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Sample records for activator receptor upar

  1. Tumour microenvironments induce expression of urokinase plasminogen activator receptor (uPAR and concomitant activation of gelatinolytic enzymes.

    Directory of Open Access Journals (Sweden)

    Synnøve Magnussen

    Full Text Available The urokinase plasminogen activator receptor (uPAR is associated with poor prognosis in oral squamous cell carcinoma (OSCC, and increased expression of uPAR is often found at the invasive tumour front. The aim of the current study was to elucidate the role of uPAR in invasion and metastasis of OSCC, and the effects of various tumour microenvironments in these processes. Furthermore, we wanted to study whether the cells' expression level of uPAR affected the activity of gelatinolytic enzymes.The Plaur gene was both overexpressed and knocked-down in the murine OSCC cell line AT84. Tongue and skin tumours were established in syngeneic mice, and cells were also studied in an ex vivo leiomyoma invasion model. Soluble factors derived from leiomyoma tissue, as well as purified extracellular matrix (ECM proteins, were assessed for their ability to affect uPAR expression, glycosylation and cleavage. Activity of gelatinolytic enzymes in the tissues were assessed by in situ zymography.We found that increased levels of uPAR did not induce tumour invasion or metastasis. However, cells expressing low endogenous levels of uPAR in vitro up-regulated uPAR expression both in tongue, skin and leiomyoma tissue. Various ECM proteins had no effect on uPAR expression, while soluble factors originating from the leiomyoma tissue increased both the expression and glycosylation of uPAR, and possibly also affected the proteolytic processing of uPAR. Tumours with high levels of uPAR, as well as cells invading leiomyoma tissue with up-regulated uPAR expression, all displayed enhanced activity of gelatinolytic enzymes.Although high levels of uPAR are not sufficient to induce invasion and metastasis, the activity of gelatinolytic enzymes was increased. Furthermore, several tumour microenvironments have the capacity to induce up-regulation of uPAR expression, and soluble factors in the tumour microenvironment may have an important role in the regulation of posttranslational

  2. Mapping the topographic epitope landscape on the urokinase plasminogen activator receptor (uPAR) by surface plasmon resonance and X-ray crystallography

    DEFF Research Database (Denmark)

    Zhao, Baoyu; Gandhi, Sonu; Yuan, Cai;

    2015-01-01

    The urokinase-type plasminogen activator receptor (uPAR or CD87) is a glycolipid-anchored membrane protein often expressed in the microenvironment of invasive solid cancers and high levels are generally associated with poor patient prognosis (Kriegbaum et al., 2011 [1]). uPAR is organized as a dy...... of these mAbs by X-ray crystallography alone and in complex with uPAR [deposited in the PDB database as 4QTH and 4QTI, respectively]....

  3. Mapping the topographic epitope landscape on the urokinase plasminogen activator receptor (uPAR) by surface plasmon resonance and X-ray crystallography

    OpenAIRE

    Baoyu Zhao; Sonu Gandhi; Cai Yuan; Zhipu Luo; Rui Li; Henrik Gårdsvoll; Valentina de Lorenzi; Nicolai Sidenius; Mingdong Huang; Michael Ploug

    2015-01-01

    The urokinase-type plasminogen activator receptor (uPAR or CD87) is a glycolipid-anchored membrane protein often expressed in the microenvironment of invasive solid cancers and high levels are generally associated with poor patient prognosis (Kriegbaum et al., 2011 [1]). uPAR is organized as a dynamic modular protein structure composed of three homologous Ly6/uPAR domains (LU).This internally flexible protein structure of uPAR enables an allosteric regulation of the interactions with its two ...

  4. Structure, function and expression on blood and bone marrow cells of the urokinase-type plasminogen activator receptor, uPAR

    DEFF Research Database (Denmark)

    Plesner, T; Behrendt, N; Ploug, M

    1997-01-01

    Several important functions have been assigned to the receptor for urokinase-type plasminogen activator, uPAR. As implied by the name, uPAR was first identified as a high affinity cellular receptor for urokinase plasminogen activator (uPA). It mediates the binding of the zymogen, pro-uPA, to the...... shown that urokinase-type plasminogen activator (uPAR) is expressed as a differentiation antigen on cells of the myelomonocytic lineage and as an activation antigen on monocytes and T lymphocytes. Neutrophils contain intracellular reservoirs of uPAR that are translocated to the plasma membrane upon...... activation, and neutrophils from patients with the rare blood disease paroxysmal nocturnal hemoglobinuria (PNH) that fail to express glycosyl-phosphatidylinositol-anchored proteins including uPAR, show a very significantly reduced transmigration over an endothelial barrier. Cell-associated plasminogen...

  5. Flow cytometry evaluation of urokinase-type plasminogen activator receptor (UPA-R) in acute myeloid leukemia cells.

    Science.gov (United States)

    Castagnari, B; Moretti, S; Latorraca, A; Rigolin, G M; Balsamo, R; Lanza, F; Castoldi, G L

    1995-01-01

    The aim of this study was to investigate by flow cytometry the expression of the UPA-R (Urokinase type plasminogen activator receptor-CD87) on the blastic population of AML and ALL patients in order to evaluate whether the presence of this molecule could be associated with peculiar clinical and biologic features of leukemic cells. Five different monoclonal antibodies (MoAbs) (clones: 3B10#; VIM5*; 109#; 68#; 100#) were used in order to detect the distinct forms of this cellular receptor. Cell reactivity varied significantly from case to case, also depending on the MoAb used for the flow cytometry analysis. In brief, 3B10# and VIM5* MoAbs were found to be positive in more than 90% of monocytes and neutrophils from healthy subjects, while the number of positive cells was decreased (60%) using the 109# MoAb. However, either 68# and 100# MoAbs recognised only a low number of blood monocytes and neutrophils (8-20%), while lymphocytes were unreactive with all the five UPA-R MoAbs. ALL cells were found to be CD87 negative in all cases. Blasts from AML showed a heterogeneous pattern of expression for the UPA-R MoAbs, being the reactivity strictly dependent on the MoAb used, and, to a higher extent, on the degree and type of maturation of the blastic cells. The number of blasts recognising 3B10# and VIM5* MoAbs was significantly higher than that reacting with the remaining MoAbs irrespective of the FAB subtype. Since proteolytic enzymes, like UPA, play a key role in the dissolution of the extracellular matrix, and in facilitating the cell egress from the bone marrow, it is conceivable that the expression of the UPA-R could contribute to the invasive properties and, possibly, metastatic potential of leukemic cells. PMID:8519488

  6. Mapping the topographic epitope landscape on the urokinase plasminogen activator receptor (uPAR by surface plasmon resonance and X-ray crystallography

    Directory of Open Access Journals (Sweden)

    Baoyu Zhao

    2015-12-01

    Full Text Available The urokinase-type plasminogen activator receptor (uPAR or CD87 is a glycolipid-anchored membrane protein often expressed in the microenvironment of invasive solid cancers and high levels are generally associated with poor patient prognosis (Kriegbaum et al., 2011 [1]. uPAR is organized as a dynamic modular protein structure composed of three homologous Ly6/uPAR domains (LU.This internally flexible protein structure of uPAR enables an allosteric regulation of the interactions with its two principal ligands: the serine protease urokinase-type plasminogen activator (uPA and the provisional matrix protein vitronectin (Vn (Mertens et al., 2012; Gårdsvoll et al., 2011; Madsen et al., 2007 [2–4]. The data presented here relates to the non-covalent trapping of one of these biologically relevant uPAR-conformations by a novel class of monoclonal antibodies (Zhao et al., 2015 [5] and to the general mapping of the topographic epitope landscape on uPAR. The methods required to achieve these data include: (1 recombinant expression and purification of a uPAR-hybrid protein trapped in the desired conformation [patent; WO 2013/020898 A12013]; (2 developing monoclonal antibodies with unique specificities using this protein as antigen; (3 mapping the functional epitope on uPAR for these mAbs by surface plasmon resonance with a complete library of purified single-site uPAR mutants (Zhao et al., 2015; Gårdsvoll et al., 2006 [5,6]; and finally (4 solving the three-dimensional structures for one of these mAbs by X-ray crystallography alone and in complex with uPAR [deposited in the PDB database as 4QTH and 4QTI, respectively].

  7. Mapping the topographic epitope landscape on the urokinase plasminogen activator receptor (uPAR) by surface plasmon resonance and X-ray crystallography.

    Science.gov (United States)

    Zhao, Baoyu; Gandhi, Sonu; Yuan, Cai; Luo, Zhipu; Li, Rui; Gårdsvoll, Henrik; de Lorenzi, Valentina; Sidenius, Nicolai; Huang, Mingdong; Ploug, Michael

    2015-12-01

    The urokinase-type plasminogen activator receptor (uPAR or CD87) is a glycolipid-anchored membrane protein often expressed in the microenvironment of invasive solid cancers and high levels are generally associated with poor patient prognosis (Kriegbaum et al., 2011 [1]). uPAR is organized as a dynamic modular protein structure composed of three homologous Ly6/uPAR domains (LU).This internally flexible protein structure of uPAR enables an allosteric regulation of the interactions with its two principal ligands: the serine protease urokinase-type plasminogen activator (uPA) and the provisional matrix protein vitronectin (Vn) (Mertens et al., 2012; Gårdsvoll et al., 2011; Madsen et al., 2007 [2-4]). The data presented here relates to the non-covalent trapping of one of these biologically relevant uPAR-conformations by a novel class of monoclonal antibodies (Zhao et al., 2015 [5]) and to the general mapping of the topographic epitope landscape on uPAR. The methods required to achieve these data include: (1) recombinant expression and purification of a uPAR-hybrid protein trapped in the desired conformation [patent; WO 2013/020898 A12013]; (2) developing monoclonal antibodies with unique specificities using this protein as antigen; (3) mapping the functional epitope on uPAR for these mAbs by surface plasmon resonance with a complete library of purified single-site uPAR mutants (Zhao et al., 2015; Gårdsvoll et al., 2006 [5,6]); and finally (4) solving the three-dimensional structures for one of these mAbs by X-ray crystallography alone and in complex with uPAR [deposited in the PDB database as 4QTH and 4QTI, respectively]. PMID:26504891

  8. The receptor for urokinase-plasminogen activator (uPAR) controls plasticity of cancer cell movement in mesenchymal and amoeboid migration style.

    Science.gov (United States)

    Margheri, Francesca; Luciani, Cristina; Taddei, Maria Letizia; Giannoni, Elisa; Laurenzana, Anna; Biagioni, Alessio; Chillà, Anastasia; Chiarugi, Paola; Fibbi, Gabriella; Del Rosso, Mario

    2014-03-30

    The receptor for the urokinase plasminogen activator (uPAR) is up-regulated in malignant tumors. Historically the function of uPAR in cancer cell invasion is strictly related to its property to promote uPA-dependent proteolysis of extracellular matrix and to open a path to malignant cells. These features are typical of mesenchymal motility. Here we show that the full-length form of uPAR is required when prostate and melanoma cancer cells convert their migration style from the "path generating" mesenchymal to the "path finding" amoeboid one, thus conferring a plasticity to tumor cell invasiveness across three-dimensional matrices. Indeed, in response to a protease inhibitors-rich milieu, prostate and melanoma cells activated an amoeboid invasion program connoted by retraction of cell protrusions, RhoA-mediated rounding of the cell body, formation of a cortical ring of actin and a reduction of Rac-1 activation. While the mesenchymal movement was reduced upon silencing of uPAR expression, the amoeboid one was almost completely abolished, in parallel with a deregulation of small Rho-GTPases activity. In melanoma and prostate cancer cells we have shown uPAR colocalization with β1/β3 integrins and actin cytoskeleton, as well integrins-actin co-localization under both mesenchymal and amoeboid conditions. Such co-localizations were lost upon treatment of cells with a peptide that inhibits uPAR-integrin interactions. Similarly to uPAR silencing, the peptide reduced mesenchymal invasion and almost abolished the amoeboid one. These results indicate that full-length uPAR bridges the mesenchymal and amoeboid style of movement by an inward-oriented activity based on its property to promote integrin-actin interactions and the following cytoskeleton assembly. PMID:24681666

  9. Targeting of peptide conjugated magnetic nanoparticles to urokinase plasminogen activator receptor (uPAR) expressing cells

    DEFF Research Database (Denmark)

    Hansen, Line; Larsen, Esben Kjær Unmack; Nielsen, Erik Holm;

    2013-01-01

    patient prognosis shared by several cancers including breast, colorectal, and gastric cancers. Conjugation of a uPAR specific targeting peptide onto polyethylene glycol (PEG) coated USPIO nanoparticles by click chemistry resulted in a five times higher uptake in vitro in a uPAR positive cell line compared...

  10. The urokinase receptor (uPAR facilitates clearance of Borrelia burgdorferi.

    Directory of Open Access Journals (Sweden)

    Joppe W R Hovius

    2009-05-01

    Full Text Available The causative agent of Lyme borreliosis, the spirochete Borrelia burgdorferi, has been shown to induce expression of the urokinase receptor (uPAR; however, the role of uPAR in the immune response against Borrelia has never been investigated. uPAR not only acts as a proteinase receptor, but can also, dependently or independently of ligation to uPA, directly affect leukocyte function. We here demonstrate that uPAR is upregulated on murine and human leukocytes upon exposure to B. burgdorferi both in vitro as well as in vivo. Notably, B. burgdorferi-inoculated C57BL/6 uPAR knock-out mice harbored significantly higher Borrelia numbers compared to WT controls. This was associated with impaired phagocytotic capacity of B. burgdorferi by uPAR knock-out leukocytes in vitro. B. burgdorferi numbers in vivo, and phagocytotic capacity in vitro, were unaltered in uPA, tPA (low fibrinolytic activity and PAI-1 (high fibrinolytic activity knock-out mice compared to WT controls. Strikingly, in uPAR knock-out mice partially backcrossed to a B. burgdorferi susceptible C3H/HeN background, higher B. burgdorferi numbers were associated with more severe carditis and increased local TLR2 and IL-1beta mRNA expression. In conclusion, in B. burgdorferi infection, uPAR is required for phagocytosis and adequate eradication of the spirochete from the heart by a mechanism that is independent of binding of uPAR to uPA or its role in the fibrinolytic system.

  11. The urokinase receptor (uPAR) facilitates clearance of Borrelia burgdorferi

    NARCIS (Netherlands)

    J.W.R. Hovius; M.F. Bijlsma; G.J.W. van der Windt; W.J. Wiersinga; B.J.D. Boukens; J. Coumou; A. Oei; R. de Beer; A.F. de Vos; C. van 't Veer; A.P. van Dam; P. Wang; E. Fikrig; M.M. Levi; J.J.T.H. Roelofs; T. van der Poll

    2009-01-01

    The causative agent of Lyme borreliosis, the spirochete Borrelia burgdorferi, has been shown to induce expression of the urokinase receptor (uPAR); however, the role of uPAR in the immune response against Borrelia has never been investigated. uPAR not only acts as a proteinase receptor, but can also

  12. Urokinase-type plasminogen activator receptor (uPAR) expression is associated with T-stage and survival in urothelial carcinoma of the bladder

    DEFF Research Database (Denmark)

    Dohn, Line Hammer; Illemann, Martin; Høyer-Hansen, Gunilla;

    2015-01-01

    myofibroblasts at the invasive front and tumor core, respectively. Statistical analyses were performed to evaluate the association of uPAR localization and score with clinicopathologic covariates and survival. RESULTS: uPAR positivity was seen in 122/137 (89%) and 118/149 (74%) of the neoplasias at the invasive...... invasive tumors that could benefit from additional chemotherapy or more intensive follow-up after cystectomy....

  13. The superoxide scavenger TEMPOL induces urokinase receptor (uPAR expression in human prostate cancer cells

    Directory of Open Access Journals (Sweden)

    Francis Joseph

    2006-06-01

    Full Text Available Abstract There is little understanding of the effect that reactive oxygen metabolites have on cellular behavior during the processes of invasion and metastasis. These oxygen metabolites could interact with a number of targets modulating their function such as enzymes involved in basement membrane dissolution, adhesion molecules involved in motility or receptors involved in proliferation. We investigated the effect of increased scavenging of superoxide anions on the expression of the urokinase receptor (uPAR in PC-3M human prostate cancer cells. Urokinase receptor is a GPI-linked cell surface molecule which mediates multiple functions including adhesion, proliferation and pericellular proteolysis. Addition of the superoxide scavenger 4-hydroxy-2,2,6,6-tetramethylpiperidinyloxy (TEMPOL to PC-3M cultures stimulated expression of uPAR protein peaking between 48 and 72 hours. Cell surface expression of the uPAR was also increased. Surprisingly, uPAR transcript levels increased only slightly and this mild increase did not coincide with the striking degree of protein increase. This disparity indicates that the TEMPOL effect on uPAR occurs through a post-transcriptional mechanism. TEMPOL presence in PC-3M cultures reduced intracellular superoxide-type species by 75% as assayed by NBT dye conversion; however this reduction significantly diminished within hours following TEMPOL removal. The time gap between TEMPOL treatment and peak uPAR protein expression suggests that reduction of reactive oxygen metabolites in prostate cancer cells initiates a multistep pathway which requires several hours to culminate in uPAR induction. These findings reveal a novel pathway for uPAR regulation involving reactive oxygens such as superoxide anion.

  14. First (18)F-labeled ligand for PET imaging of uPAR

    DEFF Research Database (Denmark)

    Persson, Morten; Liu, Hongguang; Madsen, Jacob;

    2013-01-01

    Urokinase-type plasminogen activator receptor (uPAR) is overexpressed in human prostate cancer and uPAR has been found to be associated with metastatic disease and poor prognosis. AE105 is a small linear peptide with high binding affinity to uPAR. We synthesized an N-terminal NOTA...

  15. The intact urokinase receptor is required for efficient vitronectin binding

    DEFF Research Database (Denmark)

    Høyer-Hansen, G; Behrendt, N; Ploug, M;

    1997-01-01

    The urokinase receptor (uPAR) is a receptor for both urokinase plasminogen activator (uPA) and the adhesion protein vitronectin. There are two forms of cell surface-bound uPAR; intact uPAR and a cleaved form, uPAR(2+3), which is formed by uPA-catalyzed cleavage of uPAR. In ligand-blotting experim...

  16. Structure and ligand interactions of the urokinase receptor (uPAR)

    DEFF Research Database (Denmark)

    Kjærgaard, Magnus; Hansen, Line; Jacobsen, Benedikte;

    2008-01-01

    on disulfide connectivities, and they adopt a characteristic three-finger fold. Interestingly, the gene for uPAR is localized in a cluster of 6 homologous genes encoding proteins with multiple LU-domains. The structural biology of uPAR will be reviewed with special emphasis on its multidomain composition...

  17. Rational targeting of the urokinase receptor (uPAR): development of antagonists and non-invasive imaging probes

    DEFF Research Database (Denmark)

    Kriegbaum, M C; Persson, M; Haldager, L;

    2011-01-01

    PA-mediated plasminogen activation at the cell surface, which is accomplished by its high-affinity interaction with the growth factor-like domain of uPA. Detailed insights into the molecular basis underlying the interactions between uPAR and its two bona fide ligands, uPA and vitronectin, have been obtained recently by X...

