine-rich-repeat domain (LRRD), as this binding is similar for the two hormones. We therefore speculate that there are previously unknown differences in the hormone/receptor interaction at the extracellular hinge region, which might help to understand functional differences between the two hormones. We have therefore performed a detailed study of the binding and action of LH and CG at the LHCGR hinge region. We focused on a primate-specific additional exon in the hinge region, which is located between LRRD and the serpentine domain. The segment of the hinge region encoded by exon10 was previously reported to be only relevant to hLH signaling, as the exon10-deletion receptor exhibits decreased hLH signaling, but unchanged hCG signaling. We designed an advanced homology model of the hormone/LHCGR complex, followed by experimental characterization of relevant fragments in the hinge region. In addition, we examined predictions of a helical exon10-encoded conformation by block-wise polyalanine (helix supporting) mutations. These helix preserving modifications showed no effect on hormone-induced signaling. However, introduction of a structure-disturbing double-proline mutant LHCGR-Q303P/E305P within the exon10-helix has, in contrast to exon10-deletion, no impact on hLH, but only on hCG signaling. This opposite effect on signaling by hLH and hCG can be explained by distinct sites of hormone interaction in the hinge region. In conclusion, our analysis provides details of the differences between hLH- and hCG-induced signaling that are mainly determined in the L2-beta loop of the hormones and in the hinge

  2. A gate-latch-lock mechanism for hormone signalling by abscisic acid receptors

    KAUST Repository

    Melcher, Karsten

    2009-12-03

    Abscisic acid (ABA) is a ubiquitous hormone that regulates plant growth, development and responses to environmental stresses. Its action is mediated by the PYR/PYL/RCAR family of START proteins, but it remains unclear how these receptors bind ABA and, in turn, how hormone binding leads to inhibition of the downstream type 2C protein phosphatase (PP2C) effectors. Here we report crystal structures of apo and ABA-bound receptors as well as a ternary PYL2-ABA-PP2C complex. The apo receptors contain an open ligand-binding pocket flanked by a gate that closes in response to ABA by way of conformational changes in two highly conserved ?-loops that serve as a gate and latch. Moreover, ABA-induced closure of the gate creates a surface that enables the receptor to dock into and competitively inhibit the PP2C active site. A conserved tryptophan in the PP2C inserts directly between the gate and latch, which functions to further lock the receptor in a closed conformation. Together, our results identify a conserved gate-latch-lock mechanism underlying ABA signalling. © 2009 Macmillan Publishers Limited. All rights reserved.

  3. A gate-latch-lock mechanism for hormone signalling by abscisic acid receptors

    KAUST Repository

    Melcher, Karsten; Ng, Ley-Moy; Zhou, X. Edward; Soon, Fen-Fen; Xu, Yong; Suino-Powell, Kelly M.; Park, Sang-Youl; Weiner, Joshua J.; Fujii, Hiroaki; Chinnusamy, Viswanathan; Kovach, Amanda; Li, Jun; Wang, Yonghong; Li, Jiayang; Peterson, Francis C.; Jensen, Davin R.; Yong, Eu-Leong; Volkman, Brian F.; Cutler, Sean R.; Zhu, Jian-Kang; Xu, H. Eric

    2009-01-01

    Abscisic acid (ABA) is a ubiquitous hormone that regulates plant growth, development and responses to environmental stresses. Its action is mediated by the PYR/PYL/RCAR family of START proteins, but it remains unclear how these receptors bind ABA and, in turn, how hormone binding leads to inhibition of the downstream type 2C protein phosphatase (PP2C) effectors. Here we report crystal structures of apo and ABA-bound receptors as well as a ternary PYL2-ABA-PP2C complex. The apo receptors contain an open ligand-binding pocket flanked by a gate that closes in response to ABA by way of conformational changes in two highly conserved ?-loops that serve as a gate and latch. Moreover, ABA-induced closure of the gate creates a surface that enables the receptor to dock into and competitively inhibit the PP2C active site. A conserved tryptophan in the PP2C inserts directly between the gate and latch, which functions to further lock the receptor in a closed conformation. Together, our results identify a conserved gate-latch-lock mechanism underlying ABA signalling. © 2009 Macmillan Publishers Limited. All rights reserved.

  4. β-Adrenergic receptor signaling and modulation of long-term potentiation in the mammalian hippocampus

    OpenAIRE

    O'Dell, Thomas J.; Connor, Steven A.; Guglietta, Ryan; Nguyen, Peter V.

    2015-01-01

    Encoding new information in the brain requires changes in synaptic strength. Neuromodulatory transmitters can facilitate synaptic plasticity by modifying the actions and expression of specific signaling cascades, transmitter receptors and their associated signaling complexes, genes, and effector proteins. One critical neuromodulator in the mammalian brain is norepinephrine (NE), which regulates multiple brain functions such as attention, perception, arousal, sleep, learning, and memory. The m...

  5. Cardiac Alpha1-Adrenergic Receptors: Novel Aspects of Expression, Signaling Mechanisms, Physiologic Function, and Clinical Importance

    Science.gov (United States)

    O’Connell, Timothy D.; Jensen, Brian C.; Baker, Anthony J.

    2014-01-01

    Adrenergic receptors (AR) are G-protein-coupled receptors (GPCRs) that have a crucial role in cardiac physiology in health and disease. Alpha1-ARs signal through Gαq, and signaling through Gq, for example, by endothelin and angiotensin receptors, is thought to be detrimental to the heart. In contrast, cardiac alpha1-ARs mediate important protective and adaptive functions in the heart, although alpha1-ARs are only a minor fraction of total cardiac ARs. Cardiac alpha1-ARs activate pleiotropic downstream signaling to prevent pathologic remodeling in heart failure. Mechanisms defined in animal and cell models include activation of adaptive hypertrophy, prevention of cardiac myocyte death, augmentation of contractility, and induction of ischemic preconditioning. Surprisingly, at the molecular level, alpha1-ARs localize to and signal at the nucleus in cardiac myocytes, and, unlike most GPCRs, activate “inside-out” signaling to cause cardioprotection. Contrary to past opinion, human cardiac alpha1-AR expression is similar to that in the mouse, where alpha1-AR effects are seen most convincingly in knockout models. Human clinical studies show that alpha1-blockade worsens heart failure in hypertension and does not improve outcomes in heart failure, implying a cardioprotective role for human alpha1-ARs. In summary, these findings identify novel functional and mechanistic aspects of cardiac alpha1-AR function and suggest that activation of cardiac alpha1-AR might be a viable therapeutic strategy in heart failure. PMID:24368739

  6. Connexins and M3 Muscarinic Receptors Contribute to Heterogeneous Ca2+ Signaling in Mouse Aortic Endothelium

    Directory of Open Access Journals (Sweden)

    François-Xavier Boittin

    2013-02-01

    Full Text Available Background/Aims: Smooth muscle tone is controlled by Ca2+ signaling in the endothelial layer. Mouse endothelial cells are interconnected by gap junctions made of Connexin40 (Cx40 and Cx37, which allow the exchange of signaling molecules to coordinate their activity. Here, we investigated the role of Cx40 in the endothelial Ca2+ signaling of the mouse aorta. Methods: Ca2+ imaging was performed on intact aortic endothelium from both wild type (Cx40+/+ and Connexin40-deficient (Cx40 -/- mice. Results: Acetylcholine (ACh induced early fast and high amplitude Ca2+ transients in a fraction of endothelial cells expressing the M3 muscarinic receptors. Inhibition of intercellular communication using carbenoxolone or octanol fully blocked the propagation of ACh-induced Ca2+ transients toward adjacent cells in WT and Cx40-/- mice. As compared to WT, Cx40-/- mice displayed a reduced propagation of ACh-induced Ca2+ waves, indicating that Cx40 contributes to the spreading of Ca2+ signals. The propagation of those Ca2+ responses was not blocked by suramin, a blocker of purinergic ATP receptors, indicating that there is no paracrine effect of ATP release on the Ca2+ waves. Conclusions: Altogether our data show that Cx40 and Cx37 contribute to the propagation and amplification of the Ca2+ signaling triggered by ACh in endothelial cells expressing the M3 muscarinic receptors.

  7. Sortilin Associates with Trk Receptors to Enhance Anterograde Transport and Signaling by Neurotrophins

    DEFF Research Database (Denmark)

    Vægter, Christian Bjerggaard

      Neurotrophins (NT) are neuronal growth factors essential for development and maintenance of the nervous system. They are released in two forms with opposing biological functions. The proforms induce apoptosis by engaging a death signaling complex comprising the p75NTR neurotrophin receptor...

  8. Biased signaling of G protein-coupled receptors - From a chemokine receptor CCR7 perspective

    DEFF Research Database (Denmark)

    Jørgensen, Astrid Sissel; Rosenkilde, Mette M; Hjortø, Gertrud M

    2018-01-01

    of CCL21 displays an extraordinarily strong glycosaminoglycan (GAG) binding, CCR7 plays a central role in coordinating the meeting between mature antigen presenting DCs and naïve T-cells which normally takes place in the lymph nodes (LNs). This process is a prerequisite for the initiation of an antigen...... the cell-based immune system is controlled. Bias comes in three forms; ligand-, receptor- and tissue-bias. Biased signaling is increasingly being recognized as playing an important role in contributing to the fine-tuned coordination of immune cell chemotaxis. In the current review we discuss the recent...

  9. Curcumin blocks interleukin (IL)-2 signaling in T-lymphocytes by inhibiting IL-2 synthesis, CD25 expression, and IL-2 receptor signaling

    Energy Technology Data Exchange (ETDEWEB)

    Forward, Nicholas A.; Conrad, David M. [Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia (Canada); Power Coombs, Melanie R.; Doucette, Carolyn D. [Department of Pathology, Dalhousie University, Halifax, Nova Scotia (Canada); Furlong, Suzanne J. [Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia (Canada); Lin, Tong-Jun [Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia (Canada); Department of Pediatrics, Dalhousie University, Halifax, Nova Scotia (Canada); Hoskin, David W., E-mail: d.w.hoskin@dal.ca [Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia (Canada); Department of Pathology, Dalhousie University, Halifax, Nova Scotia (Canada); Department of Surgery, Dalhousie University, Halifax, Nova Scotia (Canada)

    2011-04-22

    Highlights: {yields} Curcumin inhibits CD4{sup +} T-lymphocyte proliferation. {yields} Curcumin inhibits interleukin-2 (IL-2) synthesis and CD25 expression by CD4{sup +} T-lymphocytes. {yields} Curcumin interferes with IL-2 receptor signaling by inhibiting JAK3 and STAT5 phosphorylation. {yields} IL-2-dependent regulatory T-lymphocyte function and Foxp3 expression is downregulated by curcumin. -- Abstract: Curcumin (diferulomethane) is the principal curcuminoid in the spice tumeric and a potent inhibitor of activation-induced T-lymphocyte proliferation; however, the molecular basis of this immunosuppressive effect has not been well studied. Here we show that micromolar concentrations of curcumin inhibited DNA synthesis by mouse CD4{sup +} T-lymphocytes, as well as interleukin-2 (IL-2) and CD25 ({alpha} chain of the high affinity IL-2 receptor) expression in response to antibody-mediated cross-linking of CD3 and CD28. Curcumin acted downstream of protein kinase C activation and intracellular Ca{sup 2+} release to inhibit I{kappa}B phosphorylation, which is required for nuclear translocation of the transcription factor NF{kappa}B. In addition, IL-2-dependent DNA synthesis by mouse CTLL-2 cells, but not constitutive CD25 expression, was impaired in the presence of curcumin, which demonstrated an inhibitory effect on IL-2 receptor (IL-2R) signaling. IL-2-induced phosphorylation of STAT5A and JAK3, but not JAK1, was diminished in the presence of curcumin, indicating inhibition of critical proximal events in IL-2R signaling. In line with the inhibitory action of curcumin on IL-2R signaling, pretreatment of CD4{sup +}CD25{sup +} regulatory T-cells with curcumin downregulated suppressor function, as well as forkhead box p3 (Foxp3) expression. We conclude that curcumin inhibits IL-2 signaling by reducing available IL-2 and high affinity IL-2R, as well as interfering with IL-2R signaling.

  10. Bcr-aBL1 kinase domain mutation analysis in chronic myeloid leukaemia patients with suboptimal response to tyrosine kinase inhibitors

    Directory of Open Access Journals (Sweden)

    Tadej Pajič

    2012-12-01

    Conclusions: It seems that the BCR-ABl1 mutations are rare in patients who do not achieve a MMR by 18 months or more or who have lost MMR. The T315I mutation detected in one patient in our cohort of CML patients indicates that the BCR-ABL1 mutation analysis could be recommended in these cases. The silent mutation detected did not lead to amino acid change, however, it is listed in major single nucleotide polymorphisms databases (SNP, rs2227985. The role of the SNP in the resistance to TKIs is not clear.

  11. Assembly of Oligomeric Death Domain Complexes during Toll Receptor Signaling*

    OpenAIRE

    Moncrieffe, Martin C.; Grossmann, J. Günter; Gay, Nicholas J.

    2008-01-01

    The Drosophila Toll receptor is activated by the endogenous protein ligand Spätzle in response to microbial stimuli in immunity and spatial cues during embryonic development. Downstream signaling is mediated by the adaptor proteins Tube, the kinase Pelle, and the Drosophila homologue of myeloid differentiation primary response protein (dMyD88). Here we have characterized heterodimeric (dMyD88-Tube) and heterotrimeric (dMyD88-Tube-Pelle) death domain complexes. We show ...

  12. Progesterone receptors (PR) mediate STAT actions: PR and prolactin receptor signaling crosstalk in breast cancer models.

    Science.gov (United States)

    Leehy, Katherine A; Truong, Thu H; Mauro, Laura J; Lange, Carol A

    2018-02-01

    Estrogen is the major mitogenic stimulus of mammary gland development during puberty wherein ER signaling acts to induce abundant PR expression. PR signaling, in contrast, is the primary driver of mammary epithelial cell proliferation in adulthood. The high circulating levels of progesterone during pregnancy signal through PR, inducing expression of the prolactin receptor (PRLR). Cooperation between PR and prolactin (PRL) signaling, via regulation of downstream components in the PRL signaling pathway including JAKs and STATs, facilitates the alveolar morphogenesis observed during pregnancy. Indeed, these pathways are fully integrated via activation of shared signaling pathways (i.e. JAKs, MAPKs) as well as by the convergence of PRs and STATs at target genes relevant to both mammary gland biology and breast cancer progression (i.e. proliferation, stem cell outgrowth, tissue cell type heterogeneity). Thus, rather than a single mediator such as ER, transcription factor cascades (ER>PR>STATs) are responsible for rapid proliferative and developmental programming in the normal mammary gland. It is not surprising that these same mediators typify uncontrolled proliferation in a majority of breast cancers, where ER and PR are most often co-expressed and may cooperate to drive malignant tumor progression. This review will primarily focus on the integration of PR and PRL signaling in breast cancer models and the importance of this cross-talk in cancer progression in the context of mammographic density. Components of these PR/PRL signaling pathways could offer alternative drug targets and logical complements to anti-ER or anti-estrogen-based endocrine therapies. Copyright © 2017 Elsevier Ltd. All rights reserved.