  18. First-in-human uPAR PET

    DEFF Research Database (Denmark)

    Persson, Morten; Skovgaard, Dorthe; Brandt-Larsen, Malene;

    2015-01-01

    A first-in-human clinical trial with Positron Emission Tomography (PET) imaging of the urokinase-type plasminogen activator receptor (uPAR) in patients with breast, prostate and bladder cancer, is described. uPAR is expressed in many types of human cancers and the expression is predictive...... of invasion, metastasis and indicates poor prognosis. uPAR PET imaging therefore holds promise to be a new and innovative method for improved cancer diagnosis, staging and individual risk stratification. The uPAR specific peptide AE105 was conjugated to the macrocyclic chelator DOTA and labeled with (64)Cu...... for targeted molecular imaging with PET. The safety, pharmacokinetic, biodistribution profile and radiation dosimetry after a single intravenous dose of (64)Cu-DOTA-AE105 were assessed by serial PET and computed tomography (CT) in 4 prostate, 3 breast and 3 bladder cancer patients. Safety assessment...

  19. uPAR as anti-cancer target

    DEFF Research Database (Denmark)

    Lund, Ida K; Illemann, Martin; Thurison, Tine;

    2011-01-01

    , and a potential diagnostic and predictive impact of the different uPAR forms has been reported. Hence, pericellular proteolysis seems to be a suitable target for anti-cancer therapy and numerous approaches have been pursued. Targeting of this process may be achieved by preventing the binding of uPA to u...... using mouse monoclonal antibodies (mAbs) against mouse uPA or uPAR. These reagents will target uPA and uPAR in both stromal cells and cancer cells, and their therapeutic potential can now be assessed in syngenic mouse cancer models.......Degradation of proteins in the extracellular matrix is crucial for the multistep process of cancer invasion and metastasis. Compelling evidence has demonstrated the urokinase receptor (uPAR) and its cognate ligand, the urokinase plasminogen activator (uPA), to play critical roles in the concerted...

  20. Identification of a new epitope in uPAR as a target for the cancer therapeutic monoclonal antibody ATN-658, a structural homolog of the uPAR binding integrin CD11b (αM.

    Directory of Open Access Journals (Sweden)

    Xiang Xu

    Full Text Available The urokinase plasminogen activator receptor (uPAR plays a role in tumor progression and has been proposed as a target for the treatment of cancer. We recently described the development of a novel humanized monoclonal antibody that targets uPAR and has anti-tumor activity in multiple xenograft animal tumor models. This antibody, ATN-658, does not inhibit ligand binding (i.e. uPA and vitronectin to uPAR and its mechanism of action remains unclear. As a first step in understanding the anti-tumor activity of ATN-658, we set out to identify the epitope on uPAR to which ATN-658 binds. Guided by comparisons between primate and human uPAR, epitope mapping studies were performed using several orthogonal techniques. Systematic site directed and alanine scanning mutagenesis identified the region of aa 268-275 of uPAR as the epitope for ATN-658. No known function has previously been attributed to this epitope Structural insights into epitope recognition were obtained from structural studies of the Fab fragment of ATN-658 bound to uPAR. The structure shows that the ATN-658 binds to the DIII domain of uPAR, close to the C-terminus of the receptor, corroborating the epitope mapping results. Intriguingly, when bound to uPAR, the complementarity determining region (CDR regions of ATN-658 closely mimic the binding regions of the integrin CD11b (αM, a previously identified uPAR ligand thought to be involved in leukocyte rolling, migration and complement fixation with no known role in tumor progression of solid tumors. These studies reveal a new functional epitope on uPAR involved in tumor progression and demonstrate a previously unrecognized strategy for the therapeutic targeting of uPAR.

  1. Circulating intact and cleaved forms of the urokinase-type plasminogen activator receptor

    DEFF Research Database (Denmark)

    Røndbjerg, Tine Thurison; Christensen, Ib J; Lund, Ida K;

    2015-01-01

    BACKGROUND: High levels of circulating forms of the urokinase-type plasminogen activator receptor (uPAR) are significantly associated to poor prognosis in cancer patients. Our aim was to determine biological variations and reference intervals of the uPAR forms in blood, and in addition, to test t...

  2. Quantitative PET of human urokinase-type plasminogen activator receptor with 64Cu-DOTA-AE105

    DEFF Research Database (Denmark)

    Persson, Morten; Madsen, Jacob; Østergaard, Søren;

    2012-01-01

    Expression levels of the urokinase-type plasminogen activator receptor (uPAR) represent an established biomarker for poor prognosis in a variety of human cancers. The objective of the present study was to explore whether noninvasive PET can be used to perform a quantitative assessment of expression...... levels of uPAR across different human cancer xenograft models in mice and to illustrate the clinical potential of uPAR PET in future settings for individualized therapy....

  3. 68Ga-labeling and in vivo evaluation of a uPAR binding DOTA- and NODAGA-conjugated peptide for PET imaging of invasive cancers

    DEFF Research Database (Denmark)

    Persson, Morten; Madsen, Jacob; Østergaard, Søren;

    2012-01-01

    INTRODUCTION: The urokinase-type plasminogen activator receptor (uPAR) is a well-established biomarker for tumor aggressiveness and metastatic potential. DOTA-AE105 and DOTA-AE105-NH(2) labeled with (64)Cu have previously been demonstrated to be able to noninvasively monitor uPAR expression using...

  4. Expression of uPAR in tumor-associated stromal cells is associated with colorectal cancer patient prognosis: a TMA study

    International Nuclear Information System (INIS)

    The receptor for urokinase-type plasminogen activator (uPAR) is associated with cancer development and progression. Within the tumor microenvironment uPAR is expressed by malignant cells as well as tumor-associated stromal cells. However, the contribution of uPAR expression in these stromal cells to malignancy and patient survival in colorectal cancer is still unclear. This study compares the association of uPAR expression in both colorectal tumor-associated stromal cells and neoplastic cells with clinico-pathological characteristics and patient survival using tissue micro arrays (TMA). Immunohistochemical staining of uPAR expression was performed on tumor tissue from 262 colorectal cancer patients. Kaplan-Meier, log rank, and uni- and multivariate Cox’s regression analyses were used to calculate associations between uPAR expression and patient survival. In the colorectal tumor-associated stromal microenvironment, uPAR is expressed in macrophages, (neoangiogenic) endothelial cells and myofibroblasts. uPAR expression in tumor-associated stromal cells and neoplastic cells (and both combined) were negatively associated with overall survival (OS) and Disease Free Survival (DFS). Uni- and multivariate Cox’s regression analysis for combined uPAR expression in tumor-associated stromal and neoplastic cells showed significant and independent negative associations with OS and DFS. Only uPAR expression in tumor-associated stromal cells showed independent significance in the uni- and multivariate analysis for DFS. This study demonstrates a significant independent negative association between colorectal cancer patient survival and uPAR expression in especially tumor-associated stromal cells

  5. Differential uPAR recruitment in caveolar-lipid rafts by GM1 and GM3 gangliosides regulates endothelial progenitor cells angiogenesis.

    Science.gov (United States)

    Margheri, Francesca; Papucci, Laura; Schiavone, Nicola; D'Agostino, Riccardo; Trigari, Silvana; Serratì, Simona; Laurenzana, Anna; Biagioni, Alessio; Luciani, Cristina; Chillà, Anastasia; Andreucci, Elena; Del Rosso, Tommaso; Margheri, Giancarlo; Del Rosso, Mario; Fibbi, Gabriella

    2015-01-01

    Gangliosides and the urokinase plasminogen activator receptor (uPAR) tipically partition in specialized membrane microdomains called lipid-rafts. uPAR becomes functionally important in fostering angiogenesis in endothelial progenitor cells (EPCs) upon recruitment in caveolar-lipid rafts. Moreover, cell membrane enrichment with exogenous GM1 ganglioside is pro-angiogenic and opposite to the activity of GM3 ganglioside. On these basis, we first checked the interaction of uPAR with membrane models enriched with GM1 or GM3, relying on the adoption of solid-supported mobile bilayer lipid membranes with raft-like composition formed onto solid hydrophilic surfaces, and evaluated by surface plasmon resonance (SPR) the extent of uPAR recruitment. We estimated the apparent dissociation constants of uPAR-GM1/GM3 complexes. These preliminary observations, indicating that uPAR binds preferentially to GM1-enriched biomimetic membranes, were validated by identifying a pro-angiogenic activity of GM1-enriched EPCs, based on GM1-dependent uPAR recruitment in caveolar rafts. We have observed that addition of GM1 to EPCs culture medium promotes matrigel invasion and capillary morphogenesis, as opposed to the anti-angiogenesis activity of GM3. Moreover, GM1 also stimulates MAPKinases signalling pathways, typically associated with an angiogenesis program. Caveolar-raft isolation and Western blotting of uPAR showed that GM1 promotes caveolar-raft partitioning of uPAR, as opposed to control and GM3-challenged EPCs. By confocal microscopy, we have shown that in EPCs uPAR is present on the surface in at least three compartments, respectively, associated to GM1, GM3 and caveolar rafts. Following GM1 exogenous addition, the GM3 compartment is depleted of uPAR which is recruited within caveolar rafts thereby triggering angiogenesis.

  6. Differential uPAR recruitment in caveolar-lipid rafts by GM1 and GM3 gangliosides regulates endothelial progenitor cells angiogenesis.

    Science.gov (United States)

    Margheri, Francesca; Papucci, Laura; Schiavone, Nicola; D'Agostino, Riccardo; Trigari, Silvana; Serratì, Simona; Laurenzana, Anna; Biagioni, Alessio; Luciani, Cristina; Chillà, Anastasia; Andreucci, Elena; Del Rosso, Tommaso; Margheri, Giancarlo; Del Rosso, Mario; Fibbi, Gabriella

    2015-01-01

    Gangliosides and the urokinase plasminogen activator receptor (uPAR) tipically partition in specialized membrane microdomains called lipid-rafts. uPAR becomes functionally important in fostering angiogenesis in endothelial progenitor cells (EPCs) upon recruitment in caveolar-lipid rafts. Moreover, cell membrane enrichment with exogenous GM1 ganglioside is pro-angiogenic and opposite to the activity of GM3 ganglioside. On these basis, we first checked the interaction of uPAR with membrane models enriched with GM1 or GM3, relying on the adoption of solid-supported mobile bilayer lipid membranes with raft-like composition formed onto solid hydrophilic surfaces, and evaluated by surface plasmon resonance (SPR) the extent of uPAR recruitment. We estimated the apparent dissociation constants of uPAR-GM1/GM3 complexes. These preliminary observations, indicating that uPAR binds preferentially to GM1-enriched biomimetic membranes, were validated by identifying a pro-angiogenic activity of GM1-enriched EPCs, based on GM1-dependent uPAR recruitment in caveolar rafts. We have observed that addition of GM1 to EPCs culture medium promotes matrigel invasion and capillary morphogenesis, as opposed to the anti-angiogenesis activity of GM3. Moreover, GM1 also stimulates MAPKinases signalling pathways, typically associated with an angiogenesis program. Caveolar-raft isolation and Western blotting of uPAR showed that GM1 promotes caveolar-raft partitioning of uPAR, as opposed to control and GM3-challenged EPCs. By confocal microscopy, we have shown that in EPCs uPAR is present on the surface in at least three compartments, respectively, associated to GM1, GM3 and caveolar rafts. Following GM1 exogenous addition, the GM3 compartment is depleted of uPAR which is recruited within caveolar rafts thereby triggering angiogenesis. PMID:25313007

  7. uPAR as anti-cancer target: evaluation of biomarker potential, histological localization, and antibody-based therapy

    DEFF Research Database (Denmark)

    Lund, Ida K; Illemann, Martin; Sørensen, Tine Thurison;

    2011-01-01

    , and a potential diagnostic and predictive impact of the different uPAR forms has been reported. Hence, pericellular proteolysis seems to be a suitable target for anti-cancer therapy and numerous approaches have been pursued. Targeting of this process may be achieved by preventing the binding of uPA to u...... using mouse monoclonal antibodies (mAbs) against mouse uPA or uPAR. These reagents will target uPA and uPAR in both stromal cells and cancer cells, and their therapeutic potential can now be assessed in syngenic mouse cancer models.......Degradation of proteins in the extracellular matrix is crucial for the multistep process of cancer invasion and metastasis. Compelling evidence has demonstrated the urokinase receptor (uPAR) and its cognate ligand, the urokinase plasminogen activator (uPA), to play critical roles in the concerted...

  8. The urokinase plasminogen activator receptor-associated protein/endo180 is coexpressed with its interaction partners urokinase plasminogen activator receptor and matrix metalloprotease-13 during osteogenesis

    DEFF Research Database (Denmark)

    Engelholm, L H; Nielsen, B S; Netzel-Arnett, S;

    2001-01-01

    The urokinase plasminogen activator receptor-associated protein/Endo180 (uPARAP/Endo180) is a newly discovered member of the macrophage mannose receptor family that was reported to interact with ligand-bound urokinase plasminogen activator receptor (uPAR), matrix metalloprotease-13 (MMP-13), and ......, uPARAP/Endo180 expression was detected only in a mesenchymal condensation of the midbrain and in the developing lungs. The data suggest a function of this novel protease receptor in bone development, possibly mediated through its interactions with uPAR, MMP-13, or collagen V....

  9. Human Secreted Ly-6/uPAR Related Protein-1 (SLURP-1 Is a Selective Allosteric Antagonist of α7 Nicotinic Acetylcholine Receptor.

    Directory of Open Access Journals (Sweden)

    Ekaterina N Lyukmanova

    Full Text Available SLURP-1 is a secreted toxin-like Ly-6/uPAR protein found in epithelium, sensory neurons and immune cells. Point mutations in the slurp-1 gene cause the autosomal inflammation skin disease Mal de Meleda. SLURP-1 is considered an autocrine/paracrine hormone that regulates growth and differentiation of keratinocytes and controls inflammation and malignant cell transformation. The majority of previous studies of SLURP-1 have been made using fusion constructs containing, in addition to the native protein, extra polypeptide sequences. Here we describe the activity and pharmacological profile of a recombinant analogue of human SLURP-1 (rSLURP-1 differing from the native protein only by one additional N-terminal Met residue. rSLURP-1 significantly inhibited proliferation (up to ~ 40%, EC50 ~ 4 nM of human oral keratinocytes (Het-1A cells. Application of mecamylamine and atropine,--non-selective inhibitors of nicotinic acetylcholine receptors (nAChRs and muscarinic acetylcholine receptors, respectively, and anti-α7-nAChRs antibodies revealed α7 type nAChRs as an rSLURP-1 target in keratinocytes. Using affinity purification from human cortical extracts, we confirmed that rSLURP-1 binds selectively to the α7-nAChRs. Exposure of Xenopus oocytes expressing α7-nAChRs to rSLURP-1 caused a significant non-competitive inhibition of the response to acetylcholine (up to ~ 70%, IC50 ~ 1 μM. It was shown that rSLURP-1 binds to α7-nAChRs overexpressed in GH4Cl cells, but does not compete with 125I-α-bungarotoxin for binding to the receptor. These findings imply an allosteric antagonist-like mode of SLURP-1 interaction with α7-nAChRs outside the classical ligand-binding site. Contrary to rSLURP-1, other inhibitors of α7-nAChRs (mecamylamine, α-bungarotoxin and Lynx1 did not suppress the proliferation of keratinocytes. Moreover, the co-application of α-bungarotoxin with rSLURP-1 did not influence antiproliferative activity of the latter. This supports the

  10. Estradiol attenuates EGF-induced rapid uPAR mobilization and cell migration via the G-protein-coupled receptor 30 in ovarian cancer cells

    DEFF Research Database (Denmark)

    Henic, Emir; Noskova, Vera; Høyer-Hansen, Gunilla;

    2009-01-01

    mobilization of uPAR from detergent-resistant domains such as lipid rafts. Estradiol influenced neither the amount of uPAR mRNA nor the rate of uPAR degradation or solubilization. The nuclear ER antagonists ICI 182780 and tamoxifen, which are GPR30 agonists, as well as the specifically constructed GPR30...

  11. uPAR Expression Pattern in Patients with Urothelial Carcinoma of the Bladder

    DEFF Research Database (Denmark)

    Dohn, Line Hammer; Pappot, Helle; Iversen, Benedikte Richter;

    2015-01-01

    The objective of the present study was to confirm the expression and localisation pattern of the urokinase-type plasminogen activator receptor (uPAR) focusing on its possible clinical relevance in patients with urothelial neoplasia of the bladder. uPAR is a central molecule in tissue remodelling...... or positive as well as by the actual score. Separate scores were obtained for cancer cells, macrophages and myofibroblasts at the invasive front and in tumour core. We were able to confirm, in an independent patient cohort, the tissue expression and localisation pattern of uPAR as investigated...... investigations have generated new and valuable biological information about the cell types being involved in tumour invasion and progression through the plasminogen activation system....

  12. Urokinase plasminogen activator receptor on invasive cancer cells: A prognostic factor in distal gastric adenocarcinoma

    DEFF Research Database (Denmark)

    Alpizar, Warner Enrique Alpizar; Christensen, Ib Jarle; Santoni-Rugiu, Eric;

    2012-01-01

    Gastric cancer is the second cancer causing death worldwide. The five-year survival for this malignancy is below 25% and few parameters have shown an impact on the prognosis of the disease. The receptor for urokinase plasminogen activator (uPAR) is involved in extracellular matrix degradation...... by mediating cell surface associated plasminogen activation, and its presence on gastric cancer cells is linked to micrometastasis and poor prognosis. Using immunohistochemistry, the prognostic significance of uPAR was evaluated in tissue samples from a retrospective series of 95 gastric cancer patients. u...... association between the expression of uPAR on tumor cells in the peripheral invasion zone and overall survival of gastric cancer patients (HR = 2.16; 95% CI: 1.13-4.14; p = 0.02). Multivariate analysis showed that uPAR immunoreactivity in cancer cells at the invasive front is an independent prognostic factor...

  13. Urokinase-type plasminogen activator receptor as a predictor of poor outcome in patients with systemic inflammatory response syndrome

    OpenAIRE

    Wu, Xiao-Ling; Long, Ding; Yu, Li; Yang, Jun-hui; Zhang, Yuan-chao; Geng, Feng

    2013-01-01

    BACKGROUND: Urokinase-type plasminogen activator (uPA) and urokinase-type plasminogen activator receptor (uPAR) are known as important factors, which mediate a variety of functions in terms of vascular homeostasis, inflammation and tissue repair. However, their role in systemic inflammatory response syndrome (SIRS) has been less well studied. This study aimed to test the hypothesis that the abnormalities of fibrinolysis and degradation of extracellular matrix mediated by uPA and uPAR are dire...

  14. Urokinase plasminogen activator receptor affects bone homeostasis by regulating osteoblast and osteoclast function

    DEFF Research Database (Denmark)

    Furlan, Federico; Galbiati, Clara; Jørgensen, Niklas R;

    2007-01-01

    reorganization in mature osteoclasts. INTRODUCTION: Urokinase receptor (uPAR) is actively involved in the regulation of important cell functions, such as proliferation, adhesion, and migration. It was previously shown that the major players in bone remodeling, osteoblasts and osteoclasts, express u...... with other osteoblasts markers. On the resorptive side, the number of osteoclasts formed in vitro from uPAR KO monocytes was decreased. Podosome imaging in uPAR KO osteoclasts revealed a defect in actin ring formation. CONCLUSIONS: The defective proliferation and differentiation of bone cells, coincident......The uPAR and its ligand uPA are expressed by both osteoblasts and osteoclasts. Their function in bone remodeling is unknown. We report that uPAR-lacking mice display increased BMD, increased osteogenic potential of osteoblasts, decreased osteoclasts formation, and altered cytoskeletal...