  13. Embelin and its derivatives unravel the signaling, proinflammatory and antiatherogenic properties of GPR84 receptor.

    Science.gov (United States)

    Gaidarov, Ibragim; Anthony, Todd; Gatlin, Joel; Chen, Xiaohua; Mills, David; Solomon, Michelle; Han, Sangdon; Semple, Graeme; Unett, David J

    2018-05-01

    GPR84 is an orphan G-protein coupled receptor, expressed on monocytes, macrophages and neutrophils and is significantly upregulated by inflammatory stimuli. The physiological role of GPR84 remains largely unknown. Medium chain fatty acids (MCFA) activate the receptor and have been proposed to be its endogenous ligands, although the high concentrations of MCFAs required for receptor activation generally exceed normal physiological levels. We identified the natural product embelin as a highly potent and selective surrogate GPR84 agonist (originally disclosed in patent application WO2007027661A2, 2007) and synthesized close structural analogs with widely varying receptor activities. These tools were used to perform a comprehensive study of GPR84 signaling and function in recombinant cells and in primary human macrophages and neutrophils. Activation of recombinant GPR84 by embelin in HEK293 cells results in G i/o as well as G12/13-Rho signaling. In human macrophages, GPR84 initiates PTX sensitive Erk1/2 and Akt phosphorylation, PI-3 kinase activation, calcium flux, and release of prostaglandin E2. In addition, GPR84 signaling in macrophages elicits G i Gβγ-mediated augmentation of intracellular cAMP, rather than the decrease expected from G iα engagement. GPR84 activation drives human neutrophil chemotaxis and primes them for amplification of oxidative burst induced by FMLP and C5A. Loss of GPR84 is associated with attenuated LPS-induced release of proinflammatory mediators IL-6, KC-GROα, VEGF, MIP-2 and NGAL from peritoneal exudates. While initiating numerous proinflammatory activities in macrophages and neutrophils, GPR84 also possesses GPR109A-like antiatherosclerotic properties in macrophages. Macrophage receptor activation leads to upregulation of cholesterol transporters ABCA1 and ABCG1 and stimulates reverse cholesterol transport. These data suggest that GPR84 may be a target of therapeutic value and that distinct modes of receptor modulation (inhibition vs

  14. [JAK2 V617F and exon 12 genetic variations in Korean patients with BCR/ABL1-negative myeloproliferative neoplasms].

    Science.gov (United States)

    Kim, Jeong Tae; Cho, Yong Gon; Choi, Sam Im; Lee, Young Jin; Kim, Hye Ran; Jang, Sook Jin; Moon, Dae Soo; Park, Young Jin; Park, Geon

    2010-12-01

    JAK2 genetic variations have been described in a high proportion of patients with BCR/ABL1-negative myeloproliferative neoplasms (MPN). This study was designed to analyze the frequencies of JAK2 V617F and exon 12 variations, and their correlations with clinical characteristics of Korean patients with BCR/ABL1-negative MPN. We examined a total of 154 patients with BCR/ABL1-negative MPN that included 24, 26, 89, and 15 patients with polycythemia vera (PV), primary myelofibrosis (PMF), essential thrombocythemia (ET), and unclassified myeloproliferative neoplasms (MPNU), respectively. We performed allele-specific PCR to detect V617F in all BCR/ABL1-negative patients, and performed direct sequencing to detect exon 12 variations in 47 V617F-negative MPN patients. JAK2 c.1641+179_183del5 variation was detected by restriction fragment length polymorphism assay in 176 healthy subjects. JAK2 V617F was detected in 91 patients (59.1%): PV (91.6%), PMF (46.2%), ET (52.8%), and MPNU (66.7%). In V617F-negative MPN patients, no mutations were found in exon 12. The c.1641+179_183del5 was detected in 68.1% of V617F-negative MPN patients and 45.4% of healthy subjects (P=0.008). JAK2 V617F was closely correlated with age and leukocytosis in BCR/ABL1-negative MPN patients (P<0.05). However, c.1641+179_183del5 was not related to age, sex, or complete blood cell count parameters in V617F-negative MPN patients and healthy subjects. The c.1641+179_183del5 was associated with an increased odds ratio for MPN (odds ratio, 2.6; 95% confidences interval, 1.3-5.1; P=0.007). Frequencies of V617F are similar to reported results. JAK2 exon 12 mutations may be rare and c.1641+179_183del5 may influence the occurrence of MPN in Korean patients with V6 17F-negative MPN.

  15. Receptor activity modifying proteins (RAMPs) interact with the VPAC1 receptor: evidence for differential RAMP modulation of multiple signalling pathways

    International Nuclear Information System (INIS)

    Christopoulos, G.; Morfis, M.; Sexton, P.M.; Christopoulos, A.; Laburthe, M.; Couvineau, A.

    2001-01-01

    Full text: Receptor activity modifying proteins (RAMP) constitute a family of three accessory proteins that affect the expression and/or phenotype of the calcitonin receptor (CTR) or CTR-like receptor (CRLR). In this study we screened a range of class II G protein-coupled receptors (PTH1, PTH2, GHRH, VPAC1, VPAC2 receptors) for possible RAMP interactions by measurement of receptor-induced translocation of c-myc tagged RAMP1 or HA tagged RAMP3. Of these, only the VPAC1 receptor caused significant translocation of c-myc-RAMP1 or HA-RAMP3 to the cell surface. Co-transfection of VPAC1 and RAMPs did not alter 125 I-VIP binding and specificity. VPAC1 receptor function was subsequently analyzed through parallel determinations of cAMP accumulation and phosphoinositide (PI) hydrolysis in the presence and absence of each of the three RAMPs. In contrast to CTR-RAMP interaction, where there was an increase in cAMP Pharmacologisand a decrease in PI hydrolysis, VPAC1-RAMP interaction was characterized by a specific increase in agonist-mediated PI hydrolysis when co-transfected with RAMP2. This change was due to an enhancement of Emax with no change in EC 50 value for VIP. No significant change in cAMP accumulation was observed. This is the first demonstration of an interaction of RAMPs with a G protein-coupled receptor outside the CTR family and may suggest a more generalized role for RAMPs in modulating G protein-coupled receptor signaling. Copyright (2001) Australasian Society of Clinical and Experimental Pharmacologists and Toxicologists

  16. Molecular Mechanisms of SH2- and PTB-Domain-Containing Proteins in Receptor Tyrosine Kinase Signaling

    Science.gov (United States)