  15. Clathrin and LRP-1-independent constitutive endocytosis and recycling of uPAR.

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    Katia Cortese

    Full Text Available BACKGROUND: The urokinase receptor (uPAR/CD87 is highly expressed in malignant tumours. uPAR, as a GPI anchored protein, is preferentially located at the cell surface, where it interacts with its ligands urokinase (uPA and the extracellular matrix protein vitronectin, thus promoting plasmin generation, cell-matrix interactions and intracellular signalling events. Interaction with a complex formed by uPA and its inhibitor PAI-1 induces cell surface down regulation and recycling of the receptor via the clathrin-coated pathway, a process dependent on the association to LRP-1. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we have found that along with the ligand-induced down-regulation, uPAR also internalizes and recycles constitutively through a second pathway that is independent of LRP-1 and clathrin but shares some properties with macropinocytosis. The ligand-independent route is amiloride-sensitive, does not require uPAR partitioning into lipid rafts, is independent of the activity of small GTPases RhoA, Rac1 and Cdc42, and does not require PI3K activity. Constitutively endocytosed uPAR is found in EEA1 positive early/recycling endosomes but does not reach lysosomes in the absence of ligands. Electron microscopy analysis reveals the presence of uPAR in ruffling domains at the cell surface, in macropinosome-like vesicles and in endosomal compartments. CONCLUSIONS/SIGNIFICANCE: These results indicate that, in addition to the ligand-induced endocytosis of uPAR, efficient surface expression and membrane trafficking might also be driven by an uncommon macropinocytic mechanism coupled with rapid recycling to the cell surface.

  16. Stabilizing a Flexible Interdomain Hinge Region Harboring the SMB Binding Site Drives uPAR into Its Closed Conformation

    DEFF Research Database (Denmark)

    Zhao, Baoyu; Gandhi, Sonu; Yuan, Cai;

    2015-01-01

    The urokinase-type plasminogen activator receptor (uPAR) is a multidomain glycolipid-anchored membrane protein, which facilitates extracellular matrix remodeling by focalizing plasminogen activation to cell surfaces via its high-affinity interaction with uPA. The modular assembly of its three LU...

  17. Urokinase receptor forms in serum from non-small cell lung cancer patients

    DEFF Research Database (Denmark)

    Almasi, Charlotte Elberling; Christensen, Ib Jarle; Høyer-Hansen, Gunilla;

    2011-01-01

    To study the prognostic impact of the different forms of the receptor for urokinase plasminogen activator (uPAR) in serum from 171 non-small cell lung cancer (NSCLC) patients.......To study the prognostic impact of the different forms of the receptor for urokinase plasminogen activator (uPAR) in serum from 171 non-small cell lung cancer (NSCLC) patients....

  18. Cathepsin B and uPAR Knockdown Inhibits Tumor-induced Angiogenesis by Modulating VEGF Expression in Glioma

    OpenAIRE

    MALLA, RAMA RAO; Gopinath, Sreelatha; Christopher S Gondi; Alapati, Kiranmai; Dinh, Dzung H.; Gujrati, Meena; Rao, Jasti S.

    2011-01-01

    Angiogenesis, which is the process of sprouting of new blood vessels from pre-existing vessels, is vital for tumor progression. Proteolytic remodeling of extracellular matrix is a key event in vessel sprouting during angiogenesis. Urokinase plasminogen activator receptor (uPAR) and cathepsin B are both known to be overexpressed and implicated in tumor angiogenesis. In the present study, we observed that knockdown of uPAR and cathepsin B using puPAR (pU), pCathepsin B (pC), and a bicistronic c...

  19. uPAR EXPRESSION IN CANINE NORMAL PROSTATE AND WITH PROLIFERATIVE DISORDERS

    Directory of Open Access Journals (Sweden)

    Mariana Rodrigues Faleiro

    2013-06-01

    Full Text Available Prostatic lesions such as prostatic intraepithelial neoplasia (PIN and proliferative inflammatory atrophy (PIA are studied in human and canine species due to their malignance potential. The plasminogen activator (PA system has been suggested to play a central role in cell adhesion, angiogenesis, inflammation, and tumor invasion. The urokinase-type plasminogen activator receptor (uPAR is a component of the PA, with a range of expression in tumor and stromal cells. In this study, uPAR expression in both canine normal prostates and with proliferative disorders (benign prostatic hyperplasia-BPH, proliferative inflammatory atrophy-PIA, prostatic intraepithelial neoplasia-PIN, and carcinoma-PC was evaluated by immunohistochemistry in a tissue microarray (TMA slide to establish the role of this enzyme in extracellular matrix (ECM remodeling and in the processes of tissue invasion. A total of 298 cores and 355 diagnoses were obtained, with 36 (10.1% normal prostates, 46 (13.0% with BPH, 128 (36.1% with PIA, 74 (20.8% with PIN and 71 (20.0% with PC. There is variation in the expression of uPAR in canine prostate according to the lesion, with lower expression in normal tissue and with BPH, and higher expression in tissue with PIA, PIN and PC. The high expression of uPAR in inflammatory and neoplastic microenvironment indicates increased proteolytic activity in canine prostates with PIA, PIN, and PC.

  20. Expression and functional role of urokinase-type plasminogen activator receptor in normal and acute leukaemic cells.

    Science.gov (United States)

    Lanza, F; Castoldi, G L; Castagnari, B; Todd, R F; Moretti, S; Spisani, S; Latorraca, A; Focarile, E; Roberti, M G; Traniello, S

    1998-10-01

    Urokinase-type plasminogen activator receptor (UPA-R-CD87) is a GPI-anchored membrane protein which promotes the generation of plasmin on the surface of many cell types, probably facilitating cellular extravasation and tissue invasion. A flow cytometric quantitative analysis of expression levels for UPA-R was performed on fresh blast cells from patients with acute myeloid leukaemia (AML, n = 74), acute lymphoblastic leukaemia (ALL, n = 24), and biphenotypic leukaemia (BAL, n = 3) using two CD87 monoclonal antibodies (McAbs) (3B10 and VIM5). Peripheral blood and bone marrow (BM) cells from 15 healthy adults served as controls. Using 3B10 McAb, UPA-R was expressed (>99%) by blood monocytes, neutrophils, and BM myelomonocytic precursors in controls, whereas resting T and B lymphocytes, and CD34+ cells were UPA-R negative. We also attempted to clarify whether UPA-R has a role in mediating neutrophil functions. Oriented locomotion induced by different chemotaxins and lysozyme release by granules stimulated with fMLP or PMA were significantly decreased when UPA-R was neutralized by CD87 McAb. In contrast, the anti-UPA-R McAb had no effect on superoxide anion generation of normal neutrophils. Blasts from AML showed a heterogenous pattern of expression for the UPA-R McAbs, with reactivity strictly dependent on FAB subtype. The highest UPA-R expression was seen in the M5 group: all patients tested (n = 20) showed strong positivity for the UPA-R McAb whereas only 12% (3/24) of ALL patients were CD87 positive, and 2/3 of BAL patients showed a dim expression for CD87. The number of receptors expressed by blast cells in 6/74 (8.1%) AML patients was higher than those of normal samples: in addition, since co-expression of UPA-R and CD34 was not found in normal haemopoietic cells, it may be postulated that CD87 can be used alone (when overexpressed) or in combination with CD34 for the detection of minimal residual disease. Results also indicated that patients with UPA-receptors

  1. Factor XII stimulates ERK1/2 and Akt through uPAR, integrins, and the EGFR to initiate angiogenesis

    Science.gov (United States)

    LaRusch, Gretchen A.; Mahdi, Fakhri; Shariat-Madar, Zia; Adams, Gregory; Sitrin, Robert G.; Zhang, Wan Ming; McCrae, Keith R.

    2010-01-01

    Factor XII (FXII) and high molecular weight kininogen (HK) mutually block each other's binding to the urokinase plasminogen activator receptor (uPAR). We investigated if FXII stimulates cells by interacting with uPAR. FXII (3-62nM) with 0.05mM Zn2+ induces extracellular signal-related kinase 1/2 (ERK1/2; mitogen-activated protein kinase 44 [MAPK44] andMAPK42) and Akt (Ser473) phosphorylation in endothelial cells. FXII-induced phosphorylation of ERK1/2 or Akt is a zymogen activity, not an enzymatic event. ERK1/2 or Akt phosphorylation is blocked upstream by PD98059 or Wortmannin or LY294002, respectively. An uPAR signaling region for FXII is on domain 2 adjacent to uPAR's integrin binding site. Cleaved HK or peptides from HK's domain 5 blocks FXII-induced ERK1/2 and Akt phosphorylation. A β1 integrin peptide that binds uPAR, antibody 6S6 to β1 integrin, or the epidermal growth factor receptor (EGFR) inhibitor AG1478 blocks FXII-induced phosphorylation of ERK1/2 and Akt. FXII induces endothelial cell proliferation and 5-bromo-2′deoxy-uridine incorporation. FXII stimulates aortic sprouting in normal but not uPAR-deficient mouse aorta. FXII produces angiogenesis in matrigel plugs in normal but not uPAR-deficient mice. FXII knockout mice have reduced constitutive and wound-induced blood vessel number. In sum, FXII initiates signaling mediated by uPAR, β1 integrin, and the EGFR to induce human umbilical vein endothelial cell proliferation, growth, and angiogenesis. PMID:20228268

  2. Measurement of plasminogen activator system components in plasma and tumor tissue extracts obtained from patients with breast cancer: an EORTC Receptor and Biomarker Group collaboration.

    NARCIS (Netherlands)

    Grebenchtchikov, N.I.; Maguire, T.M.; Riisbro, R.; Geurts-Moespot, A.; O'Donovan, N.; Schmitt, M.; McGreal, G.; McDermott, E.; O'Higgins, N.; Brunner, N.; Sweep, C.G.J.; Duffy, M.J.

    2005-01-01

    The plasminogen activator (PA) system comprises the 2 serine proteases, urokinase PA (uPA) and tissue PA (tPA), the 2 serpin inhibitors, PAI-1 and PAI-2 and the uPA receptor (uPAR; CD87). High levels of uPA, PAI-1, uPA-PAI-1 complex and uPAR in breast cancer tissue are associated with poor prognosis

  3. The receptor for urokinase regulates TLR2 mediated inflammatory responses in neutrophils.

    Directory of Open Access Journals (Sweden)

    Gang Liu

    Full Text Available The urokinase-type plasminogen activator receptor (uPAR, a glycosylphosphatidylinositol (GPI anchored membrane protein, regulates urokinase (uPA protease activity, chemotaxis, cell-cell interactions, and phagocytosis of apoptotic cells. uPAR expression is increased in cytokine or bacteria activated cell populations, including macrophages and monocytes. However, it is unclear if uPAR has direct involvement in the response of inflammatory cells, such as neutrophils and macrophages, to Toll like receptor (TLR stimulation. In this study, we found that uPAR is required for optimal neutrophil activation after TLR2, but not TLR4 stimulation. We found that the expression of TNF-α and IL-6 induced by TLR2 engagement in uPAR-/- neutrophils was less than that in uPAR+/+ (WT neutrophils. Pretreatment of neutrophils with PI-PLC, which cleaves GPI moieties, significantly decreased TLR2 induced expression of TNF-α in WT neutrophils, but demonstrated only marginal effects on TNF-α expression in PAM treated uPAR-/- neutrophils. IκB-α degradation and NF-κB activation were not different in uPAR-/- or WT neutrophils after TLR2 stimulation. However, uPAR is required for optimal p38 MAPK activation after TLR2 engagement. Consistent with the in vitro findings that uPAR modulates TLR2 engagement induced neutrophil activation, we found that pulmonary and systemic inflammation induced by TLR2, but not TLR4 stimulation is reduced in uPAR-/- mice compared to WT counterparts. Therefore, our data suggest that neutrophil associated uPAR could be a potential target for treating acute inflammation, sepsis, and organ injury related to severe bacterial and other microbial infections in which TLR2 engagement plays a major role.

  4. Cadmium induces urokinase-type plasminogen activator receptor expression and the cell invasiveness of human gastric cancer cells via the ERK-1/2, NF-κB, and AP-1 signaling pathways.

    Science.gov (United States)

    Khoi, Pham Ngoc; Xia, Yong; Lian, Sen; Kim, Ho Dong; Kim, Do Hyun; Joo, Young Eun; Chay, Kee-Oh; Kim, Kyung Keun; Jung, Young Do

    2014-10-01

    Cadmium exposure has been linked to human cancers, including stomach cancer. In this study, the effects of cadmium on urokinase-type plasminogen activator receptor (uPAR) expression in human gastric cancer cells and the underlying signal transduction pathways were investigated. Cadmium induced uPAR expression in a time- and concentration-dependent manner. Cadmium also induced uPAR promoter activity. Additionally, cadmium induced the activation of extracellular signal regulated kinase-1/2 (ERK-1/2), p38 mitogen-activated protein kinase (MAPK), and the activation of c-Jun amino terminal kinase (JNK). A specific inhibitor of MEK-1 (PD98059) inhibited cadmium-induced uPAR expression, while JNK and p38 MAPK inhibitors did not. Expression vectors encoding dominant-negative MEK-1 (pMCL-K97M) also prevented cadmium-induced uPAR promoter activity. Site-directed mutagenesis and electrophoretic mobility shift studies showed that sites for the transcription factors nuclear factor (NF)-κB and activator protein-1 (AP-1) were involved in cadmium-induced uPAR transcription. Suppression of the cadmium-induced uPAR promoter activity by a mutated-type NF-κB-inducing kinase and I-κB and an AP-1 decoy oligonucleotide confirmed that the activation of NF-κB and AP-1 are essential for cadmium-induced uPAR upregulation. Cells pretreated with cadmium showed markedly enhanced invasiveness and this effect was partially abrogated by uPAR-neutralizing antibodies and by inhibitors of ERK-1/2, NF-κB, and AP-1. These results suggest that cadmium induces uPAR expression via ERK-1/2, NF-κB, and AP-1 signaling pathways and, in turn, stimulates cell invasiveness in human gastric cancer AGS cells.

  5. Cigarette Smoke and the Induction of Urokinase Plasminogen Activator Receptor In Vivo: Selective Contribution of Isoforms to Bronchial Epithelial Phenotype.

    Science.gov (United States)

    Portelli, Michael A; Stewart, Ceri E; Hall, Ian P; Brightling, Christopher E; Sayers, Ian

    2015-08-01

    The urokinase plasminogen activator receptor (uPAR) gene (PLAUR) has been identified as an asthma susceptibility gene, with polymorphisms within that gene being associated with baseline lung function, lung function decline, and lung function in a smoking population. Soluble cleaved uPAR (scuPAR), a molecule identified as a marker of increased morbidity and mortality in a number of diseases, has been shown to be elevated in the airways of patients with asthma and in patients with chronic obstructive pulmonary disease. However, the functionality of soluble receptor isoforms and their relationship with an important initiator for obstructive lung disease, cigarette smoke, remains undefined. In this study, we set out to determine the effect of cigarette smoke on soluble uPAR isoforms, its regulatory pathway and the resultant effect on bronchial epithelial cell function. We identified a positive association between cigarette pack-years and uPAR expression in the airway bronchial epithelium of biopsies from patients with asthma (n = 27; P = 0.0485). In vitro, cigarette smoke promoted cleavage of uPAR from the surface of bronchial epithelial cells (1.5× induction; P bronchial epithelial cells. This suggests that cigarette smoke elevates soluble receptor isoforms in bronchial epithelial cells through direct (cleavage) and indirect (messenger RNA expression) means. These findings provide further insight into how cigarette smoke may influence changes in the airways of importance to airway remodeling and obstructive lung disease progression. PMID:25490122

  6. Cell-surface acceleration of urokinase-catalyzed receptor cleavage

    DEFF Research Database (Denmark)

    Høyer-Hansen, G; Ploug, M; Behrendt, N;

    1997-01-01

    The urokinase-type plasminogen activator (uPA) binds to a specific cell-surface receptor, uPAR. On several cell types uPAR is present both in the full-length form and a cleaved form, uPAR(2+3), which is devoid of binding activity. The formation of uPAR(2+3) on cultured U937 cells is either directly...... by a prior incubation of the cells with uPA inactivated by diisopropyl fluorophosphate, demonstrating a requirement for specific receptor binding of the active uPA to obtain the high-efficiency cleavage of cell-bound uPAR. Furthermore, amino-terminal sequence analysis revealed that uPAR(2+3), purified from U......937 cell lysates, had the same amino termini as uPAR(2+3), generated by uPA in a purified system. In both cases cleavage had occurred at two positions in the hinge region connecting domain 1 and 2, between Arg83-Ala84 and Arg89-Ser90, respectively. The uPA-catalyzed cleavage of uPAR is a new negative...

  7. Transcriptional Regulation of Urokinase-type Plasminogen Activator Receptor by Hypoxia-Inducible Factor 1 Is Crucial for Invasion of Pancreatic and Liver Cancer

    Directory of Open Access Journals (Sweden)

    Peter Büchler

    2009-02-01

    Full Text Available Angioinvasion is critical for metastasis with urokinase-type plasminogen activator receptor (uPAR and tumor hypoxia-activated hypoxia-inducible factor 1 (HIF-1 as key players. Transcriptional control of uPAR expression by HIF has never been reported. The aim of the present study, therefore, was to test whether tumor hypoxia-induced HIF expression may be linked to transcriptional activation of uPAR and dependent angioinvasion. We used human pancreatic cancer cells and a model of parental and derived HIF-1β-deficient mouse liver cancer cell lines and performed Northern blot analysis, nuclear runoff assays, electrophoretic mobility shift assay, polymerase chain reaction-generated deletion mutants, luciferase assays, Matrigel invasion assays, and in vivo angioinvasion assays in the chorioallantoic membrane of fertilized chicken eggs. Urokinase-type plasminogen activator receptor promoter analysis resulted in four putative HIF binding sites. Hypoxia strongly induced de novo transcription of uPAR mRNA. With sequential deletion mutants of the uPAR promoter, it was possible to identify one HIF binding site causing a nearly 200-fold increase in luciferase activity. Hypoxia enhanced the number of invading tumor cells in vitro and in vivo. In contrast, HIF-1β-deficient cells failed to upregulate uPAR expression, to activate luciferase activity, and to invade on hypoxia. Taken together, we show for the first time that uPAR is under transcriptional control of HIF and that this is important for hypoxia-induced metastasis.

  8. The low density lipoprotein receptor-related protein/alpha2-macroglobulin receptor regulates cell surface plasminogen activator activity on human trophoblast cells.