    Wagner, Melany J.; Stacey, Melissa M.; Liu, Bernard A.; Pawson, Tony

    2013-01-01

    Intracellular signaling is mediated by reversible posttranslational modifications (PTMs) that include phosphorylation, ubiquitination, and acetylation, among others. In response to extracellular stimuli such as growth factors, receptor tyrosine kinases (RTKs) typically dimerize and initiate signaling through phosphorylation of their cytoplasmic tails and downstream scaffolds. Signaling effectors are recruited to these phosphotyrosine (pTyr) sites primarily through Src homology 2 (SH2) domains and pTyr-binding (PTB) domains. This review describes how these conserved domains specifically recognize pTyr residues and play a major role in mediating precise downstream signaling events. PMID:24296166

  17. Molecular mechanisms of SH2- and PTB-domain-containing proteins in receptor tyrosine kinase signaling.

    Science.gov (United States)

    Wagner, Melany J; Stacey, Melissa M; Liu, Bernard A; Pawson, Tony

    2013-12-01

    Intracellular signaling is mediated by reversible posttranslational modifications (PTMs) that include phosphorylation, ubiquitination, and acetylation, among others. In response to extracellular stimuli such as growth factors, receptor tyrosine kinases (RTKs) typically dimerize and initiate signaling through phosphorylation of their cytoplasmic tails and downstream scaffolds. Signaling effectors are recruited to these phosphotyrosine (pTyr) sites primarily through Src homology 2 (SH2) domains and pTyr-binding (PTB) domains. This review describes how these conserved domains specifically recognize pTyr residues and play a major role in mediating precise downstream signaling events.

  18. Orexin/Hypocretin Signaling.

    Science.gov (United States)

    Kukkonen, Jyrki P

    Orexin/hypocretin peptide (orexin-A and orexin-B) signaling is believed to take place via the two G-protein-coupled receptors (GPCRs), named OX 1 and OX 2 orexin receptors, as described in the previous chapters. Signaling of orexin peptides has been investigated in diverse endogenously orexin receptor-expressing cells - mainly neurons but also other types of cells - and in recombinant cells expressing the receptors in a heterologous manner. Findings in the different systems are partially convergent but also indicate cellular background-specific signaling. The general picture suggests an inherently high degree of diversity in orexin receptor signaling.In the current chapter, I present orexin signaling on the cellular and molecular levels. Discussion of the connection to (potential) physiological orexin responses is only brief since these are in focus of other chapters in this book. The same goes for the post-synaptic signaling mechanisms, which are dealt with in Burdakov: Postsynaptic actions of orexin. The current chapter is organized according to the tissue type, starting from the central nervous system. Finally, receptor signaling pathways are discussed across tissues, cell types, and even species.

  19. Serotonin 2A Receptor Signaling Underlies LSD-induced Alteration of the Neural Response to Dynamic Changes in Music.

    Science.gov (United States)

    Barrett, Frederick S; Preller, Katrin H; Herdener, Marcus; Janata, Petr; Vollenweider, Franz X

    2017-09-28

    Classic psychedelic drugs (serotonin 2A, or 5HT2A, receptor agonists) have notable effects on music listening. In the current report, blood oxygen level-dependent (BOLD) signal was collected during music listening in 25 healthy adults after administration of placebo, lysergic acid diethylamide (LSD), and LSD pretreated with the 5HT2A antagonist ketanserin, to investigate the role of 5HT2A receptor signaling in the neural response to the time-varying tonal structure of music. Tonality-tracking analysis of BOLD data revealed that 5HT2A receptor signaling alters the neural response to music in brain regions supporting basic and higher-level musical and auditory processing, and areas involved in memory, emotion, and self-referential processing. This suggests a critical role of 5HT2A receptor signaling in supporting the neural tracking of dynamic tonal structure in music, as well as in supporting the associated increases in emotionality, connectedness, and meaningfulness in response to music that are commonly observed after the administration of LSD and other psychedelics. Together, these findings inform the neuropsychopharmacology of music perception and cognition, meaningful music listening experiences, and altered perception of music during psychedelic experiences. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  20. GPR30 is necessary for estradiol-induced desensitization of 5-HT1A receptor signaling in the paraventricular nucleus of the rat hypothalamus.

    Science.gov (United States)

    McAllister, C E; Creech, R D; Kimball, P A; Muma, N A; Li, Q

    2012-08-01

    Estrogen therapy used in combination with selective serotonin reuptake inhibitor (SSRI) treatment improves SSRI efficacy for the treatment of mood disorders. Desensitization of serotonin 1A (5-HT(1A)) receptors, which takes one to two weeks to develop in animals, is necessary for SSRI therapeutic efficacy. Estradiol modifies 5-HT(1A) receptor signaling and induces a partial desensitization in the paraventricular nucleus (PVN) of the rat within two days, but the mechanisms underlying this effect are currently unknown. The purpose of this study was to identify the estrogen receptor necessary for estradiol-induced 5-HT(1A) receptor desensitization. We previously showed that estrogen receptor β is not necessary for 5-HT(1A) receptor desensitization and that selective activation of estrogen receptor GPR30 mimics the effects of estradiol in rat PVN. Here, we used a recombinant adenovirus containing GPR30 siRNAs to decrease GPR30 expression in the PVN. Reduction of GPR30 prevented estradiol-induced desensitization of 5-HT(1A) receptor as measured by hormonal responses to the selective 5-HT(1A) receptor agonist, (+)8-OH-DPAT. To determine the possible mechanisms underlying these effects, we investigated protein and mRNA levels of 5-HT(1A) receptor signaling components including 5-HT(1A) receptor, Gαz, and RGSz1. We found that two days of estradiol increased protein and mRNA expression of RGSz1, and decreased 5-HT(1A) receptor protein but increased 5-HT(1A) mRNA; GPR30 knockdown prevented the estradiol-induced changes in 5-HT(1A) receptor protein in the PVN. Taken together, these data demonstrate that GPR30 is necessary for estradiol-induced changes in the 5-HT(1A) receptor signaling pathway and desensitization of 5-HT(1A) receptor signaling. Copyright © 2012 Elsevier Ltd. All rights reserved.

  1. P2X7 receptor activates extracellular signal-regulated kinases ERK1 and ERK2 independently of Ca2+ influx

    DEFF Research Database (Denmark)

    Amstrup, Jan; Novak, Ivana

    2003-01-01

    P2X7 nucleotide receptors modulate a spectrum of cellular events in various cells including epithelia, such as exocrine pancreas. Although the pharmacology and channel properties of the P2X7 receptors have been studied intensively, signal transduction pathways are relatively unknown. In this study...... we applied a heterologous expression system of rat P2X7 receptors in HEK-293 cells. We followed the receptor expression and function using the enhanced green fluorescent protein (EGFP) tag, activation of intracellular proteins and increases in cellular Ca2+. EGFP-P2X7 receptors localized...... to the plasma membrane, clusters within the membrane and intracellularly. Stimulation of P2X7 receptors in HEK-293 cells led to an activation of extracellular signal-regulated kinases ERK1 and ERK2 and this activation was seen after just 1 min of stimulation with ATP. Using C- and N-terminal P2X7-receptor...