    Science.gov (United States)

    Zhang, J C; Sakthivel, R; Kniss, D; Graham, C H; Strickland, D K; McCrae, K R

    1998-11-27

    The low density lipoprotein receptor-related protein/alpha2-macroglobulin receptor (LRP/alpha2MR) mediates the internalization of numerous ligands, including prourokinase (pro-UK) and complexes between two-chain urokinase (tc-u-PA) and plasminogen activator inhibitor type-1 (PAI-1). It has been suggested that through its ability to internalize these ligands, LRP/alpha2MR may regulate the expression of plasminogen activator activity on cell surfaces; this hypothesis, however, has not been experimentally confirmed. To address this issue, we assessed the ability of LRP/alpha2MR to regulate plasminogen activator activity on human trophoblast cells, which express both LRP/alpha2MR and the urokinase receptor (uPAR). Trophoblasts internalized and degraded exogenous 125I-pro-UK (primarily following its conversion to tc-u-PA and incorporation into tc-u-PA.PAI complexes) in an LRP/alpha2MR-dependent manner, which was inhibited by the LRP/alpha2MR receptor-associated protein. Receptor-associated protein also caused a approximately 50% reduction in cell surface plasminogen activator activity and delayed the regeneration of unoccupied uPAR by cells on which uPAR were initially saturated with pro-UK. Identical effects were caused by anti-LRP/alpha2MR antibodies. These results demonstrate that LRP/alpha2MR promotes the expression of cell surface plasminogen activator activity on trophoblasts by facilitating the clearance of tc-u-PA.PAI complexes and regeneration of unoccupied cell surface uPAR. PMID:9822706

  9. Urokinase receptor mRNA level and gene transcription are strongly and rapidly increased by phorbol myristate acetate in human monocyte-like U937 cells

    DEFF Research Database (Denmark)

    Lund, L R; Rønne, E; Roldan, A L;

    1991-01-01

    We have studied the effect of the tumor promotor phorbol myristate acetate (PMA) on the level of mRNA for the receptor for urokinase-type plasminogen activator (u-PAR) in the human monocyte-like cell line U937. PMA causes an early increase in the u-PAR mRNA level which reaches a maximal 50-fold e...

  10. The liberated domain I of urokinase plasminogen activator receptor--a new tumour marker in small cell lung cancer

    DEFF Research Database (Denmark)

    Almasi, Charlotte E; Drivsholm, Lars; Pappot, Helle;

    2013-01-01

    The prognosis of small cell lung cancer (SCLC) remains poor with a 5-year survival rate of 4-6%. In non-small cell lung cancer (NSCLC), high levels of intact and cleaved forms of the receptor for urokinase plasminogen activator (uPAR) are significantly associated with short overall survival. Our...

  11. Cellular receptor for urokinase plasminogen activator. Carboxyl-terminal processing and membrane anchoring by glycosyl-phosphatidylinositol

    DEFF Research Database (Denmark)

    Ploug, M; Rønne, E; Behrendt, N;

    1991-01-01

    The cellular receptor for human urokinase-type plasminogen activator (u-PAR) is shown by several independent criteria to be a true member of a family of integral membrane proteins, anchored to the plasma membrane exclusively by a COOH-terminal glycosyl-phosphatidylinositol moiety. 1) Amino acid...... acid compositions derived from cDNA sequence and amino acid analysis shows that a polypeptide of medium hydrophobicity is excised from the COOH terminus of the nascent u-PAR. A similar proteolytic processing has been reported for other proteins that are linked to the plasma membrane by a glycosyl...

  12. Stabilizing a flexible interdomain hinge region harboring the SMB binding site drives uPAR into its closed conformation.

    Science.gov (United States)

    Zhao, Baoyu; Gandhi, Sonu; Yuan, Cai; Luo, Zhipu; Li, Rui; Gårdsvoll, Henrik; de Lorenzi, Valentina; Sidenius, Nicolai; Huang, Mingdong; Ploug, Michael

    2015-03-27

    The urokinase-type plasminogen activator receptor (uPAR) is a multidomain glycolipid-anchored membrane protein, which facilitates extracellular matrix remodeling by focalizing plasminogen activation to cell surfaces via its high-affinity interaction with uPA. The modular assembly of its three LU (Ly6/uPAR-like) domains is inherently flexible and binding of uPA drives uPAR into its closed conformation, which presents the higher-affinity state for vitronectin thus providing an allosteric regulatory mechanism. Using a new class of epitope-mapped anti-uPAR monoclonal antibodies (mAbs), we now demonstrate that the reciprocal stabilization is indeed also possible. By surface plasmon resonance studies, we show that these mAbs and vitronectin have overlapping binding sites on uPAR and that they share Arg91 as hotspot residue in their binding interfaces. The crystal structure solved for one of these uPAR·mAb complexes at 3.0Å clearly shows that this mAb preselects the closed uPAR conformation with an empty but correctly assembled large hydrophobic binding cavity for uPA. Accordingly, these mAbs inhibit the uPAR-dependent lamellipodia formation and migration on vitronectin-coated matrices irrespective of the conformational status of uPAR and its occupancy with uPA. This is the first study to the best of our knowledge, showing that the dynamic assembly of the three LU domains in uPARwt can be driven toward the closed form by an external ligand, which is not engaging the hydrophobic uPA binding cavity. As this binding interface is also exploited by the somatomedin B domain of vitronectin, therefore, this relationship should be taken into consideration when exploring uPAR-dependent cell adhesion and migration in vitronectin-rich environments. PMID:25659907

  13. Prognostic value analysis of urokinase-type plasminogen activator receptor in oral squamous cell carcinoma: an immunohistochemical study

    Directory of Open Access Journals (Sweden)

    Rocchetti Romina

    2008-08-01

    Full Text Available Abstract Background Oral squamous cell carcinoma (OSCC represents the most common oral malignancy. Despite recent advances in therapy, up to 50% of the cases have relapse and/or metastasis. There is therefore a strong need for the identification of new biological markers able to predict the clinical behaviour of these lesions in order to improve quality of life and overall survival. Among tumour progression biomarkers, already known for their involvement in other neoplasia, a crucial role is ascribed to the urokinase-type plasminogen activator receptor (uPAR, which plays a multiple role in extracellular proteolysis, cell migration and tissue remodelling not only as a receptor for the zymogen pro-uPA but also as a component for cell adhesion and as a chemoattractant. The purpose of this study was to gain information on the expression of uPAR in OSCC and to verify whether this molecule can have a role as a prognostic/predictive marker for this neoplasia. Methods In a retrospective study, a cohort of 189 OSCC patients was investigated for uPAR expression and its cellular localization by immunohistochemistry. As standard controls, 8 normal oral mucosal tissues free of malignancy, obtained from patients with no evidence or history of oral cavity tumours, were similarly investigated. After grouping for uPAR expression, OSCCs were statistically analyzed for the variables age, gender, histological grading (G, tumour size, recurrence, TNM staging and overall survival rate. Results In our immunohistochemical study, 74 cases (39.1% of OSCC showed a mostly cytoplasmic positivity for uPAR, whereas 115 were negative. uPAR expression correlated with tumour differentiation grade and prognosis: percentage of positive cases was the greatest in G3 (70.4% and patients positives for uPAR expression had an expectation of life lower than those for uPAR negatives. Conclusion The results obtained in this study suggest a role of uPAR as a potential biomarker useful to

  14. Human Secreted Ly-6/uPAR Related Protein-1 (SLURP-1) Is a Selective Allosteric Antagonist of α7 Nicotinic Acetylcholine Receptor

    OpenAIRE

    Lyukmanova, Ekaterina N.; Mikhail A Shulepko; Denis Kudryavtsev; Bychkov, Maxim L.; Kulbatskii, Dmitrii S.; Kasheverov, Igor E.; Astapova, Maria V.; Feofanov, Alexey V; Thomsen, Morten S.; Jens D Mikkelsen; Shenkarev, Zakhar O.; Tsetlin, Victor I.; Dolgikh, Dmitry A.; Mikhail P Kirpichnikov

    2016-01-01

    SLURP-1 is a secreted toxin-like Ly-6/uPAR protein found in epithelium, sensory neurons and immune cells. Point mutations in the slurp-1 gene cause the autosomal inflammation skin disease Mal de Meleda. SLURP-1 is considered an autocrine/paracrine hormone that regulates growth and differentiation of keratinocytes and controls inflammation and malignant cell transformation. The majority of previous studies of SLURP-1 have been made using fusion constructs containing, in addition to the native ...

  15. Hypoxia stimulates urokinase receptor expression through a heme protein-dependent pathway.

    Science.gov (United States)

    Graham, C H; Fitzpatrick, T E; McCrae, K R

    1998-05-01

    Hypoxia underlies a number of biologic processes in which cellular migration and invasion occur. Because earlier studies have shown that the receptor for urokinase-type plasminogen activator (uPAR) may facilitate such events, we studied the effect of hypoxia on the expression of uPAR by first trimester human trophoblasts (HTR-8/SVneo) and human umbilical vein endothelial cells (HUVEC). Compared with control cells cultured under standard conditions (20% O2), HTR-8/SVneo cells and HUVEC cultured in 1% O2 expressed more uPAR, as determined by flow cytometric and [125I]-prourokinase ligand binding analyses. Increased uPAR expression paralleled increases in uPAR mRNA. The involvement of a heme protein in the hypoxia-induced expression of uPAR was suggested by the observations that culture of cells with cobalt chloride, or sodium 4, 5-dihydroxybenzene-1,3-disulfonate (Tiron), an iron-chelating agent, also stimulated uPAR expression, and that the hypoxia-induced uPAR expression was inhibited by adding carbon monoxide to the hypoxic atmosphere. Culture of HTR-8/SVneo cells with vascular endothelial growth factor (VEGF) did not increase uPAR mRNA levels, suggesting that the hypoxia-mediated effect on uPAR expression by these cells did not occur through a VEGF-dependent mechanism. The functional importance of these findings is suggested by the fact that HTR-8/SVneo cells cultured under hypoxia displayed higher levels of cell surface plasminogen activator activity and greater invasion through a reconstituted basement membrane. These results suggest that hypoxia may promote cellular invasion by stimulating the expression of uPAR through a heme protein-dependent pathway. PMID:9558386

  16. Synthesis of New Ligands of Urokinase Receptor and New Urotensin-II Derivatives

    OpenAIRE

    Yousif, Ali Munaim

    2015-01-01

    Unit 1: The urokinase-type plasminogen activator receptor (uPAR) is involved in the regulation of cell migration. The uPAR is formed by three domains connected by short linker region. The Ser88-Arg-Ser- Arg-Tyr92 is the minimum chemotactic sequence of uPAR required to induce the same intracellular signaling as result of its binding with formyl peptide receptor (FPRs). With aim to perform a SAR study on this sequence we have synthesized a large peptide library using phosphorylated ...

  17. uPAR and cathepsin B inhibition enhanced radiation-induced apoptosis in gliomainitiating cells

    OpenAIRE

    MALLA, RAMA RAO; Gopinath, Sreelatha; Alapati, Kiranmai; Gorantla, Bharathi; Christopher S Gondi; Rao, Jasti S.

    2012-01-01

    Glioblastomas present as diffuse tumors with invasion into normal brain tissue and frequently recur or progress after radiation as focal masses because of glioma-initiating cells. The role of the urokinase-type plasminogen activator receptor (uPAR) and cathepsin B in stem-like phenotype has been extensively studied in several solid tumors. In the present study, we demonstrated that selection of glioma-initiating cells using CD133 expression leads to a specific enrichment of CD133+ cells in bo...

  18. Direct interaction of the kringle domain of urokinase-type plasminogen activator (uPA) and integrin alpha v beta 3 induces signal transduction and enhances plasminogen activation

    OpenAIRE

    Tarui, Takehiko; Akakura, Nobuaki; Majumdar, Mousumi; Andronicos, Nicholas; Takagi, Junichi; Mazar, Andrew P.; Bdeir, Khalil; Kuo, Alice; Yarovoi, Serge V.; Cines, Douglas B.; Takada, Yoshikazu

    2006-01-01

    It has been questioned whether there are receptors for urokinase-type plasminogen activator (uPA) that facilitate plasminogen activation other than the high affinity uPA receptor (uPAR/CD87) since studies of uPAR knockout mice did not support a major role of uPAR in plasminogen activation. uPA also promotes cell adhesion, chemotaxis, and proliferation besides plasminogen activation. These uPA-induced signaling events are not mediated by uPAR, but mediated by unidentified, lower-affinity recep...

  19. New peptide receptor radionuclide therapy of invasive cancer cells: in vivo studies using 177Lu-DOTA-AE105 targeting uPAR in human colorectal cancer xenografts

    International Nuclear Information System (INIS)

    The proposition of uPAR as a potential target in cancer therapy is advanced by its predominant expression at the invasive front of colorectal cancer (CRC) and its value as prognostic biomarker for poor survival in this disease. In this study, we provide the first in vivo proof-of-concept for a theranostic approach as treatment modality in a human xenograft colorectal cancer model. Methods: A DOTA-conjugated 9-mer high affinity uPAR binding peptide (DOTA-AE105) was radiolabeled with 64Cu and 177Lu, for PET imaging and targeted radionuclide therapy study, respectively. Human uPAR-positive CRC HT-29 cells were inoculated in Nude mice and treated with 177Lu-DOTA-AE105 once a visible tumor had formed. To evaluate the true effect of the targeted radiotherapy, two controls groups were included in this study, one receiving a 177Lu-labeled non-binding control peptide and one receiving vehicle. All animals were treated day 0 and 7. A parallel 18F-FLT PET/CT study was performed on day 0, 1, 3 and 6. Dosimetry calculations were based on a biodistribution study, where organs and tissue of interest were collected 0.5, 1.0, 2.0, 4.0 and 24 h post injection of 177Lu-DOTA-AE105. Toxicity was assessed by recording mouse weight and by H and E staining of kidneys in each treatment group. Results: uPAR-positive HT-29 xenograft was clearly visualized by PET/CT imaging using 64Cu-DOTA-AE105. Subsequently, these xenograft transplants were locally irradiated using 177Lu-DOTA-AE105, where a significant effect on tumor size and the number of uPAR-positive cells in the tumor was found (p 18F-FLT PET/CT imaging study revealed a significant correlation between 18F-FLT tumor uptake and efficacy of the radionuclide therapy. A histological examination of the kidneys from one animal in each treatment group did not reveal any gross abnormalities and the general performance of all treated animals also showed no indications of radioactivity-induced toxicity. Conclusion: These findings document for the

  20. Urokinase plasminogen receptor and the fibrinolytic complex play a role in nerve repair after nerve crush in mice, and in human neuropathies.

    Directory of Open Access Journals (Sweden)

    Cristina Rivellini

    Full Text Available Remodeling of extracellular matrix (ECM is a critical step in peripheral nerve regeneration. In fact, in human neuropathies, endoneurial ECM enriched in fibrin and vitronectin associates with poor regeneration and worse clinical prognosis. Accordingly in animal models, modification of the fibrinolytic complex activity has profound effects on nerve regeneration: high fibrinolytic activity and low levels of fibrin correlate with better nerve regeneration. The urokinase plasminogen receptor (uPAR is a major component of the fibrinolytic complex, and binding to urokinase plasminogen activator (uPA promotes fibrinolysis and cell movement. uPAR is expressed in peripheral nerves, however, little is known on its potential function on nerve development and regeneration. Thus, we investigated uPAR null mice and observed that uPAR is dispensable for nerve development, whereas, loss of uPAR affects nerve regeneration. uPAR null mice showed reduced nerve repair after sciatic nerve crush. This was a consequence of reduced fibrinolytic activity and increased deposition of endoneurial fibrin and vitronectin. Exogenous fibrinolysis in uPAR null mice rescued nerve repair after sciatic nerve crush. Finally, we measured the fibrinolytic activity in sural nerve biopsies from patients with peripheral neuropathies. We showed that neuropathies with defective regeneration had reduced fibrinolytic activity. On the contrary, neuropathies with signs of active regeneration displayed higher fibrinolytic activity. Overall, our results suggest that enforced fibrinolysis may facilitate regeneration and outcome of peripheral neuropathies.

  1. Immunoglobulin and enzyme-conjugated dextran polymers enhance u-PAR staining intensity of carcinoma cells in peripheral blood smears

    DEFF Research Database (Denmark)

    Werther, K; Normark, M; Hansen, B F;

    1999-01-01

    phenotyping of disseminated carcinoma cells in bone marrow and peripheral blood smears. In the first step, the cells were incubated with antibodies against urokinase plasminogen activator receptor (u-PAR) and subsequently with secondary antibodies conjugated to peroxidase-labeled dextran polymers. A brown...... color reaction was developed with diaminobenzidine as chromogen. In the second step, the cells were incubated with alkaline phosphatase-conjugated murine monoclonal antibodies against a common cytokeratin epitope and a red color reaction was developed with new fuchsin as substrate. This method allows...

  2. uPA, uPAR and TGFβ₁ expression during early and late post myocardial infarction period in rat myocardium.

    Science.gov (United States)

    Stavropoulou, Anastasia; Philippou, Anastassios; Halapas, Antonios; Sourla, Antigone; Pissimissis, Nikolaos; Koutsilieris, Michael

    2010-01-01

    The expression patterns of transforming growth factor beta 1 (TGFβ₁), urokinase-type plasminogen activator (uPA) and uPA receptor (uPAR) were analysed after artery ligation-induced myocardial infarction (MI) in the rat myocardium. uPA and uPAR expressions were significantly increased both at transcriptional and protein level during early phase post MI period (uPA at 1 hour and uPAR at 24 hours post infarction). TGFβ1 mRNA expression profile revealed a significant increase of TGFβ1 expression from day 4 up to 8 weeks post infarction. These data suggest that the need for an increasing TGFβ₁ bioavailability during the post-infarction period in rat myocardium is achieved in the early post MI period by an increased expression of uPA/uPAR proteolytic system (indirect activation of latent TGFβ₁) and in the late post MI period by direct regulation of TGFβ₁ expression. It is therefore concluded that differential regulation of the TGFβ₁ bioavailability may be a crucial step of the repair mechanisms during the post MI infarction period in the rat myocardium.

  3. Immunocytochemical Phenotyping of Disseminated Tumor Cells in Bone Marrow by uPA Receptor and CK18: Investigation of Sensitivity and Specificity of an Immunogold/Alkaline Phosphatase Double Staining Protocol

    OpenAIRE

    Allgayer, Heike; Heiss, Markus Maria; Riesenberg, Rainer; Babic, Rudolf; Jauch, Karl Walter; Schildberg, Friedrich Wilhelm

    1997-01-01

    Phenotyping of cytokeratin (CK) 18-positive cells in bone marrow is gaining increasing importance for future prognostic screening of carcinoma patients. Urokinase-type plasminogen activator receptor (uPA-R) is one example of a potential aggressive marker for those cells. However, a valid and reliable double staining method is needed. Using monoclonal antibodies against uPA-R and CK18, we modified an immunogold/alkaline phosphatase double staining protocol. UPA-R/CK18-positive tumor cell contr...

  4. Conformational regulation of urokinase receptor function

    DEFF Research Database (Denmark)

    Gårdsvoll, Henrik; Jacobsen, Benedikte; Kriegbaum, Mette C;

    2011-01-01

    PA per se into the hydrophobic ligand binding cavity of uPAR that modulates the function of this receptor. Based on these data, we now propose a model in which the inherent interdomain mobility in uPAR plays a major role in modulating its function. Particularly one uPAR conformation, which is stabilized...