  2. Differential actions of orexin receptors in brainstem cholinergic and monoaminergic neurons revealed by receptor knockouts: implications for orexinergic signaling in arousal and narcolepsy

    Directory of Open Access Journals (Sweden)

    Kristi A Kohlmeier

    2013-12-01

    Full Text Available Orexin neuropeptides influence multiple homeostatic functions and play an essential role in the expression of normal sleep-wake behavior. While their two known receptors (OX1 and OX2 are targets for novel pharmacotherapeutics, the actions mediated by each receptor remain largely unexplored. Using brain slices from mice constitutively lacking either receptor, we used whole-cell and Ca2+ imaging methods to delineate the cellular actions of each receptor within cholinergic (laterodorsal tegmental nucleus; LDT and monoaminergic (dorsal raphe; DR and locus coeruleus; LC brainstem nuclei – where orexins promote arousal and suppress REM sleep. In slices from OX2-/- mice, orexin-A (300 nM elicited wild-type responses in LDT, DR and LC neurons consisting of a depolarizing current and augmented voltage-dependent Ca2+ transients. In slices from OX1-/- mice, the depolarizing current was absent in LDT and LC neurons and was attenuated in DR neurons, although Ca2+-transients were still augmented. Since orexin-A produced neither of these actions in slices lacking both receptors, our findings suggest that orexin-mediated depolarization is mediated by both receptors in DR, but is exclusively mediated by OX1 in LDT and LC neurons, even though OX2 is present and OX2 mRNA appears elevated in brainstems from OX1-/- mice. Considering published behavioral data, these findings support a model in which orexin-mediated excitation of mesopontine cholinergic and monoaminergic neurons contributes little to stabilizing spontaneous waking and sleep bouts, but functions in context-dependent arousal and helps restrict muscle atonia to REM sleep. The augmented Ca2± transients mediated by both receptors appeared mediated by influx via L-type Ca2+ channels, which is often linked to transcriptional signaling. This could provide an adaptive signal to compensate for receptor loss or prolonged antagonism and may contribute to the reduced severity of narcolepsy in single receptor

  3. BCR-crosslinking induces a transcription of protein phosphatase component G5PR that is required for mature B-cell survival

    International Nuclear Information System (INIS)

    Huq Ronny, Faisal Mahmudul; Igarashi, Hideya; Sakaguchi, Nobuo

    2006-01-01

    BCR-crosslinking triggers activation-induced cell death (AICD) selectively in the restricted stage of B-cell differentiation. We examined the transcription of a protein phosphatase subunit G5PR in immature and mature B-cells, because absence of this factor augmented cell sensitivity to AICD, associated with increased activation of JNK and Bim. BCR-crosslinking-induced G5pr transcription in AICD-resistant mature splenic IgM lo IgD hi B-cells but not in AICD susceptible immature IgM hi IgD lo B-cells. Thus, G5pr induction correlated with the prevention of AICD; High in mature splenic CD23 hi B-cells but low in immature B-cells of neonatal mice, sub-lethally irradiated mice, or xid mice. Lack of G5pr upregulation was associated with the prolonged activation of JNK. The G5pr cDNA transfection protected an immature B-cell line WEHI-231 from BCR-mediated AICD. The differential expression of G5PR might be responsible for the antigen-dependent selection of B-cells

  4. Use of the modified BCR three-step sequential extraction procedure for the study of trace element dynamics in contaminated soils

    International Nuclear Information System (INIS)

    Pueyo, M.; Mateu, J.; Rigol, A.; Vidal, M.; Lopez-Sanchez, J.F.; Rauret, G.

    2008-01-01

    The modified BCR three-step sequential extraction procedure was used to examine the temporal dynamics of trace elements in soils contaminated by an accidental spill from an opencast mine in south-west Spain. Soils were mainly contaminated with pyritic sludge and acidic wastewater, whereas some soils were affected only by acidic wastewater. The distributions obtained for both some major (Ca, Fe and Mn) and trace elements (As, Cd, Cu, Pb and Zn) in the sludge and soil samples taken at different times after the accident, 1-3 months and 21 months, were compared. Sequential extractions were useful in identifying different sources of contamination, and in obtaining additional information on the solubility of secondary minerals formed by pyrite oxidation. Thus, the effectiveness of the BCR procedure has proved to be a useful tool for predicting short- and long-term mobility of trace elements, even in complex environmental scenarios. - The modified BCR three-step sequential extraction procedure has proved a useful prediction tool for short- and long-term mobility of trace elements in contaminated soils

  5. ON012380: A Non-ATP Competitive Inhibitor of BCR-ABL for the Therapy of Imatinib-Resistant CMLs

    National Research Council Canada - National Science Library

    Reddy, E. P

    2007-01-01

    Because it is now apparent that a significant proportion of patients chronically treated with imatinib develop resistance due to the acquisition of mutations in the kinase domain of BCR-ABL our aim...

  6. In Vivo Phosphoproteomics Analysis Reveals the Cardiac Targets of β-Adrenergic Receptor Signaling

    DEFF Research Database (Denmark)

    Lundby, Alicia; Andersen, Martin N; Steffensen, Annette B

    2013-01-01

    β-Blockers are widely used to prevent cardiac arrhythmias and to treat hypertension by inhibiting β-adrenergic receptors (βARs) and thus decreasing contractility and heart rate. βARs initiate phosphorylation-dependent signaling cascades, but only a small number of the target proteins are known. We...

  7. Regulation of EGF receptor signaling by the MARVEL domain-containing protein CKLFSF8.

    Science.gov (United States)

    Jin, Caining; Ding, Peiguo; Wang, Ying; Ma, Dalong

    2005-11-21

    It is known that chemokine-like factor superfamily 8 (CKLFSF8), a member of the CKLF superfamily, has four putative transmembrane regions and a MARVEL domain. Its structure is similar to TM4SF11 (plasmolipin) and widely distributed in normal tissue. However, its function is not yet known. We show here that CKLFSF8 is associated with the epidermal growth factor receptor (EGFR) and that ectopic expression of CKLFSF8 in several cell lines suppresses EGF-induced cell proliferation, whereas knockdown of CKLFSF8 by siRNA promotes cell proliferation. In cells overexpressing CKLFSF8, the initial activation of EGFR was not affected, but subsequent desensitization of EGF-induced signaling occurred rapidly. This attenuation was correlated with an increased rate of receptor endocytosis. In contrast, knockdown of CKLFSF8 by siCKLFSF8 delayed EGFR endocytosis. These results identify CKLFSF8 as a novel regulator of EGF-induced signaling and indicate that the association of EGFR with four transmembrane proteins is critical for EGFR desensitization.

  8. Distinct Signaling Cascades Elicited by Different Formyl Peptide Receptor 2 (FPR2 Agonists

    Directory of Open Access Journals (Sweden)

    Fabio Cattaneo

    2013-04-01

    Full Text Available The formyl peptide receptor 2 (FPR2 is a remarkably versatile transmembrane protein belonging to the G-protein coupled receptor (GPCR family. FPR2 is activated by an array of ligands, which include structurally unrelated lipids and peptide/proteins agonists, resulting in different intracellular responses in a ligand-specific fashion. In addition to the anti-inflammatory lipid, lipoxin A4, several other endogenous agonists also bind FPR2, including serum amyloid A, glucocorticoid-induced annexin 1, urokinase and its receptor, suggesting that the activation of FPR2 may result in potent pro- or anti-inflammatory responses. Other endogenous ligands, also present in biological samples, include resolvins, amyloidogenic proteins, such as beta amyloid (Aβ-42 and prion protein (Prp106–126, the neuroprotective peptide, humanin, antibacterial peptides, annexin 1-derived peptides, chemokine variants, the neuropeptides, vasoactive intestinal peptide (VIP and pituitary adenylate cyclase activating polypeptide (PACAP-27, and mitochondrial peptides. Upon activation, intracellular domains of FPR2 mediate signaling to G-proteins, which trigger several agonist-dependent signal transduction pathways, including activation of phospholipase C (PLC, protein kinase C (PKC isoforms, the phosphoinositide 3-kinase (PI3K/protein kinase B (Akt pathway, the mitogen-activated protein kinase (MAPK pathway, p38MAPK, as well as the phosphorylation of cytosolic tyrosine kinases, tyrosine kinase receptor transactivation, phosphorylation and nuclear translocation of regulatory transcriptional factors, release of calcium and production of oxidants. FPR2 is an attractive therapeutic target, because of its involvement in a range of normal physiological processes and pathological diseases. Here, we review and discuss the most significant findings on the intracellular pathways and on the cross-communication between FPR2 and tyrosine kinase receptors triggered by different FPR2

  9. CALR, JAK2 and MPL mutation status in Argentinean patients with BCR-ABL1- negative myeloproliferative neoplasms.

    Science.gov (United States)

    Ojeda, Mara Jorgelina; Bragós, Irma Margarita; Calvo, Karina Lucrecia; Williams, Gladis Marcela; Carbonell, María Magdalena; Pratti, Arianna Flavia

    2018-05-01

    To establish the frequency of JAK2, MPL and CALR mutations in Argentinean patients with BCR-ABL1-negative  myeloproliferative neoplasms (MPN) and to compare their clinical and haematological features. Mutations of JAK2V617F, JAK2 exon 12, MPL W515L/K and CALR were analysed in 439 Argentinean patients with BCR-ABL1-negative MPN, including 176 polycythemia vera (PV), 214 essential thrombocythemia (ET) and 49 primary myelofibrosis (PMF). In 94.9% of PV, 85.5% ET and 85.2% PMF, we found mutations in JAK2, MPL or CALR. 74.9% carried JAK2V617F, 12.3% CALR mutations, 2.1% MPL mutations and 10.7% were triple negative. In ET, nine types of CALR mutations were identified, four of which were novel. PMF patients were limited to types 1 and 2, type 2 being more frequent. In ET, patients with CALR mutation were younger and had higher platelet counts than those with JAK2V617F and triple negative. In addition, JAK2V617F patients had high leucocyte and haemoglobin values compared with CALR-mutated and triple-negative patients. In PMF, patients with mutant CALR were associated with higher platelet counts. Our study underscores the importance of JAK2, MPL and CALR genotyping for accurate diagnosis of patients with BCR-ABL1-negative MPN.

  10. Atrial natriuretic factor receptor guanylate cyclase, ANF-RGC, transduces two independent signals, ANF and Ca2+

    Directory of Open Access Journals (Sweden)

    Teresa eDuda

    2014-03-01

    Full Text Available Atrial natriuretic factor receptor guanylate cyclase, ANF-RGC, was the first discovered member of the mammalian membrane guanylate cyclase family. The hallmark feature of the family is that a single protein contains both the site for recognition of the regulatory signal and the ability to transduce it into the production of the second messenger, cyclic GMP. For over two decades, the family has been classified into two subfamilies, the hormone receptor subfamily with ANF-RGC being its paramount member, and the Ca2+ modulated subfamily, which includes the rod outer segment guanylate cyclases, ROS-GC1 and 2, and the olfactory neuroepithelial guanylate cyclase, ONE-GC. ANF-RGC is the receptor and the signal transducer of the most hypotensive hormones, atrial natriuretic factor (ANF and B-type natriuretic peptide (BNP. After binding these hormones at the extracellular domain it, at its intracellular domain, signals activation of the C-terminal catalytic module and accelerates the production of cyclic GMP. Cyclic GMP then serves the second messenger role in biological responses of ANF and BNP such as natriuresis, diuresis, vasorelaxation and anti-proliferation. Very recently another modus operandi for ANF-RGC was revealed. Its crux is that ANF-RGC activity is also regulated by Ca2+. The Ca2+ sensor neurocalcin  mediates this signaling mechanism. Strikingly, the Ca2+ and ANF signaling mechanisms employ separate structural motifs of ANF-RGC in modulating its core catalytic domain in accelerating the production of cyclic GMP. In this review the biochemistry and physiology of these mechanisms with emphasis on cardiovascular regulation will be discussed.

  11. Determination of lactate dehydrogenase (LDH and Bcr-Abl transcript in the follow-up of patients with chronic myeloid leukemia = Determinação da lactate desidrogenase (LDH e do transcrito Bcr-Abl em pacientes com leucemia mielóide crônica

    Directory of Open Access Journals (Sweden)

    Roberto Iemitsu Tatakihara

    2010-07-01

    Full Text Available Chronic myeloid leukemia (CML is a malignant myeloproliferative disorder that originates from a pluripotent stem cell characterized by abnormal release of the expanded, malignant stem cell clone from the bone marrow into the bloodstream. The vast majority of patients with CML present Bcr-Abl transcripts. Lactate dehydrogenase (LDH is considered a biochemical marker common for tumor growth, anaerobic glycolysis and has been considered a poor prognostic factor for acute myeloid leukemia. Therefore, this study aimed to evaluate the concentration of LDH in plasma and the detection of the Bcr-Abl transcripts in patients with CML and healthy donors. We analyzed 22 patients demonstrably diagnosed with CML and 56 healthy donors. LDH concentration in plasma was higher in patients with CML. All patients with CML in this study were under treatment, but even so four patients had the Bcr-Abl (b3a2 transcript in peripheral blood. Two out of the four patients with b3a2 showed higher LDH (486 U L-1 and 589 U L-1. Thus, although the study was conducted with small numbers of samples, it is possible to suggest therapy alteration for two patients who presented transcript b3a2 in the peripheral blood samples and whose LDH concentration was high, in order to improve the disease. Leucemia mieloide crônica (LMC é uma desordem mieloproliferativa maligna que é originada de célula-tronco pluripotente caracterizada por expansão anormal, maligna de clones de células tronco da medula óssea na circulação. A grande maioria dos pacientes com LMC apresentam transcritos Bcr-Abl. Lactato desidrogenase (LDH,considerado um marcador bioquímico para crescimento tumoral, glicólise anaeróbica, e tem sido considerado um fator de pior prognóstico da LMC. Portanto, este estudo visa avaliar a concentraçãode LDH no plasma e a detecção do transcrito Bcr-Abl em 22 pacientes com LMC e 56 indivíduos saudáveis. Foram avaliados 22 pacientes com LMC e 56 doadores saudáveis. A

  12. Successful treatment with allogeneic stem cell transplantation followed by DLI and TKIs for e6a2 BCR-ABL-positive acute myeloid leukaemia

    Science.gov (United States)

    Harada, Yasuhiko; Nishiwaki, Satoshi; Sugimoto, Takumi; Onodera, Koichi; Goto, Tatsunori; Sato, Takahiko; Kamoshita, Sonoko; Kawashima, Naomi; Seto, Aika; Okuno, Shingo; Yamamoto, Satomi; Iwasaki, Toshihiro; Ozawa, Yukiyasu; Miyamura, Koichi; Akatsuka, Yoshiki; Sugiura, Isamu