  5. Human Secreted Ly-6/uPAR Related Protein-1 (SLURP-1) Is a Selective Allosteric Antagonist of α7 Nicotinic Acetylcholine Receptor

    DEFF Research Database (Denmark)

    Lyukmanova, Ekaterina N; Shulepko, Mikhail A; Kudryavtsev, Denis;

    2016-01-01

    of nicotinic acetylcholine receptors (nAChRs) and muscarinic acetylcholine receptors, respectively, and anti-α7-nAChRs antibodies revealed α7 type nAChRs as an rSLURP-1 target in keratinocytes. Using affinity purification from human cortical extracts, we confirmed that rSLURP-1 binds selectively to the α7-n......AChRs. Exposure of Xenopus oocytes expressing α7-nAChRs to rSLURP-1 caused a significant non-competitive inhibition of the response to acetylcholine (up to ~ 70%, IC50 ~ 1 μM). It was shown that rSLURP-1 binds to α7-nAChRs overexpressed in GH4Cl cells, but does not compete with 125I-α-bungarotoxin for binding...

  6. Urokinase receptor cleavage correlates with tumor volume in a transgenic mouse model of breast cancer

    DEFF Research Database (Denmark)

    Thurison, Tine; Almholt, Kasper; Gårdsvoll, Henrik;

    2015-01-01

    marker for cancer than increased expression of muPAR per se. The levels of the muPAR forms are dramatically affected by in vivo challenge with a urokinase -blocking antibody, demonstrating a functional role of uPA in uPAR cleavage. The levels of the muPAR forms are, however, unaffected by u......The urokinase plasminogen activator system plays a key role in tissue degradation during cancer invasion. The linker region between domains I and II of the intact, three domain urokinase receptor uPAR(I-III) is highly susceptible to proteolytic cleavage and the resulting cleaved uPAR forms...... are strong prognostic biomarkers in several types of cancer, i.e., high levels of the cleaved uPAR forms indicate poor survival. To better understand the role of uPAR cleavage in cancer, we have designed immunoassays for specific quantification of intact mouse uPAR [muPAR(I-III)] and mouse uPAR domain I [mu...

  7. ELISA for complexes between urokinase-type plasminogen activator and its receptor in lung cancer tissue extracts

    DEFF Research Database (Denmark)

    de Witte, H; Pappot, H; Brünner, N;

    1997-01-01

    A sandwich-type ELISA has been developed for the assessment of complexes between urokinase-type plasminogen activator (uPA) and its receptor (uPAR) in extracts of squamous cell lung carcinomas. The assay is based on a combination of rabbit polyclonal anti-uPA antibodies and a biotinylated mouse...... extraction of uPAR yields the highest amounts of uPA:uPAR complexes. Absorption of tumor extracts with anti-uPA or anti-uPAR MAbs results in a complete disappearance of the ELISA signal, demonstrating the specificity of the ELISA. The recovery of chemically cross-linked uPA:uPAR complexes added to tumor...... extracts varies between 80% and 105%. The intra- and inter-assay variation coefficients are 5.3% and 9.8%, respectively. Furthermore, a peptide antagonist for uPAR was employed to evaluate de novo uPA:uPAR complex formation during tumor tissue extraction and the immunoassay procedure. Our results strongly...

  8. Prognostic significance of circulating intact and cleaved forms of urokinase plasminogen activator receptor in inoperable chemotherapy treated cholangiocarcinoma patients

    DEFF Research Database (Denmark)

    Grunnet, M; Christensen, I J; Lassen, U;

    2014-01-01

    determine if pre-treatment serum levels of uPAR forms and a decrease in levels during chemotherapy are predictive of survival in patients with inoperable cholangiocarcinoma. DESIGN AND METHODS: Patients with inoperable cholangiocarcinoma were consecutively included in the training set (n=108). A test set......PAR(I-III)+uPAR(II-III) after 2cycles of chemotherapy was associated with poor survival (HR=1.79, 95% CI:1.08-2.97, p=0.023, n=57). This predictor, however, was not significant in the test set (p=0.21, 26 events in 27 patients). CONCLUSION: The baseline level of uPAR(I-III)+uPAR(II-III) is a predictor of survival in inoperable......BACKGROUND: High levels of intact and cleaved forms of the urokinase-type plasminogen activator receptor (uPAR) in both tissue and blood are associated with poor survival in several cancer diseases. The prognostic significance of uPAR in cholangiocarcinoma is unknown. The aims of this study were to...

  9. Signaling through urokinase and urokinase receptor in lung cancer cells requires interactions with beta1 integrins.

    Science.gov (United States)

    Tang, Chi-Hui; Hill, Marla L; Brumwell, Alexis N; Chapman, Harold A; Wei, Ying

    2008-11-15

    The urokinase receptor (uPAR) is upregulated upon tumor cell invasion and correlates with poor lung cancer survival. Although a cis-interaction with integrins has been ascribed to uPAR, whether this interaction alone is critical to urokinase (uPA)- and uPAR-dependent signaling and tumor promotion is unclear. Here we report the functional consequences of point mutations of uPAR (H249A-D262A) that eliminate beta1 integrin interactions but maintain uPA binding, vitronectin attachment and association with alphaV integrins, caveolin and epidermal growth factor receptor. Disruption of uPAR interactions with beta1 integrins recapitulated previously reported findings with beta1-integrin-derived peptides that attenuated matrix-dependent ERK activation, MMP expression and in vitro migration by human lung adenocarcinoma cell lines. The uPAR mutant cells acquired enhanced capacity to adhere to vitronectin via uPAR-alphaVbeta5-integrin, rather than through the uPAR-alpha3beta1-integrin complex and they were unable to initiate uPA signaling to activate ERK, Akt or Stat1. In an orthotopic lung cancer model, uPAR mutant cells exhibited reduced tumor size compared with cells expressing wild-type uPAR. Taken together, the results indicate that uPAR-beta1-integrin interactions are essential to signals induced by integrin matrix ligands or uPA that support lung cancer cell invasion in vitro and progression in vivo. PMID:18940913

  10. Urokinase plasminogen activator receptor affects bone homeostasis by regulating osteoblast and osteoclast function

    DEFF Research Database (Denmark)

    Furlan, Federico; Galbiati, Clara; Jørgensen, Niklas R;

    2007-01-01

    The uPAR and its ligand uPA are expressed by both osteoblasts and osteoclasts. Their function in bone remodeling is unknown. We report that uPAR-lacking mice display increased BMD, increased osteogenic potential of osteoblasts, decreased osteoclasts formation, and altered cytoskeletal reorganizat......The uPAR and its ligand uPA are expressed by both osteoblasts and osteoclasts. Their function in bone remodeling is unknown. We report that uPAR-lacking mice display increased BMD, increased osteogenic potential of osteoblasts, decreased osteoclasts formation, and altered cytoskeletal...... to mechanical tests. UPAR KO calvaria osteoblasts were characterized by proliferation assays, RT-PCR for important proteins secreted during differentiation, and immunoblot for activator protein 1 (AP-1) family members. In vitro osteoclast formation was tested with uPAR KO bone marrow monocytes in the presence...

  11. Urokinase-type Plasminogen Activator-like Proteases in Teleosts Lack Genuine Receptor-binding Epidermal Growth Factor-like Domains*

    Science.gov (United States)

    Bager, René; Kristensen, Thomas K.; Jensen, Jan K.; Szczur, Agnieszka; Christensen, Anni; Andersen, Lisbeth M.; Johansen, Jesper S.; Larsen, Niels; Baatrup, Erik; Huang, Mingdong; Ploug, Michael; Andreasen, Peter A.

    2012-01-01

    Plasminogen activation catalyzed by urokinase-type plasminogen activator (uPA) plays an important role in normal and pathological tissue remodeling processes. Since its discovery in the mid-1980s, the cell membrane-anchored urokinase-type plasminogen activator receptor (uPAR) has been believed to be central to the functions of uPA, as uPA-catalyzed plasminogen activation activity appeared to be confined to cell surfaces through the binding of uPA to uPAR. However, a functional uPAR has so far only been identified in mammals. We have now cloned, recombinantly produced, and characterized two zebrafish proteases, zfuPA-a and zfuPA-b, which by several criteria are the fish orthologs of mammalian uPA. Thus, both proteases catalyze the activation of fish plasminogen efficiently and both proteases are inhibited rapidly by plasminogen activator inhibitor-1 (PAI-1). But zfuPA-a differs from mammalian uPA by lacking the exon encoding the uPAR-binding epidermal growth factor-like domain; zfuPA-b differs from mammalian uPA by lacking two cysteines of the epidermal growth factor-like domain and a uPAR-binding sequence comparable with that found in mammalian uPA. Accordingly, no zfuPA-b binding activity could be found in fish white blood cells or fish cell lines. We therefore propose that the current consensus of uPA-catalyzed plasminogen activation taking place on cell surfaces, derived from observations with mammals, is too narrow. Fish uPAs appear incapable of receptor binding in the manner known from mammals and uPA-catalyzed plasminogen activation in fish may occur mainly in solution. Studies with nonmammalian vertebrate species are needed to obtain a comprehensive understanding of the mechanism of plasminogen activation. PMID:22733817

  12. Structure-based Engineering of Species Selectivity in the Interaction Between Urokinase and its Receptor: Implication for Preclinical Cancer Therapy

    International Nuclear Information System (INIS)

    The high affinity interaction between the urokinase-type plasminogen activator (uPA) and its glycolipid-anchored receptor (uPAR) is decisive for cell surface-associated plasminogen activation. Because plasmin activity controls fibrinolysis in a variety of pathological conditions, including cancer and wound healing, several intervention studies have focused on targeting the uPA · uPAR interaction in vivo. Evaluations of such studies in xenotransplanted tumor models are, however, complicated by the pronounced species selectivity in this interaction. We now report the molecular basis underlying this difference by solving the crystal structure for the murine uPA · uPAR complex and demonstrate by extensive surface plasmon resonance studies that the kinetic rate constants for this interaction can be swapped completely between these orthologs by exchanging only two residues. This study not only discloses the structural basis required for a successful rational design of the species selectivity in the uPA · uPAR interaction, which is highly relevant for functional studies in mouse models, but it also suggests the possible development of general inhibitors that will target the uPA · uPAR interaction across species barriers.

  13. The Inhibitory Effects of an Antisense u-PAR Vector on Invasion of Highly Invasive Human Prostate Carcinoma PC-3M Cell Subclones

    Institute of Scientific and Technical Information of China (English)

    廖国宁; 李清芬; 冯友梅; 邓耀祖; 李卓娅; 龚非力; 马丁

    2003-01-01

    Summary: To observe the inhibitory effects of an antisense u-PAR vector on invasion of highly inva-sive PC-3M cell subclones, the effects of the antisense u-PAR on activity of MMP-9 in those highlyinvasive cell subclones were detected by a quantitative RT-PCR and zymography. The monolayer in-vasion assay and colony formation assay in soft agar were used. And tumorigenesis rate and invasionsby the cell subclones with or without the antisense u-PAR were observed in nude mice. It was foundthat in vitro growth of highly invasive PC-3M cell subclones transfected with the antisense u-PARwas declined, and the ability of anchorage-independent growth of those cell subclones was found de-creased sharply, with the inhibiting rate becoming 79 % and 60 %, respectively. Although the anti-sense u-PAR didn't change MMP-9 gene transcription, they could inhibit the activation of MMP-9 ofhighly invasive PC-3M cell subclones. Moreover, the tumorigenesis rate of the cell subclones with theantisense u-PAR decreased and the growth of a neoplasm also slowed down. Thet tests showed thedifference between experimental and control groups was statistically significant (P<0. 01). The anti-sense u-PAR vector could not only inhibit the invasion ability of highly invasive PC-3M cell subclonesin vitro but also restrain the growth of those cell subclones in vivo.

  14. 68Ga-labeling and in vivo evaluation of a uPAR binding DOTA- and NODAGA-conjugated peptide for PET imaging of invasive cancers

    International Nuclear Information System (INIS)

    Introduction: The urokinase-type plasminogen activator receptor (uPAR) is a well-established biomarker for tumor aggressiveness and metastatic potential. DOTA-AE105 and DOTA-AE105-NH2 labeled with 64Cu have previously been demonstrated to be able to noninvasively monitor uPAR expression using positron emission tomography (PET) in human cancer xenograft mice models. Here we introduce 68Ga-DOTA-AE105-NH2 and 68Ga-NODAGA-AE105-NH2 and evaluate their imaging properties using small-animal PET. Methods: Synthesis of DOTA-AE105-NH2 and NODAGA-AE105-NH2 was based on solid-phase peptide synthesis protocols using the Fmoc strategy. 68GaCl3 was eluted from a 68Ge/68Ga generator. The eluate was either concentrated on a cation-exchange column or fractionated and used directly for labeling. For in vitro characterization of both tracers, partition coefficient, buffer and plasma stability, uPAR binding affinity and cell uptake were determined. To characterize the in vivo properties, dynamic microPET imaging was carried out in nude mice bearing human glioma U87MG tumor xenograft. Results: In vitro experiments revealed uPAR binding affinities in the lower nM range for both conjugated peptides and identical to AE105. Labeling of DOTA-AE105-NH2 and NODAGA-AE105-NH2 with 68Ga was done at 95°C and room temperature, respectively. The highest radiochemical yield and purity were obtained using fractionated elution, whereas a negative effect of acetone on labeling efficiency for NODAGA-AE105-NH2 was observed. Good stability in phosphate-buffered saline and mouse plasma was observed. High cell uptake was found for both tracers in U87MG tumor cells. Dynamic microPET imaging demonstrated good tumor-to-background ratio for both tracers. Tumor uptake was 2.1% ID/g and 1.3% ID/g 30 min postinjection and 2.0% ID/g and 1.1% ID/g 60 min postinjection for 68Ga-NODAGA-AE105-NH2 and 68Ga-DOTA-AE105-NH2, respectively. A significantly higher tumor-to-muscle ratio (P68Ga-NODAGA-AE105-NH2 60 min

  15. Reduced release of intact and cleaved urokinase receptor in stimulated whole-blood cultures from human immunodeficiency virus-1-infected patients

    DEFF Research Database (Denmark)

    Ostrowski, S R; Piironen, T; Høyer-Hansen, G;

    2005-01-01

    The blood levels of the soluble forms of the urokinase receptor (suPAR) are increased in human immunodeficiency virus (HIV)-1-infected patients. This study investigated whether the release of urokinase-type plasminogen activator receptor (uPAR) in whole-blood cultures was affected by HIV infection...... controls, whereas the correlation was weaker to leucocytes and nonexisting to circulating suPAR levels in HIV patients. These findings demonstrate that HIV infection affects stimulated and spontaneous uPAR release in whole-blood cultures. Given that high blood levels of suPAR in HIV patients are linked to....... The release of different uPAR forms in whole-blood cultures incubated 24 h with or without phytohemagglutinin and lipopolysaccharide was analysed in 47 HIV patients and 19 controls. suPAR was measured by enzyme-linked immunosorbent assay (ELISA) (bulk-suPAR) and three different time...

  16. Dosimetry of 64Cu-DOTA-AE105, a PET tracer for uPAR imaging

    International Nuclear Information System (INIS)

    64Cu-DOTA-AE105 is a novel positron emission tomography (PET) tracer specific to the human urokinase-type plasminogen activator receptor (uPAR). In preparation of using this tracer in humans, as a new promising method to distinguish between indolent and aggressive cancers, we have performed PET studies in mice to evaluate the in vivo biodistribution and estimate human dosimetry of 64Cu-DOTA-AE105. Methods: Five mice received iv tail injection of 64Cu-DOTA-AE105 and were PET/CT scanned 1, 4.5 and 22 h post injection. Volume-of-interest (VOI) were manually drawn on the following organs: heart, lung, liver, kidney, spleen, intestine, muscle, bone and bladder. The activity concentrations in the mentioned organs [%ID/g] were used for the dosimetry calculation. The %ID/g of each organ at 1, 4.5 and 22 h was scaled to human value based on a difference between organ and body weights. The scaled values were then exported to OLINDA software for computation of the human absorbed doses. The residence times as well as effective dose equivalent for male and female could be obtained for each organ. To validate this approach, of human projection using mouse data, five mice received iv tail injection of another 64Cu-DOTA peptide-based tracer, 64Cu-DOTA-TATE, and underwent same procedure as just described. The human dosimetry estimates were then compared with observed human dosimetry estimate recently found in a first-in-man study using 64Cu-DOTA-TATE. Results: Human estimates of 64Cu-DOTA-AE105 revealed the heart wall to receive the highest dose (0.0918 mSv/MBq) followed by the liver (0.0815 mSv/MBq), All other organs/tissue were estimated to receive doses in the range of 0.02–0.04 mSv/MBq. The mean effective whole-body dose of 64Cu-DOTA-AE105 was estimated to be 0.0317 mSv/MBq. Relatively good correlation between human predicted and observed dosimetry estimates for 64Cu-DOTA-TATE was found. Importantly, the effective whole body dose was predicted with very high precision

  17. Administration of Recombinant Soluble Urokinase Receptor Per Se Is Not Sufficient to Induce Podocyte Alterations and Proteinuria in Mice

    DEFF Research Database (Denmark)

    Cathelin, Dominique; Placier, Sandrine; Ploug, Michael;

    2014-01-01

    Circulating levels of soluble forms of urokinase-type plasminogen activator receptor (suPAR) are generally elevated in sera from children and adults with FSGS compared with levels in healthy persons or those with other types of kidney disease. In mice lacking the gene encoding uPAR, forced increa...

  18. Identification and characterization of the murine cell surface receptor for the urokinase-type plasminogen activator

    DEFF Research Database (Denmark)

    Solberg, H; Løber, D; Eriksen, J;

    1992-01-01

    -hybridization and pronounced sequence similarity with human u-PAR cDNA [Kristensen, P., Eriksen, J., Blasi, F. & Danø, K. (1991) J. Cell Biol. 115, 1763-1771]. A rabbit antiserum raised against this peptide specifically recognized two polypeptide bands with electrophoretic mobilities identical to those identified by ligand...... of this variant yielded a polypeptide with an apparent M(r) of about 30,000, which corresponds to the Mr calculated from the cDNA derived protein sequence of mouse u-PAR. Receptor-bound mouse u-PA could be released by phosphatidylinositol-specific phospholipase C treatment, indicating that mouse u-PAR is attached...... by ligand-blotting analysis.(ABSTRACT TRUNCATED AT 400 WORDS)...

  19. Prognostic value of intact and cleaved forms of the urokinase plasminogen activator receptor in a retrospective study of 518 colorectal cancer patients

    DEFF Research Database (Denmark)

    Lomholt, Anne Fog; Christensen, Ib J; Høyer-Hansen, Gunilla;

    2010-01-01

    The levels of the soluble urokinase plasminogen activator receptor (suPAR) in blood have been shown to correlate with prognosis in various cancers. Plasma levels of the combined suPAR forms have previously shown to be a strong prognostic marker in the present cohort of CRC patients and could...... potentially identify high-risk patients among those with early stage disease. In order to investigate whether the individual suPAR forms are stronger prognostic markers than the combined amount we measured the different uPAR forms in serum from the same cohort and evaluated their prognostic significance....

  20. Markers of angiogenesis and epidermal growth factor receptor signalling in patients with pancreatic and gastroesophageal junction cancer.

    Science.gov (United States)

    Rohrberg, Kristoffer Staal; Skov, Birgit Guldhammer; Lassen, Ulrik; Christensen, Ib Jarle; Høyer-Hansen, Gunilla; Buysschaert, Ian; Pappot, Helle

    2010-01-01

    The epidermal growth factor receptor (EGFR) and angiogenesis are well established targets in anti-cancer therapy. Several targeted anti-cancer therapies are in clinical trials in pancreatic and gastroesophageal (GEJ) cancer. However, many patients do not respond to these targeted therapies and there is therefore an increasing need for biomarkers for selection of patients to these therapies. We investigated the expression of EGFR, vascular endothelial growth factor A (VEGF-A), and VEGF receptor 2 (VEGFR-2) in tumour tissue by immunohistochemistry, and soluble EGFR (sEGFR), soluble VEGFR-2 (sVEGFR-2), basic fibroblast growth factor (bFGF), placental growth factor (PlGF), plasminogen activator inhibitor 1 (PAI-1), and different forms of the urokinase plasminogen activator receptor (uPAR): uPAR (I), uPAR (I-III), and uPAR (I-III)+(II-III) in plasma by quantitative immunoassays in 14 patients with pancreatic and GEJ cancer. We found expression in tumour tissue and the plasma levels to be similar to those found in patients with other tumour types. No correlation was found between the blood levels of soluble receptors and the corresponding tumour tissue levels. We conclude that these markers are present in pancreatic and GEJ cancer patients, and could be investigated further as predictive biomarkers in such patients treated with EGFR or angiogenesis targeted therapies.

  1. Mimicry of the regulatory role of urokinase in lamellipodia formation by introduction of a non-native interdomain disulfide bond in its receptor

    DEFF Research Database (Denmark)

    Gårdsvoll, Henrik; Kjærgaard, Magnus; Jacobsen, Benedikte;

    2011-01-01

    The high-affinity interaction between the urokinase-type plasminogen activator (uPA) and its glycolipid-anchored receptor (uPAR) plays a regulatory role for both extravascular fibrinolysis and uPAR-mediated adhesion and migration on vitronectin-coated surfaces. We have recently proposed that the ...

  2. Differential binding of urokinase and peptide antagonists to the urokinase receptor

    DEFF Research Database (Denmark)

    Engelholm, L H; Behrendt, N

    2001-01-01

    though these sequences contain very few substitutions relative to the human uPAR, the receptor protein products differ markedly in terms of ligand selectivity. Thus, a well described competitive peptide antagonist directed against the human uPAR reacts with only one of the monkey receptors (chimpanzee u......PAR), in spite of the fact that uPAR from all of the four species cross-reacts with human uPA. Notably, uPAR from African green monkey, which is completely devoid of reactivity with the peptide, contains only three substitutions relative to chimpanzee uPAR in the molecular regions critical for binding....... These findings aid the elucidation of the structure/function relationship of uPAR and, unexpectedly, identify a structural distinction governing the binding of uPA and a very similar peptide antagonist....

  3. HIV-1 infected lymphoid organs upregulate expression and release of the cleaved form of uPAR that modulates chemotaxis and virus expression.

    Directory of Open Access Journals (Sweden)

    Manuela Nebuloni

    Full Text Available Cell-associated receptor for urokinase plasminogen activator (uPAR is released as both full-length soluble uPAR (suPAR and cleaved (c-suPAR form that maintain ability to bind to integrins and other receptors, thus triggering and modulating cell signaling responses. Concerning HIV-1 infection, plasma levels of suPAR have been correlated with the severity of disease, levels of immune activation and ineffective immune recovery also in individuals receiving combination anti-retroviral therapy (cART. However, it is unknown whether and which suPAR forms might contribute to HIV-1 induced pathogenesis and to the related state of immune activation. In this regard, lymphoid organs represent an import site of chronic immune activation and virus persistence even in individuals receiving cART. Lymphoid organs of HIV-1(+ individuals showed an enhanced number of follicular dendritic cells, macrophages and endothelial cells expressing the cell-associated uPAR in comparison to those of uninfected individuals. In order to investigate the potential role of suPAR forms in HIV-1 infection of secondary lymphoid organs, tonsil histocultures were established from HIV-1 seronegative individuals and infected ex vivo with CCR5- and CXCR4-dependent HIV-1 strains. The levels of suPAR and c-suPAR were significantly increased in HIV-infected tonsil histocultures supernatants in comparison to autologous uninfected histocultures. Supernatants from infected and uninfected cultures before and after immunodepletion of suPAR forms were incubated with the chronically infected promonocytic U1 cell line characterized by a state of proviral latency in unstimulated conditions. In the contest of HIV-conditioned supernatants we established that c-suPAR, but not suPAR, inhibited chemotaxis and induced virus expression in U1 cells. In conclusion, lymphoid organs are an important site of production and release of both suPAR and c-suPAR, this latter form being endowed with the capacity of

  4. Levels of plasminogen activator inhibitor type 1 and urokinase plasminogen activator receptor in non-small cell lung cancer as measured by quantitative ELISA and semiquantitative immunohistochemistry

    DEFF Research Database (Denmark)

    Pappot, Helle; Skov, Birgit Guldhammer; Pyke, Charles;

    1997-01-01

    , results in non-small cell lung cancer (NSCLC) studies obtained by quantitative ELISA and semiquantitative immunohistochemistry differ. If the prognostic value of the components of the plasminogen activation system is to be exploited clinically in the future, it is important to choose an easy and valid......PAR with the aim of predicting prognosis. In conclusion, a larger comparative study is needed to clarify the relationship between ELISA and immunohistochemical results, before a methodology for clinical use can be chosen in non-small cell lung cancer....... methodology. In the present study we investigated levels of plasminogen activator inhibitor type 1 (PAI-I) and urokinase plasminogen activator receptor (uPAR), as quantitated by ELISA in tumour extracts from 64 NSCLC patients (38 squamous cell carcinomas, 26 adenocarcinomas), and compared them to staining...

  5. Discrimination of different forms of the murine urokinase plasminogen activator receptor on the cell surface using monoclonal antibodies

    DEFF Research Database (Denmark)

    Rasch, M.G.; Pass, J.; Illemann, M.;

    2008-01-01

    of saturation with the amino-terminal fragment of uPA, ATF. However, the signal intensity obtained using another uPAR domain I specific mAb, mR1, was significantly reduced upon ATF saturation. Furthermore, when adding ATF, mR4 selectively stained the cleaved receptor. Applying these newly generated mAbs, we...... and pathological extracellular tissue remodelling processes. uPA can also cleave uPAR, resulting in liberation of the amino-terminal domain I, which encompasses binding sites for both uPA and the adhesion molecule, vitronectin. In order to localise the different uPAR forms on the plasma membrane of murine monocyte...

  6. Downregulation of uPAR and cathepsin B induces apoptosis via regulation of Bcl-2 and Bax and inhibition of the PI3K/Akt pathway in gliomas.

    Directory of Open Access Journals (Sweden)

    Ramarao Malla

    Full Text Available BACKGROUND: Glioma is the most commonly diagnosed primary brain tumor and is characterized by invasive and infiltrative behavior. uPAR and cathepsin B are known to be overexpressed in high-grade gliomas and are strongly correlated with invasive cancer phenotypes. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we observed that simultaneous downregulation of uPAR and cathepsin B induces upregulation of some pro-apoptotic genes and suppression of anti-apoptotic genes in human glioma cells. uPAR and cathepsin B (pCU-downregulated cells exhibited decreases in the Bcl-2/Bax ratio and initiated the collapse of mitochondrial membrane potential. We also observed that the broad caspase inhibitor, Z-Asp-2, 6-dichlorobenzoylmethylketone rescued pCU-induced apoptosis in U251 cells but not in 5310 cells. Immunoblot analysis of caspase-9 immunoprecipitates for Apaf-1 showed that uPAR and cathepsin B knockdown activated apoptosome complex formation in U251 cells. Downregulation of uPAR and cathepsin B also retarded nuclear translocation and interfered with DNA binding activity of CREB in both U251 and 5310 cells. Further western blotting analysis demonstrated that downregulation of uPAR and cathepsin B significantly decreased expression of the signaling molecules p-PDGFR-β, p-PI3K and p-Akt. An increase in the number of TUNEL-positive cells, increased Bax expression, and decreased Bcl-2 expression in nude mice brain tumor sections and brain tissue lysates confirm our in vitro results. CONCLUSIONS/SIGNIFICANCE: In conclusion, RNAi-mediated downregulation of uPAR and cathepsin B initiates caspase-dependent mitochondrial apoptosis in U251 cells and caspase-independent mitochondrial apoptosis in 5310 cells. Thus, targeting uPAR and cathepsin B-mediated signaling using siRNA may serve as a novel therapeutic strategy for the treatment of gliomas.

  7. TISSUE INHIBITOR OF METALLOPROTEINASE 1, MATRIX METALLOPROTEINASE 9, ALPHA-1 ANTITRYPSIN, METALLOTHIONEIN AND UROKINASE TYPE PLASMINOGEN ACTIVATOR RECEPTOR IN SKIN BIOPSIES FROM PATIENTS AFFECTED BY AUTOIMMUNE BLISTERING DISEASES

    Directory of Open Access Journals (Sweden)

    Ana Maria Abreu Velez

    2013-07-01

    Full Text Available Introduction: Proteinases and proteinase inhibitors have been described to play a role in autoimmune skin blistering diseases. We studied skin lesional biopsies from patients affected by several autoimmune skin blistering diseases for proteinases and proteinase inhibitors. Methods: We utilized immunohistochemistry to evaluate biopsies for alpha-1-antitrypsin, human matrix metalloproteinase 9 (MMP9, human tissue inhibitor of metalloproteinases 1 (TIMP-1, metallothionein and urokinase type plasminogen activator receptor (uPAR. We tested 30 patients affected by endemic pemphigus, 30 controls from the endemic area, and 15 normal controls. We also tested 30 biopsies from patients with bullous pemphigoid (BP, 20 with pemphigus vulgaris (PV, 8 with pemphigus foliaceus, and 14 with dermatitis herpetiformis (DH. Results: Contrary to findings in the current literature, most autoimmune skin blistering disease biopsies were negative for uPAR and MMP9. Only some chronic patients with El Bagre-EPF were positive to MMP9 in the dermis, in proximity to telocytes. TIMP-1 and metallothionein were positive in half of the biopsies from BP patients at the basement membrane of the skin, within several skin appendices, in areas of dermal blood vessel inflammation and within dermal mesenchymal-epithelial cell junctions.

  8. Bacterial endotoxin enhances colorectal cancer cell adhesion and invasion through TLR-4 and NF-kappaB-dependent activation of the urokinase plasminogen activator system.

    LENUS (Irish Health Repository)

    Killeen, S D

    2009-05-19

    Perioperative exposure to lipopolysaccharide (LPS) is associated with accelerated metastatic colorectal tumour growth. LPS directly affects cells through Toll-like receptor 4 (TLR-4) and the transcription factor NF-kappaB. The urokinase plasminogen activator (u-PA) system is intimately implicated in tumour cell extracellular matrix (ECM) interactions fundamental to tumour progression. Thus we sought to determine if LPS directly induces accelerated tumour cell ECM adhesion and invasion through activation of the u-PA system and to elucidate the cellular pathways involved. Human colorectal tumour cell lines were stimulated with LPS. u-PA concentration, u-PA activity, active u-PA, surface urokinase plasminogen activator receptor (u-PAR) and TLR-4 expression were assessed by ELISA, colorimetric assay, western blot analysis and flow cytometry respectively. In vitro tumour cell vitronectin adhesion and ECM invasion were analysed by vitronectin adhesion assay and ECM invasion chambers. u-PA and u-PAR function was inhibited with anti u-PA antibodies or the selective u-PA inhibitors amiloride or WXC-340, TLR-4 by TLR-4-blocking antibodies and NF-kappaB by the selective NF-kappaB inhibitor SN-50. LPS upregulates u-PA and u-PAR in a dose-dependent manner, enhancing in vitro tumour cell vitronectin adhesion and ECM invasion by >40% (P<0.01). These effects were ameliorated by u-PA and u-PAR inhibition. LPS activates NF-kappaB through TLR-4. TLR-4 and NF-kappaB inhibition ameliorated LPS-enhanced u-PA and u-PAR expression, tumour cell vitronectin adhesion and ECM invasion. LPS promotes tumour cell ECM adhesion and invasion through activation of the u-PA system in a TLR-4- and NF-kappaB-dependent manner.

  9. A composite role of vitronectin and urokinase in the modulation of cell morphology upon expression of the urokinase receptor

    DEFF Research Database (Denmark)

    Hillig, Thore; Engelholm, Lars H; Ingvarsen, Signe;

    2008-01-01

    The urokinase receptor, urokinase receptor (uPAR), is a glycosylphosphatidylinositol-anchored membrane protein engaged in pericellular proteolysis and cellular adhesion, migration, and modulation of cell morphology. A direct matrix adhesion is mediated through the binding of uPAR to vitronectin, ...

  10. A conserved TATA-less proximal promoter drives basal transcription from the urokinase-type plasminogen activator receptor gene

    DEFF Research Database (Denmark)

    Soravia, E; Grebe, A; De Luca, P;

    1995-01-01

    have cloned an uPAR DNA segment containing upstream regulatory sequences from both the human and murine genomes. We report that a proximal promoter, contained within 180 bp from the major transcription start sites of the human uPAR gene, drives basal transcription. This region lacks TATA and CAAT boxes...... and contains relatively GC-rich proximal sequences. A subregion of this sequence, highly conserved between human and murine genes, contains most of the promoter activity and is specifically bound by HeLa nuclear proteins, one of which belongs to the SP1 class....

  11. Ligand binding alters dimerization and sequestering of urokinase receptors in raft-mimicking lipid mixtures.

    Science.gov (United States)

    Ge, Yifan; Siegel, Amanda P; Jordan, Rainer; Naumann, Christoph A

    2014-11-01

    Lipid heterogeneities, such as lipid rafts, are widely considered to be important for the sequestering of membrane proteins in plasma membranes, thereby influencing membrane protein functionality. However, the underlying mechanisms of such sequestration processes remain elusive, in part, due to the small size and often transient nature of these functional membrane heterogeneities in cellular membranes. To overcome these challenges, here we report the sequestration behavior of urokinase receptor (uPAR), a glycosylphosphatidylinositol-anchored protein, in a planar model membrane platform with raft-mimicking lipid mixtures of well-defined compositions using a powerful optical imaging platform consisting of confocal spectroscopy XY-scans, photon counting histogram, and fluorescence correlation spectroscopy analyses. This methodology provides parallel information about receptor sequestration, oligomerization state, and lateral mobility with single molecule sensitivity. Most notably, our experiments demonstrate that moderate changes in uPAR sequestration are not only associated with modifications in uPAR dimerization levels, but may also be linked to ligand-mediated allosteric changes of these membrane receptors. Our data show that these modifications in uPAR sequestration can be induced by exposure to specific ligands (urokinase plasminogen activator, vitronectin), but not via adjustment of the cholesterol level in the planar model membrane system. Good agreement of our key findings with published results on cell membranes confirms the validity of our model membrane approach. We hypothesize that the observed mechanism of receptor translocation in the presence of raft-mimicking lipid mixtures is also applicable to other glycosylphosphatidylinositol-anchored proteins.

  12. Interleukin-6 infusion during human endotoxaemia inhibits in vitro release of the urokinase receptor from peripheral blood mononuclear cells

    DEFF Research Database (Denmark)

    Ostrowski, S R; Plomgaard, P; Fischer, C P;

    2005-01-01

    Leucocyte expression of the urokinase receptor [urokinase-type plasminogen activator receptor (uPAR)] is regulated by inflammatory mediators. This study investigated the in vivo effect of endotoxin, interleukin (IL)-6 and tumour necrosis factor (TNF)-alpha on uPAR-release in vivo and in vitro in...... humans. Healthy subjects received intravenous endotoxin injection [high-dose, 2 ng/kg (n=8) and low-dose, 0.06 ng/kg (n=7)], coadministration of 0.06 ng/kg endotoxin and 3 h recombinant human (rh)IL-6 infusion (n=7) or 3 h infusion of rhIL-6 (n=6), rhTNF-alpha (n=6) or NaCl (n=5). Soluble uPAR (su...

  13. Urokinase-catalysed cleavage of the urokinase receptor requires an intact glycolipid anchor

    DEFF Research Database (Denmark)

    Høyer-Hansen, G; Pessara, U; Holm, A;

    2001-01-01

    Urokinase (uPA) has the striking ability to cleave its receptor, uPAR, thereby inactivating the binding potential of this molecule. Here we demonstrate that the glycosylphosphatidylinositol (GPI) anchor of uPAR, which is attached to the third domain, is an important determinant in governing this ...

  14. Structure-driven design of radionuclide tracers for non-invasive imaging of uPAR and targeted radiotherapy. The tale of a synthetic peptide antagonist

    DEFF Research Database (Denmark)

    Ploug, Michael

    2013-01-01

    -function relationships in uPAR has been refined to such a level that a rational design of uPAR function as well as compounds specifically targeting defined functions of uPAR are now realistic options. This knowledge opens new avenues for developing therapeutic intervention regimens targeting uPAR as well...

  15. Two distinct expression patterns of urokinase, urokinase receptor and plasminogen activator inhibitor-1 in colon cancer liver metastases

    DEFF Research Database (Denmark)

    Illemann, Martin; Bird, Nigel; Majeed, Ali;

    2009-01-01

    Metastatic growth and invasion by colon cancer cells in the liver requires the ability of the cancer cells to interact with the new tissue environment. Plasmin(ogen) is activated on cell surfaces by urokinase-type PA (uPA), and is regulated by uPAR and plasminogen activator inhibitor-1 (PAI-1......). To compare the expression patterns of uPA, uPAR and PAI-1 in colon cancer with that in their liver metastases, we analysed matched samples from 14 patients. In all 14 primary colon cancers, we found upregulation of uPAR, uPA mRNA and PAI-1 in primarily stromal cells at the invasive front. In 5 of the 14......, whereas 8 of the remaining 9 showed direct contact between the cancer cells and the liver parenchyma. We conclude that there are 2 distinct patterns of expression of uPAR, uPA and PAI-1 in colon cancer liver metastases and that these correlate closely with 2 morphological growth patterns. These findings...

  16. Chapter 8. Activation mechanisms of chemokine receptors

    DEFF Research Database (Denmark)

    Jensen, Pia C; Rosenkilde, Mette M

    2009-01-01

    Chemokine receptors belong to the large family of 7-transmembrane (7TM) G-protein-coupled receptors. These receptors are targeted and activated by a variety of different ligands, indicating that activation is a result of similar molecular mechanisms but not necessarily similar modes of ligand bin...

  17. Mechanism for the activation of glutamate receptors

    Science.gov (United States)

    Scientists at the NIH have used a technique called cryo-electron microscopy to determine a molecular mechanism for the activation and desensitization of ionotropic glutamate receptors, a prominent class of neurotransmitter receptors in the brain and spina

  18. Downregulation of uPAR and Cathepsin B Retards Cofilin Dephosphorylation

    OpenAIRE

    Christopher S Gondi; Kandhukuri, Neelima; Kondraganti, Shakuntala; Gujrati, Meena; Olivero, William C.; Dinh, Dzung H.; Rao, Jasti S.