    2017-01-01

    Abstract Rationale: Patients with the e6a2 BCR-ABL transcript, 1 of the atypical transcripts, have been reported to have a poor prognosis, and allogeneic stem cell transplantation (ASCT) can be considered as additional therapy. However, long-term survival after ASCT for this disease is rare. Patient concerns: This report concerns a 55-year-old female patient with e6a2 BCR-ABL-positive acute myeloid leukemia including the outcome of ASCT followed by donor lymphocyte infusion (DLI). Diagnoses: The breakpoint was confirmed by direct sequencing. Her minimal residual disease could be detected by nested reverse-transcription polymerase chain reaction using primers for the minor BCR-ABL (e1a2) transcript. Interventions: Treatment with tyrosine kinase inhibitors (TKIs) and ASCT followed by DLI. Outcomes: Despite multiple cytogenetic and molecular relapses after ASCT, she remains in molecular remission at 46 months after ASCT. Lessons: This case indicates the efficacy of the combination of the graft-versus-leukemia effect and TKIs for e6a2 BCR-ABL-positive acute leukemia. When the Philadelphia chromosome with an unusual chromosomal breakpoint is suggested, we should clarify the breakpoint because that information can aid molecular assessments and decisions to provide an additional or alternative therapy. PMID:29390324

  13. δ-opioid receptor and somatostatin receptor-4 heterodimerization: possible implications in modulation of pain associated signaling.

    Directory of Open Access Journals (Sweden)

    Rishi K Somvanshi

    Full Text Available Pain relief is the principal action of opioids. Somatostatin (SST, a growth hormone inhibitory peptide is also known to alleviate pain even in cases when opioids fail. Recent studies have shown that mice are prone to sustained pain and devoid of analgesic effect in the absence of somatostatin receptor 4 (SSTR4. In the present study, using brain slices, cultured neurons and HEK-293 cells, we showed that SSTR4 and δ-Opioid receptor (δOR exist in a heteromeric complex and function in synergistic manner. SSTR4 and δOR co-expressed in cortical/striatal brain regions and spinal cord. Using cultured neuronal cells, we describe the heterogeneous complex formation of SSTR4 and δOR at neuronal cell body and processes. Cotransfected cells display inhibition of cAMP/PKA and co-activation of SSTR4 and δOR oppose receptor trafficking induced by individual receptor activation. Furthermore, downstream signaling pathways either associated with withdrawal or pain relief are modulated synergistically with a predominant role of SSTR4. Inhibition of cAMP/PKA and activation of ERK1/2 are the possible cellular adaptations to prevent withdrawal induced by chronic morphine use. Our results reveal direct intra-membrane interaction between SSTR4 and δOR and provide insights for the molecular mechanism for the anti-nociceptive property of SST in combination with opioids as a potential therapeutic approach to avoid undesirable withdrawal symptoms.

  14. Evolution of BCR/ABL gene mutation in CML is time dependent and dependent on the pressure exerted by tyrosine kinase inhibitor.

    Directory of Open Access Journals (Sweden)

    Shantashri Vaidya

    Full Text Available BACKGROUND: Mutations in the ABL kinase domain and SH3-SH2 domain of the BCR/ABL gene and amplification of the Philadelphia chromosome are the two important BCR/ABL dependent mechanisms of imatinib resistance. Here, we intended to study the role played by TKI, imatinib, in selection of gene mutations and development of chromosomal abnormalities in Indian CML patients. METHODS: Direct sequencing methodology was employed to detect mutations and conventional cytogenetics was done to identify Philadelphia duplication. RESULTS: Among the different mechanisms of imatinib resistance, kinase domain mutations (39% of the BCR/ABL gene were seen to be more prevalent, followed by mutations in the SH3-SH2 domain (4% and then BCR/ABL amplification with the least frequency (1%. The median duration of occurrence of mutation was significantly shorter for patients with front line imatinib than those pre-treated with hydroxyurea. Patients with high Sokal score (p = 0.003 showed significantly higher incidence of mutations, as compared to patients with low/intermediate score. Impact of mutations on the clinical outcome in AP and BC was observed to be insignificant. Of the 94 imatinib resistant patients, only 1 patient exhibited duplication of Philadelphia chromosome, suggesting a less frequent occurrence of this abnormality in Indian CML patients. CONCLUSION: Close monitoring at regular intervals and proper analysis of the disease resistance would facilitate early detection of resistance and thus aid in the selection of the most appropriate therapy.

  15. IL-6/IL-12 Cytokine Receptor Shuffling of Extra- and Intracellular Domains Reveals Canonical STAT Activation via Synthetic IL-35 and IL-39 Signaling.

    Science.gov (United States)

    Floss, D M; Schönberg, M; Franke, M; Horstmeier, F C; Engelowski, E; Schneider, A; Rosenfeldt, E M; Scheller, J

    2017-11-09

    IL-35 and IL-39 are recently discovered shared members of the IL-6- and IL-12-type cytokine family with immune-suppressive capacity. IL-35 has been reported to induce the formation of four different receptor complexes: gp130:IL-12β2, gp130:gp130, IL-12β2:IL-12β2, and IL-12β2:WSX-1. IL-39 was proposed to form a gp130:IL-23R receptor complex. IL-35, but not IL-39, has been reported to activate non-conventional STAT signaling, depending on the receptor complex and target cell. Analyses of IL-35 and IL-39 are, however, hampered by the lack of biologically active recombinant IL-35 and IL-39 proteins. Therefore, we engineered chimeric cytokine receptors to accomplish synthetic IL-35 and IL- 39 signaling by shuffling the extra- and intracellular domains of IL-6/IL-12-type cytokine receptors, resulting in biological activity for all previously described IL-35 receptor complexes. Moreover, we found that the proposed IL-39 receptor complex is biologically active and discovered two additional biologically active synthetic receptor combinations, gp130/IL-12Rβ1 and IL-23R/IL-12Rβ2. Surprisingly, synthetic IL-35 activation led to more canonical STAT signaling of all receptor complexes. In summary, our receptor shuffling approach highlights an interchangeable, modular domain structure among IL-6- and IL-12-type cytokine receptors and enabled synthetic IL-35 and IL-39 signaling.

  16. Nitric oxide from inflammatory origin impairs neural stem cell proliferation by inhibiting epidermal growth factor receptor signaling

    Directory of Open Access Journals (Sweden)

    Bruno Pereira Carreira

    2014-10-01

    Full Text Available Neuroinflammation is characterized by activation of microglial cells, followed by production of nitric oxide (NO, which may have different outcomes on neurogenesis, favoring or inhibiting this process. In the present study, we investigated how the inflammatory mediator NO can affect proliferation of neural stem cells (NSC, and explored possible mechanisms underlying this effect. We investigated which mechanisms are involved in the regulation of NSC proliferation following treatment with an inflammatory stimulus (LPS plus IFN-γ, using a culture system of subventricular zone (SVZ-derived NSC mixed with microglia cells obtained from wild-type mice (iNOS+/+ or from iNOS knockout mice (iNOS-/-. We found an impairment of NSC cell proliferation in iNOS+/+ mixed cultures, which was not observed in iNOS-/- mixed cultures. Furthermore, the increased release of NO by activated iNOS+/+ microglial cells decreased the activation of the ERK/MAPK signaling pathway, which was concomitant with an enhanced nitration of the EGF receptor. Preventing nitrogen reactive species formation with MnTBAP, a scavenger of peroxynitrite, or using the peroxynitrite degradation catalyst FeTMPyP, cell proliferation and ERK signaling were restored to basal levels in iNOS+/+ mixed cultures. Moreover, exposure to the NO donor NOC-18 (100 µM, for 48 h, inhibited SVZ-derived NSC proliferation. Regarding the antiproliferative effect of NO, we found that NOC-18 caused the impairment of signaling through the ERK/MAPK pathway, which may be related to increased nitration of the EGF receptor in NSC. Using MnTBAP nitration was prevented, maintaining ERK signaling, rescuing NSC proliferation. We show that NO from inflammatory origin leads to a decreased function of the EGF receptor, which compromised proliferation of NSC. We also demonstrated that NO-mediated nitration of the EGF receptor caused a decrease in its phosphorylation, thus preventing regular proliferation signaling through the

  17. Gene expression profiling reveals different molecular patterns in G-protein coupled receptor signaling pathways between early- and late-onset preeclampsia.