    2006-01-01

    Cathepsin B and uPAR play key roles in cancer cell migration and invasion. Here, we demonstrate that the simultaneous, siRNA-mediated downregulation of uPAR and cathepsin B inhibits glioma cell migration and is accompanied by cytoskeletal condensation. We show that the dephosphorylation of cofilin is inhibited by the downregulation of uPAR alone and, to a lesser extent, by the downregulation of cathepsin B alone, and that the effect was much higher by the downregulation of both molecules by p...

  19. CERAPP: Collaborative Estrogen Receptor Activity Prediction Project

    Data.gov (United States)

    U.S. Environmental Protection Agency — Data from a large-scale modeling project called CERAPP (Collaborative Estrogen Receptor Activity Prediction Project) demonstrating using predictive computational...

  20. The ligand-binding domain of the cell surface receptor for urokinase-type plasminogen activator

    DEFF Research Database (Denmark)

    Behrendt, N; Ploug, M; Patthy, L;

    1991-01-01

    with the internal repeats of u-PAR constitute the extracellular part of Ly-6 antigens and of the squid glycoprotein Sgp-2. Like u-PAR, these proteins are attached to the membrane by a glycosyl-phosphatidylinositol anchor. The hydrophilic, ligand-binding u-PAR domain identified in the present study has potential...

  1. Hormone activation of baculovirus expressed progesterone receptors.

    Science.gov (United States)

    Elliston, J F; Beekman, J M; Tsai, S Y; O'Malley, B W; Tsai, M J

    1992-03-15

    Human and chicken progesterone receptors (A form) were overproduced in a baculovirus expression system. These recombinant progesterone receptors were full-length bound progesterone specifically and were recognized by monoclonal antibodies, AB52 and PR22, specific for human and chicken progesterone receptor, respectively. In gel retardation studies, binding of recombinant human and chicken progesterone receptors to their progesterone response element (PRE) was specific and was enhanced in the presence of progesterone. Binding of human progesterone receptor to the PRE was also enhanced in the presence of the antiprogestin, RU486, but very little effect was observed in the presence of estradiol, dexamethasone, testosterone, and vitamin D. In our cell-free transcription system, human progesterone receptor induced transcription in a receptor-dependent and hormone-activable manner. Receptor-stimulated transcription required the presence of the PRE in the test template and could be specifically inhibited by excess PRE oligonucleotides. Furthermore, chicken progesterone receptor also induced in vitro transcription in a hormone-activable manner. These results demonstrate that steroid receptors overexpressed in a baculovirus expression system are functional and exhibit steroid-responsive binding and transcription. These observations support our present understanding of the mechanism of steroid receptor-regulated gene expression and provide a technological format for studies of the role of hormone and antihormone in altering gene expression. PMID:1544902

  2. Profiling signalling pathways in formalin-fixed and paraffin-embedded breast cancer tissues reveals cross-talk between EGFR, HER2, HER3 and uPAR.

    Science.gov (United States)

    Berg, Daniela; Wolff, Claudia; Malinowsky, Katharina; Tran, Kai; Walch, Axel; Bronger, Holger; Schuster, Tibor; Höfler, Heinz; Becker, Karl-Friedrich

    2012-01-01

    In the last few years, new approaches and developments in patient-tailored cancer therapies have raised the need to select, more precisely, those patients who will respond to personalized treatments. Therefore, the most efficient way for optimal therapy and patient selection is to provide a tumour-specific protein network portrait prior to treatment. The aim of our study was to monitor protein networks in formalin-fixed and paraffin-embedded (FFPE) breast cancer tissues, with special emphasis on epidermal growth factor receptor 2 (HER2)-mediated signalling pathways, to identify and validate new disease markers. For this purpose we used a recently developed technology to extract full-length proteins from FFPE tissues and analysed 23 molecules involved in HER2-related signalling by reverse phase protein microarray (RPPA) in a series of 106 FFPE breast cancer tissue samples. We found a significant correlation of HER2 with human epidermal growth factor receptor 3 (HER3/erbB3), epidermal growth factor receptor 1 (EGFR/HER1/erbB1) and urokinase plasminogen receptor (uPAR) in routinely used FFPE breast cancer tissues. Thus, targeting HER2, EGFR, HER3 and uPAR together may offer a more efficient treatment option for patients with breast cancer.

  3. Improved PET Imaging of uPAR expression using new Cu-64-labeled cross-bridged peptide ligands

    DEFF Research Database (Denmark)

    Persson, Morten; Hosseini, Masood; Madsen, Jacob;

    2013-01-01

    The correlation between uPAR expression, cancer cell invasion and metastases is now well-established and has prompted the development of a number of uPAR PET imaging agents, which could potentially identify cancer patients with invasive and metastatic lesions. In the present study, we synthesized...

  4. Androgen insensitivity syndrome: gonadal androgen receptor activity

    International Nuclear Information System (INIS)

    To determine whether abnormalities of the androgen receptor previously observed in skin fibroblasts from patients with androgen insensitivity syndrome also occur in the gonads of affected individuals, androgen receptor activity in the gonads of a patient with testicular feminization syndrome was investigated. Using conditions for optimal recovery of androgen receptor from human testes established by previous studies, we detected the presence of a high-affinity (dissociation constant . 3.2 X 10(-10) mol/L), low-capacity (4.2 X 10(-12) mol/mg DNA), androgen-binding protein when tritium-labeled R1881 was incubated at 4 degrees C with nuclear extracts from the gonads of control patients or from a patient with testicular feminization syndrome but not when incubated at 37 degrees C. Thus this patient has an androgen receptor with a temperature lability similar to that of receptors from normal persons

  5. Direct interaction of the kringle domain of urokinase-type plasminogen activator (uPA) and integrin alpha v beta 3 induces signal transduction and enhances plasminogen activation.

    Science.gov (United States)

    Tarui, Takehiko; Akakura, Nobuaki; Majumdar, Mousumi; Andronicos, Nicholas; Takagi, Junichi; Mazar, Andrew P; Bdeir, Khalil; Kuo, Alice; Yarovoi, Serge V; Cines, Douglas B; Takada, Yoshikazu

    2006-03-01

    It has been questioned whether there are receptors for urokinase-type plasminogen activator (uPA) that facilitate plasminogen activation other than the high affinity uPA receptor (uPAR/CD87) since studies of uPAR knockout mice did not support a major role of uPAR in plasminogen activation. uPA also promotes cell adhesion, chemotaxis, and proliferation besides plasminogen activation. These uPA-induced signaling events are not mediated by uPAR, but mediated by unidentified, lower-affinity receptors for the uPA kringle. We found that uPA binds specifically to integrin alpha v beta 3 on CHO cells depleted of uPAR. The binding of uPA to alpha v beta 3 required the uPA kringle domain. The isolated uPA kringle domain binds specifically to purified, recombinant soluble, and cell surface alpha v beta 3, and other integrins (alpha 4 beta 1 and alpha 9 beta 1), and induced migration of CHO cells in an alpha v beta 3-dependent manner. The binding of the uPA kringle to alpha v beta 3 and uPA kringle-induced alpha v beta 3-dependent cell migration were blocked by homologous plasminogen kringles 1-3 or 1-4 (angiostatin), a known integrin antagonist. We studied whether the binding of uPA to integrin alpha v beta 3 through the kringle domain plays a role in plasminogen activation. On CHO cell depleted of uPAR, uPA enhanced plasminogen activation in a kringle and alpha v beta 3-dependent manner. Endothelial cells bound to and migrated on uPA and uPA kringle in an alpha v beta 3-dependent manner. These results suggest that uPA binding to integrins through the kringle domain plays an important role in both plasminogen activation and uPA-induced intracellular signaling. The uPA kringle-integrin interaction may represent a novel therapeutic target for cancer, inflammation, and vascular remodeling. PMID:16525582

  6. NMDA receptor activity in neuropsychiatric disorders

    Directory of Open Access Journals (Sweden)

    Shaheen E Lakhan

    2013-06-01

    Full Text Available N-Methyl-D-aspartate (NMDA receptors play a variety of physiologic roles and their proper signaling is essential for cellular homeostasis. Any disruption in this pathway, leading to either enhanced or decreased activity, may result in the manifestation of neuropsychiatric pathologies such as schizophrenia, mood disorders, substance induced psychosis, Huntington's disease, Alzheimer's disease, and neuropsychiatric systemic lupus erythematosus. Here, we explore the notion that the overlap in activity of at least one biochemical pathway, the NMDA receptor pathway, may be the link to understanding the overlap in psychotic symptoms between diseases. This review intends to present a broad overview of those neuropsychiatric disorders for which alternations in NMDA receptor activity is prominent thus suggesting that continued direction of pharmaceutical intervention to this pathway may present a viable option for managing symptoms.

  7. Using Nuclear Receptor Activity to Stratify Hepatocarcinogens

    Science.gov (United States)

    Nuclear receptors (NR) are a superfamily of ligand-activated transcription factors that control a range of cellular processes. Persistent stimulation of some NR is a non-genotoxic mechanism of rodent liver cancer with unclear relevance to humans. Here we report on a systematic an...

  8. Serum uPAR as Biomarker in Breast Cancer Recurrence: A Mathematical Model.

    Science.gov (United States)

    Hao, Wenrui; Friedman, Avner

    2016-01-01

    There are currently over 2.5 million breast cancer survivors in the United States and, according to the American Cancer Society, 10 to 20 percent of these women will develop recurrent breast cancer. Early detection of recurrence can avoid unnecessary radical treatment. However, self-examination or mammography screening may not discover a recurring cancer if the number of surviving cancer cells is small, while biopsy is too invasive and cannot be frequently repeated. It is therefore important to identify non-invasive biomarkers that can detect early recurrence. The present paper develops a mathematical model of cancer recurrence. The model, based on a system of partial differential equations, focuses on tissue biomarkers that include the plasminogen system. Among them, only uPAR is known to have significant correlation to its concentration in serum and could therefore be a good candidate for serum biomarker. The model includes uPAR and other associated cytokines and cells. It is assumed that the residual cancer cells that survived primary cancer therapy are concentrated in the same location within a region with a very small diameter. Model simulations establish a quantitative relation between the diameter of the growing cancer and the total uPAR mass in the cancer. This relation is used to identify uPAR as a potential serum biomarker for breast cancer recurrence.

  9. Spongian diterpenoids inhibit androgen receptor activity

    OpenAIRE

    Yang, Yu Chi; Labros G Meimetis; Tien, Amy H; Mawji, Nasrin R.; Carr, Gavin; Wang, Jun; Andersen, Raymond J.; Sadar, Marianne D.

    2013-01-01

    Androgen receptor (AR) is a ligand-activated transcription factor and a validated drug target for all stages of prostate cancer. Antiandrogens compete with physiological ligands for AR ligand-binding domain (LBD). High-throughput screening of a marine natural product library for small molecules that inhibit AR transcriptional activity yielded the furanoditerpenoid spongia-13(16),-14-dien-19-oic acid, designated terpene 1 (T1). Characterization of T1 and the structurally related semi-synthetic...

  10. Phenobarbital and Insulin Reciprocate Activation of the Nuclear Receptor Constitutive Androstane Receptor through the Insulin Receptor.

    Science.gov (United States)

    Yasujima, Tomoya; Saito, Kosuke; Moore, Rick; Negishi, Masahiko

    2016-05-01

    Phenobarbital (PB) antagonized insulin to inactivate the insulin receptor and attenuated the insulin receptor downstream protein kinase B (AKT)-forkhead box protein O1 and extracellular signal-regulated kinase 1/2 signals in mouse primary hepatocytes and HepG2 cells. Hepatic AKT began dephosphorylation in an early stage of PB treatment, and blood glucose levels transiently increased in both wild-type and constitutive androstane receptor (CAR) knockout (KO) mice. On the other hand, blood glucose levels increased in wild-type mice, but not KO mice, in later stages of PB treatment. As a result, PB, acting as an insulin receptor antagonist, elicited CAR-independent increases and CAR-dependent decreases of blood glucose levels at these different stages of treatment, respectively. Reciprocally, insulin activation of the insulin receptor repressed CAR activation and induction of its target CYP2B6 gene in HepG2 cells. Thus, PB and insulin cross-talk through the insulin receptor to regulate glucose and drug metabolism reciprocally.

  11. Fatty acids activate a chimera of the clofibric acid-activated receptor and the glucocorticoid receptor.

    OpenAIRE

    Göttlicher, M; Widmark, E; Q. Li; Gustafsson, J.A.

    1992-01-01

    Peroxisome proliferators such as clofibric acid, nafenopin, and WY-14,643 have been shown to activate PPAR (peroxisome proliferator-activated receptor), a member of the steroid nuclear receptor superfamily. We have cloned the cDNA from the rat that is homologous to that from the mouse [Issemann, I. & Green, S. (1990) Nature (London) 347, 645-650], which encodes a 97% similar protein with a particularly well-conserved putative ligand-binding domain. To search for physiologically occurring acti...

  12. Human Receptor Activation by Aroclor 1260, a Polychlorinated Biphenyl Mixture

    OpenAIRE

    Wahlang, Banrida; Falkner, K. Cameron; Clair, Heather B.; Al-Eryani, Laila; Prough, Russell A.; States, J. Christopher; Coslo, Denise M.; Omiecinski, Curtis J.; Cave, Matthew C.

    2014-01-01

    Polychlorinated biphenyls (PCBs) are persistent environmental toxicants, present in 100% of U.S. adults and dose-dependently associated with obesity and non-alcoholic fatty liver disease (NAFLD). PCBs are predicted to interact with receptors previously implicated in xenobiotic/energy metabolism and NAFLD. These receptors include the aryl hydrocarbon receptor (AhR), pregnane xenobiotic receptor (PXR), constitutive androstane receptor (CAR), peroxisome proliferator-activated receptors (PPARs), ...

  13. RELAXIN ACTIVATES PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR GAMMA

    OpenAIRE

    Singh, Sudhir; Bennett, Robert G

    2009-01-01

    Relaxin is a polypeptide hormone that triggers multiple signaling pathways through its receptor RXFP1. Many of relaxin’s functions, including vascular and antifibrotic effects, are similar to those induced by activation of PPARγ. In this study, we tested the hypothesis that relaxin signaling through RXFP1 would activate PPARγ activity. In cells overexpressing RXFP1 (HEK-RXFP1), relaxin increased transcriptional activity through a PPAR response element (PPRE) in a concentration-dependent manne...

  14. CERAPP: Collaborative estrogen receptor activity prediction project

    DEFF Research Database (Denmark)

    Mansouri, Kamel; Abdelaziz, Ahmed; Rybacka, Aleksandra;

    2016-01-01

    Background: Humans are exposed to thousands of man-made chemicals in the environment. Some chemicals mimic natural endocrine hormones and, thus, have the potential to be endocrine disruptors. Most of these chemicals have never been tested for their ability to interact with the estrogen receptor (ER......). Risk assessors need tools to prioritize chemicals for evaluation in costly in vivo tests, for instance, within the U.S. EPA Endocrine Disruptor Screening Program. oBjectives: We describe a large-scale modeling project called CERAPP (Collaborative Estrogen Receptor Activity Prediction Project......) and demonstrate the efficacy of using predictive computational models trained on high-throughput screening data to evaluate thousands of chemicals for ER-related activity and prioritize them for further testing. Methods: CERAPP combined multiple models developed in collaboration with 17 groups in the United...

  15. Peroxisome proliferator-activated receptors for hypertension

    Institute of Scientific and Technical Information of China (English)

    Daisuke; Usuda; Tsugiyasu; Kanda

    2014-01-01

    Peroxisome proliferator-activated receptors(PPARs) are ligand-activated transcription factors belonging to the nuclear receptor superfamily, which is composed of four members encoded by distinct genes(α, β, γ, and δ). The genes undergo transactivation or transrepression under specific mechanisms that lead to the induction or repression of target gene expression. As is the case with other nuclear receptors, all four PPAR isoforms contain five or six structural regions in four functional domains; namely, A/B, C, D, and E/F. PPARs have many functions, particularly functions involving control of vascular tone, inflammation, and energy homeostasis, and are, therefore, important targets for hypertension, obesity, obesity-induced inflammation, and metabolic syndrome in general. Hence, PPARs also represent drug targets, and PPARα and PPARγ agonists are used clinically in the treatment of dyslipidemia and type 2 diabetes mellitus, respectively. Because of their pleiotropic effects, they have been identified as active in a number of diseases and are targets for the development of a broad range of therapies for a variety of diseases. It is likely that the range of PPARγ agonist therapeutic actions will result in novel approaches to lifestyle and other diseases. The combination of PPARs with reagents or with other cardiovascular drugs, such as diuretics and angiotensin Ⅱ receptor blockers, should be studied.This article provides a review of PPAR isoform characteristics, a discussion of progress in our understanding of the biological actions of PPARs, and a summary of PPAR agonist development for patient management. We also include a summary of the experimental and clinical evidence obtained from animal studies and clinical trials conducted to evaluate the usefulness and effectiveness of PPAR agonists in the treatment of lifestyle-related diseases.

  16. Activity and expression of urokinase-type plasminogen activator and matrix metalloproteinases in human colorectal cancer

    International Nuclear Information System (INIS)

    Matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9), and urokinase-type plasminogen activator (uPA) are involved in colorectal cancer invasion and metastasis. There is still debate whether the activity of MMP-2 and MMP-9 differs between tumors located in the colon and rectum. We designed this study to determine any differences in the expression of MMP-2, MMP-9 and uPA system between colon and rectal cancer tissues. Cancer tissue samples were obtained from colon carcinoma (n = 12) and rectal carcinomas (n = 10). MMP-2 and MMP-9 levels were examined using gelatin zymography and Western blotting; their endogenous inhibitors, tissue inhibitor of metalloproteinase-2 (TIMP-2) and tissue inhibitor of metalloproteinase-1 (TIMP-1), were assessed by Western blotting. uPA, uPAR and PAI-1 were examined using enzyme-linked immunosorbent assay (ELISA). The activity of uPA was assessed by casein-plasminogen zymography. In both colon and rectal tumors, MMP-2, MMP-9 and TIMP-1 protein levels were higher than in corresponding paired normal mucosa, while TIMP-2 level in tumors was significantly lower than in normal mucosa. The enzyme activities or protein levels of MMP-2, MMP-9 and their endogenous inhibitors did not reach a statistically significant difference between colon and rectal cancer compared with their normal mucosa. In rectal tumors, there was an increased activity of uPA compared with the activity in colon tumors (P = 0.0266), however urokinase-type plasminogen activator receptor (uPAR) and plasminogen activator inhibitor-1 (PAI-1) showed no significant difference between colon and rectal cancer tissues. These findings suggest that uPA may be expressed differentially in colon and rectal cancers, however, the activities or protein levels of MMP-2, MMP-9, TIMP-1, TIMP-2, PAI-1 and uPAR are not affected by tumor location in the colon or the rectum

  17. Relaxin Family Peptide Receptor 1 (RXFP1) Activation Stimulates the Peroxisome Proliferator-Activated Receptor Gamma

    OpenAIRE

    Singh, Sudhir; Bennett, Robert G

    2009-01-01

    Relaxin (Rlx) has antifibrotic effects in a number of tissues. Many of these effects are similar to those induced by the activators of peroxisome proliferator-activated receptor γ (PPARγ), raising the possibility that a mechanism for Rlx’s antifibrotic effects may involve activation of the PPARγ pathway. This study investigates the effect of Rlx on PPARs and their mechanism of upregulation. It shows that Rlx stimulates ligand-independent PPAR activation in a dose-dependent manner. The combine...