    Science.gov (United States)

    Liang, Mengmeng; Niu, Jianmin; Zhang, Liang; Deng, Hua; Ma, Jian; Zhou, Weiping; Duan, Dongmei; Zhou, Yuheng; Xu, Huikun; Chen, Longding

    2016-04-01

    Early-onset preeclampsia and late-onset preeclampsia have been regarded as two different phenotypes with heterogeneous manifestations; To gain insights into the pathogenesis of the two traits, we analyzed the gene expression profiles in preeclamptic placentas. A whole genome-wide microarray was used to determine the gene expression profiles in placental tissues from patients with early-onset (n = 7; 36 weeks) preeclampsia and their controls who delivered preterm (n = 5; 36 weeks). Genes were termed differentially expressed if they showed a fold-change ≥ 2 and q-value preeclampsia (177 genes were up-regulated and 450 were down-regulated). Gene ontology analysis identified significant alterations in several biological processes; the top two were immune response and cell surface receptor linked signal transduction. Among the cell surface receptor linked signal transduction-related, differentially expressed genes, those involved in the G-protein coupled receptor protein signaling pathway were significantly enriched. G-protein coupled receptor signaling pathway related genes, such as GPR124 and MRGPRF, were both found to be down-regulated in early-onset preeclampsia. The results were consistent with those of western blotting that the abundance of GPR124 was lower in early-onset compared with late-onset preeclampsia. The different gene expression profiles reflect the different levels of transcription regulation between the two conditions and supported the hypothesis that they are separate disease entities. Moreover, the G-protein coupled receptor signaling pathway related genes may contribute to the mechanism underlying early- and late-onset preeclampsia. Copyright © 2016 Elsevier Ltd. All rights reserved.

  18. {delta}-Opioid receptor-stimulated Akt signaling in neuroblastoma x glioma (NG108-15) hybrid cells involves receptor tyrosine kinase-mediated PI3K activation

    Energy Technology Data Exchange (ETDEWEB)

    Heiss, Anika; Ammer, Hermann [Institute of Pharmacology, Toxicology and Pharmacy Ludwig-Maximilians-University of Munich Koeniginstrasse 16 80539 Muenchen Federal Republic of Germany (Germany); Eisinger, Daniela A., E-mail: eisinger@pharmtox.vetmed.uni-muenchen.de [Institute of Pharmacology, Toxicology and Pharmacy Ludwig-Maximilians-University of Munich Koeniginstrasse 16 80539 Muenchen Federal Republic of Germany (Germany)

    2009-07-15

    {delta}-Opioid receptor (DOR) agonists possess cytoprotective properties, an effect associated with activation of the 'pro-survival' kinase Akt. Here we delineate the signal transduction pathway by which opioids induce Akt activation in neuroblastoma x glioma (NG108-15) hybrid cells. Exposure of the cells to both [D-Pen{sup 2,5}]enkephalin and etorphine resulted in a time- and dose-dependent increase in Akt activity, as measured by means of an activation-specific antibody recognizing phosphoserine-473. DOR-mediated Akt signaling is blocked by the opioid antagonist naloxone and involves inhibitory G{sub i/o} proteins, because pre-treatment with pertussis toxin, but not over-expression of the G{sub q/11} scavengers EBP50 and GRK2-K220R, prevented this effect. Further studies with Wortmannin and LY294002 revealed that phophoinositol-3-kinase (PI3K) plays a central role in opioid-induced Akt activation. Opioids stimulate Akt activity through transactivation of receptor tyrosine kinases (RTK), because pre-treatment of the cells with inhibitors for neurotrophin receptor tyrosine kinases (AG879) and the insulin-like growth factor receptor IGF-1 (AG1024), but not over-expression of the G{beta}{gamma} scavenger phosducin, abolished this effect. Activated Akt translocates to the nuclear membrane, where it promotes GSK3 phosphorylation and prevents caspase-3 cleavage, two key events mediating inhibition of cell apoptosis and enhancement of cell survival. Taken together, these results demonstrate that in NG108-15 hybrid cells DOR agonists possess cytoprotective properties mediated by activation of the RTK/PI3K/Akt signaling pathway.

  19. δ-Opioid receptor-stimulated Akt signaling in neuroblastoma x glioma (NG108-15) hybrid cells involves receptor tyrosine kinase-mediated PI3K activation

    International Nuclear Information System (INIS)

    Heiss, Anika; Ammer, Hermann; Eisinger, Daniela A.

    2009-01-01

    δ-Opioid receptor (DOR) agonists possess cytoprotective properties, an effect associated with activation of the 'pro-survival' kinase Akt. Here we delineate the signal transduction pathway by which opioids induce Akt activation in neuroblastoma x glioma (NG108-15) hybrid cells. Exposure of the cells to both [D-Pen 2,5 ]enkephalin and etorphine resulted in a time- and dose-dependent increase in Akt activity, as measured by means of an activation-specific antibody recognizing phosphoserine-473. DOR-mediated Akt signaling is blocked by the opioid antagonist naloxone and involves inhibitory G i/o proteins, because pre-treatment with pertussis toxin, but not over-expression of the G q/11 scavengers EBP50 and GRK2-K220R, prevented this effect. Further studies with Wortmannin and LY294002 revealed that phophoinositol-3-kinase (PI3K) plays a central role in opioid-induced Akt activation. Opioids stimulate Akt activity through transactivation of receptor tyrosine kinases (RTK), because pre-treatment of the cells with inhibitors for neurotrophin receptor tyrosine kinases (AG879) and the insulin-like growth factor receptor IGF-1 (AG1024), but not over-expression of the Gβγ scavenger phosducin, abolished this effect. Activated Akt translocates to the nuclear membrane, where it promotes GSK3 phosphorylation and prevents caspase-3 cleavage, two key events mediating inhibition of cell apoptosis and enhancement of cell survival. Taken together, these results demonstrate that in NG108-15 hybrid cells DOR agonists possess cytoprotective properties mediated by activation of the RTK/PI3K/Akt signaling pathway.

  20. Wnt signalling via the epidermal growth factor receptor: a role in breast cancer?

    International Nuclear Information System (INIS)

    Musgrove, Elizabeth A

    2004-01-01

    Recent data have suggested the epidermal-growth-factor receptor (EGFR) as a point of convergence for several different classes of receptor. Civenni and colleagues have now demonstrated crosstalk between Wnt signalling and the EGFR, showing that in breast epithelial cells Wnts activate downstream targets of the EGFR, including cyclin D1. Given the role of members of these pathways in the aetiology of breast cancer and as markers of outcome and potential therapeutic targets in breast cancer, this observation has a number of potential implications important for both the basic biology of breast cancer and the clinical management of the disease