  18. Intact and cleaved forms of the urokinase receptor enhance discrimination of cancer from non-malignant conditions in patients presenting with symptoms related to colorectal cancer

    DEFF Research Database (Denmark)

    Lomholt, A F; Høyer-Hansen, G; Nielsen, H J;

    2009-01-01

    plasminogen activator receptor (suPAR) was proposed as a marker in CRC patients. This study was undertaken to evaluate the individual molecular forms of suPAR as discriminators in a group of patients undergoing endoscopical examination following symptoms related to colorectal cancer. METHODS: In a case......-control study comprising 308 patients undergoing endoscopical examination following CRC-related symptoms, 77 CRC patients with adenocarcinoma were age and gender matched to: 77 patients with adenomas; 77 with other non-malignant findings, and 77 with no findings. The different uPAR forms were measured in...

  19. Monoclonal antibodies to the human insulin receptor that activate glucose transport but not insulin receptor kinase activity

    Energy Technology Data Exchange (ETDEWEB)

    Forsayeth, J.R.; Caro, J.F.; Sinha, M.K.; Maddux, B.A.; Goldfine, I.D.

    1987-05-01

    Three mouse monoclonal antibodies were produced that reacted with the ..cap alpha.. subunit of the human insulin receptor. All three both immunoprecipitated /sup 125/I-labeled insulin receptors from IM-9 lymphocytes and competitively inhibited /sup 125/I-labeled insulin binding to its receptor. Unlike insulin, the antibodies failed to stimulate receptor autophosphorylation in both intact IM-9 lymphocytes and purified human placental insulin receptors. Moreover, unlike insulin, the antibodies failed to stimulate receptor-mediated phosphorylation of exogenous substrates. However, like insulin, two of the three antibodies stimulated glucose transport in isolated human adipocytes. One antibody, on a molar basis, was as potent as insulin. These studies indicate, therefore, that monoclonal antibodies to the insulin receptor can mimic a major function of insulin without activating receptor kinase activity. They also raise the possibility that certain actions of insulin such as stimulation of glucose transport may not require the activation of receptor kinase activity.

  20. Monoclonal antibodies to the human insulin receptor that activate glucose transport but not insulin receptor kinase activity

    International Nuclear Information System (INIS)

    Three mouse monoclonal antibodies were produced that reacted with the α subunit of the human insulin receptor. All three both immunoprecipitated 125I-labeled insulin receptors from IM-9 lymphocytes and competitively inhibited 125I-labeled insulin binding to its receptor. Unlike insulin, the antibodies failed to stimulate receptor autophosphorylation in both intact IM-9 lymphocytes and purified human placental insulin receptors. Moreover, unlike insulin, the antibodies failed to stimulate receptor-mediated phosphorylation of exogenous substrates. However, like insulin, two of the three antibodies stimulated glucose transport in isolated human adipocytes. One antibody, on a molar basis, was as potent as insulin. These studies indicate, therefore, that monoclonal antibodies to the insulin receptor can mimic a major function of insulin without activating receptor kinase activity. They also raise the possibility that certain actions of insulin such as stimulation of glucose transport may not require the activation of receptor kinase activity

  1. Protease activated receptors (PARS) mediation in gyroxin biological activity

    International Nuclear Information System (INIS)

    Gyroxin is a serine protease enzyme from the South American rattlesnake (Crotalus durissus terrificus) venom; it is only partially characterized and has multiple activities. Gyroxin induces blood coagulation, blood pressure decrease and a neurotoxic behavior named barrel rotation. The mechanisms involved in this neurotoxic activity are not known. Whereas gyroxin is a member of enzymes with high potential to become a new drug with clinical applications such as thrombin, batroxobin, ancrod, tripsyn and kalicrein, it is important to find out how gyroxin works. The analysis on agarose gel electrophoresis and circular dichroism confirmed the molecules' integrity and purity. The gyroxin intravenous administration in mice proved its neurotoxicity (barrel rotation). In vivo studies employing intravital microscopy proved that gyroxin induces vasodilation with the participation of protease activated receptors (PARs), nitric oxide and Na+K+ATPase. The leukocytes' adherence and rolling counting indicated that gyroxin has no pro inflammatory activity. Gyroxin induced platelet aggregation, which was blocked by inhibitors of PAR1 and PAR4 receptors (SCH 79797 and tcY-NH2, respectively). Finally, it was proved that the gyroxin temporarily alter the permeability of the blood brain barrier (BBB). Our study has shown that both the protease-activated receptors and nitric oxide are mediators involved in the biological activities of gyroxin. (author)

  2. Model for growth hormone receptor activation based on subunit rotation within a receptor dimer

    Energy Technology Data Exchange (ETDEWEB)

    Brown, Richard J.; Adams, Julian J.; Pelekanos, Rebecca A.; Wan, Yu; McKinstry, William J.; Palethorpe, Kathryn; Seeber, Ruth M.; Monks, Thea A.; Eidne, Karin A.; Parker, Michael W.; Waters, Michael J. (UWA); (St. Vincent); (Queensland)

    2010-07-13

    Growth hormone is believed to activate the growth hormone receptor (GHR) by dimerizing two identical receptor subunits, leading to activation of JAK2 kinase associated with the cytoplasmic domain. However, we have reported previously that dimerization alone is insufficient to activate full-length GHR. By comparing the crystal structure of the liganded and unliganded human GHR extracellular domain, we show here that there is no substantial change in its conformation on ligand binding. However, the receptor can be activated by rotation without ligand by inserting a defined number of alanine residues within the transmembrane domain. Fluorescence resonance energy transfer (FRET), bioluminescence resonance energy transfer (BRET) and coimmunoprecipitation studies suggest that receptor subunits undergo specific transmembrane interactions independent of hormone binding. We propose an activation mechanism involving a relative rotation of subunits within a dimeric receptor as a result of asymmetric placement of the receptor-binding sites on the ligand.

  3. Quantitation of the receptor for urokinase plasminogen activator by enzyme-linked immunosorbent assay

    DEFF Research Database (Denmark)

    Rønne, E; Behrendt, N; Ploug, M;

    1994-01-01

    variant of uPAR, suPAR, has been constructed by recombinant technique and the protein content of a purified suPAR standard preparation was determined by amino acid composition analysis. The sensitivity of the assay (0.6 ng uPAR/ml) is strong enough to measure uPAR in extracts of cultured cells and cancer...... tissue. Recent studies have shown that a high uPA level in tumor extracts is in some cancers associated with poor prognosis. The present assay will now allow similar prognostic studies of uPAR levels....

  4. Recruitment of activation receptors at inhibitory NK cell immune synapses.

    Directory of Open Access Journals (Sweden)

    Nicolas Schleinitz

    Full Text Available Natural killer (NK cell activation receptors accumulate by an actin-dependent process at cytotoxic immune synapses where they provide synergistic signals that trigger NK cell effector functions. In contrast, NK cell inhibitory receptors, including members of the MHC class I-specific killer cell Ig-like receptor (KIR family, accumulate at inhibitory immune synapses, block actin dynamics, and prevent actin-dependent phosphorylation of activation receptors. Therefore, one would predict inhibition of actin-dependent accumulation of activation receptors when inhibitory receptors are engaged. By confocal imaging of primary human NK cells in contact with target cells expressing physiological ligands of NK cell receptors, we show here that this prediction is incorrect. Target cells included a human cell line and transfected Drosophila insect cells that expressed ligands of NK cell activation receptors in combination with an MHC class I ligand of inhibitory KIR. The two NK cell activation receptors CD2 and 2B4 accumulated and co-localized with KIR at inhibitory immune synapses. In fact, KIR promoted CD2 and 2B4 clustering, as CD2 and 2B4 accumulated more efficiently at inhibitory synapses. In contrast, accumulation of KIR and of activation receptors at inhibitory synapses correlated with reduced density of the integrin LFA-1. These results imply that inhibitory KIR does not prevent CD2 and 2B4 signaling by blocking their accumulation at NK cell immune synapses, but by blocking their ability to signal within inhibitory synapses.

  5. BioKnife, a uPA Activity-dependent Oncolytic Sendai Virus, Eliminates Pleural Spread of Malignant Mesothelioma via Simultaneous Stimulation of uPA Expression

    OpenAIRE

    Morodomi, Yosuke; Yano, Tokujiro; Kinoh, Hiroaki; Harada, Yui; Saito, Satoru; Kyuragi, Ryoichi; Yoshida, Kumi; Onimaru, Mitsuho; Shoji, Fumihiro; Yoshida, Tsukihisa; Ito, Kensaku; Shikada, Yasunori; Maruyama, Riichiroh; Hasegawa, Mamoru; Maehara, Yoshihiko

    2012-01-01

    Malignant pleural mesothelioma (MPM) is highly intractable and readily spreads throughout the surface of the pleural cavity, and these cells have been shown to express urokinase-type plasminogen activator receptor (uPAR). We here examined the potential of our new and powerful recombinant Sendai virus (rSeV), which shows uPAR-specific cell-to-cell fusion activity (rSeV/dMFct14 (uPA2), named “BioKnife”), for tumor cell killing in two independent orthotopic xenograft models of human. Multicycle ...

  6. The insulin receptor C-terminus is involved in regulation of the receptor kinase activity.

    Science.gov (United States)

    Kaliman, P; Baron, V; Alengrin, F; Takata, Y; Webster, N J; Olefsky, J M; Van Obberghen, E

    1993-09-21

    During the insulin receptor activation process, ligand binding and autophosphorylation induce two distinct conformational changes in the C-terminal domain of the receptor beta-subunit. To analyze the role of this domain and the involvement of the C-terminal autophosphorylation sites (Tyr1316 and Tyr1322) in receptor activation, we used (i) antipeptide antibodies against three different C-terminal sequences (1270-1281, 1294-1317, and 1309-1326) and (ii) an insulin receptor mutant (Y/F2) where Tyr1316 and Tyr1322 have been replaced by Phe. We show that the autophosphorylation-induced C-terminal conformational change is preserved in the Y/F2 receptor, indicating that this change is not induced by phosphorylation of the C-terminal sites but most likely by phosphorylation of the major sites in the kinase domain (Tyr1146, Tyr1150, and Tyr1151). Binding of antipeptide antibodies to the C-terminal domain modulated (activated or inhibited) both mutant and wild-type receptor-mediated phosphorylation of poly(Glu/Tyr). In contrast to the wild-type receptor, Y/F2 exhibited the same C-terminal configuration before and after insulin binding, evidencing that mutation of Tyr1316 and Tyr1322 introduced conformational changes in the C-terminus. Finally, the mutant receptor was 2-fold more active than the wild-type receptor for poly(Glu/Tyr) phosphorylation. In conclusion, the whole C-terminal region of the insulin receptor beta-subunit is likely to exert a regulatory influence on the receptor kinase activity. Perturbations of the C-terminal region, such as binding of antipeptides or mutation of Tyr1316 and Tyr1322, provoke alterations at the receptor kinase level, leading to activation or inhibition of the enzymic activity. PMID:7690586

  7. The WSXWS motif in cytokine receptors is a molecular switch involved in receptor activation

    DEFF Research Database (Denmark)

    Dagil, Robert; Knudsen, Maiken J.; Olsen, Johan Gotthardt;

    2012-01-01

    The prolactin receptor (PRLR) is activated by binding of prolactin in a 2:1 complex, but the activation mechanism is poorly understood. PRLR has a conserved WSXWS motif generic to cytokine class I receptors. We have determined the nuclear magnetic resonance solution structure of the membrane...

  8. Cell death sensitization of leukemia cells by opioid receptor activation

    Science.gov (United States)

    Friesen, Claudia; Roscher, Mareike; Hormann, Inis; Fichtner, Iduna; Alt, Andreas; Hilger, Ralf A.; Debatin, Klaus-Michael; Miltner, Erich

    2013-01-01

    Cyclic AMP (cAMP) regulates a number of cellular processes and modulates cell death induction. cAMP levels are altered upon stimulation of specific G-protein-coupled receptors inhibiting or activating adenylyl cyclases. Opioid receptor stimulation can activate inhibitory Gi-proteins which in turn block adenylyl cyclase activity reducing cAMP. Opioids such as D,L-methadone induce cell death in leukemia cells. However, the mechanism how opioids trigger apoptosis and activate caspases in leukemia cells is not understood. In this study, we demonstrate that downregulation of cAMP induced by opioid receptor activation using the opioid D,L-methadone kills and sensitizes leukemia cells for doxorubicin treatment. Enhancing cAMP levels by blocking opioid-receptor signaling strongly reduced D,L-methadone-induced apoptosis, caspase activation and doxorubicin-sensitivity. Induction of cell death in leukemia cells by activation of opioid receptors using the opioid D,L-methadone depends on critical levels of opioid receptor expression on the cell surface. Doxorubicin increased opioid receptor expression in leukemia cells. In addition, the opioid D,L-methadone increased doxorubicin uptake and decreased doxorubicin efflux in leukemia cells, suggesting that the opioid D,L-methadone as well as doxorubicin mutually increase their cytotoxic potential. Furthermore, we found that opioid receptor activation using D,L-methadone alone or in addition to doxorubicin inhibits tumor growth significantly in vivo. These results demonstrate that opioid receptor activation via triggering the downregulation of cAMP induces apoptosis, activates caspases and sensitizes leukemia cells for doxorubicin treatment. Hence, opioid receptor activation seems to be a promising strategy to improve anticancer therapies. PMID:23633472

  9. The growth hormone receptor: mechanism of activation and clinical implications.

    Science.gov (United States)

    Brooks, Andrew J; Waters, Michael J

    2010-09-01

    Growth hormone is widely used clinically to promote growth and anabolism and for other purposes. Its actions are mediated via the growth hormone receptor, both directly by tyrosine kinase activation and indirectly by induction of insulin-like growth factor 1 (IGF-1). Insensitivity to growth hormone (Laron syndrome) can result from mutations in the growth hormone receptor and can be treated with IGF-1. This treatment is, however, not fully effective owing to the loss of the direct actions of growth hormone and altered availability of exogenous IGF-1. Excessive activation of the growth hormone receptor by circulating growth hormone results in gigantism and acromegaly, whereas cell transformation and cancer can occur in response to autocrine activation of the receptor. Advances in understanding the mechanism of receptor activation have led to a model in which the growth hormone receptor exists as a constitutive dimer. Binding of the hormone realigns the subunits by rotation and closer apposition, resulting in juxtaposition of the catalytic domains of the associated tyrosine-protein kinase JAK2 below the cell membrane. This change results in activation of JAK2 by transphosphorylation, then phosphorylation of receptor tyrosines in the cytoplasmic domain, which enables binding of adaptor proteins, as well as direct phosphorylation of target proteins. This model is discussed in the light of salient information from closely related class 1 cytokine receptors, such as the erythropoietin, prolactin and thrombopoietin receptors. PMID:20664532

  10. Comparison of the activation kinetics of the M3 acetylcholine receptor and a constitutively active mutant receptor in living cells.

    Science.gov (United States)

    Hoffmann, Carsten; Nuber, Susanne; Zabel, Ulrike; Ziegler, Nicole; Winkler, Christiane; Hein, Peter; Berlot, Catherine H; Bünemann, Moritz; Lohse, Martin J

    2012-08-01

    Activation of G-protein-coupled receptors is the first step of the signaling cascade triggered by binding of an agonist. Here we compare the activation kinetics of the G(q)-coupled M(3) acetylcholine receptor (M(3)-AChR) with that of a constitutively active mutant receptor (M(3)-AChR-N514Y) using M(3)-AChR constructs that report receptor activation by changes in the fluorescence resonance energy transfer (FRET) signal. We observed a leftward shift in the concentration-dependent FRET response for acetylcholine and carbachol with M(3)-AChR-N514Y. Consistent with this result, at submaximal agonist concentrations, the activation kinetics of M(3)-AChR-N514Y were significantly faster, whereas at maximal agonist concentrations the kinetics of receptor activation were identical. Receptor deactivation was significantly faster with carbachol than with acetylcholine and was significantly delayed by the N514Y mutation. Receptor-G-protein interaction was measured by FRET between M(3)-AChR-yellow fluorescent protein (YFP) and cyan fluorescent protein (CFP)-Gγ(2). Agonist-induced receptor-G-protein coupling was of a time scale similar to that of receptor activation. As observed for receptor deactivation, receptor-G-protein dissociation was slower for acetylcholine than that for carbachol. Acetylcholine-stimulated increases in receptor-G-protein coupling of M(3)-AChR-N514Y reached only 12% of that of M(3)-AChR and thus cannot be kinetically analyzed. G-protein activation was measured using YFP-tagged Gα(q) and CFP-tagged Gγ(2). Activation of G(q) was significantly slower than receptor activation and indistinguishable for the two agonists. However, G(q) deactivation was significantly prolonged for acetylcholine compared with that for carbachol. Consistent with decreased agonist-stimulated coupling to G(q), agonist-stimulated G(q) activation by M(3)-AChR-N514Y was not detected. Taken together, these results indicate that the N514Y mutation produces constitutive activation of M(3

  11. Family C 7TM receptor dimerization and activation

    DEFF Research Database (Denmark)

    Bonde, Marie Mi; Sheikh, Søren P; Hansen, Jakob Lerche

    2006-01-01

    The family C seven transmembrane (7TM) receptors constitutes a small and especially well characterized subfamily of the large 7TM receptor superfamily. Approximately 50% of current prescription drugs target 7TM receptors, this biologically important family represents the largest class of drug......-targets today. It is well established that family C 7TM receptors form homo- or hetero-dimers on the cell surface of living cells. The large extra-cellular domains (ECD) have been crystallized as a dimer in the presence and absence of agonist. Upon agonist binding, the dimeric ECD undergoes large conformational...... to be fully defined. This review presents the biochemical support for family C 7TM receptor dimerization and discusses its importance for receptor biosynthesis, surface expression, ligand binding and activation, since lessons learnt here may well be applicable to the whole superfamily of 7TM receptors....

  12. The Orphan Nuclear Receptor TR4 Is a Vitamin A-activated Nuclear Receptor

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, X. Edward; Suino-Powell, Kelly M.; Xu, Yong; Chan, Cee-Wah; Tanabe, Osamu; Kruse, Schoen W.; Reynolds, Ross; Engel, James Douglas; Xu, H. Eric (Michigan-Med); (Van Andel)

    2015-11-30

    Testicular receptors 2 and 4 (TR2/4) constitute a subgroup of orphan nuclear receptors that play important roles in spermatogenesis, lipid and lipoprotein regulation, and the development of the central nervous system. Currently, little is known about the structural features and the ligand regulation of these receptors. Here we report the crystal structure of the ligand-free TR4 ligand binding domain, which reveals an autorepressed conformation. The ligand binding pocket of TR4 is filled by the C-terminal half of helix 10, and the cofactor binding site is occupied by the AF-2 helix, thus preventing ligand-independent activation of the receptor. However, TR4 exhibits constitutive transcriptional activity on multiple promoters, which can be further potentiated by nuclear receptor coactivators. Mutations designed to disrupt cofactor binding, dimerization, or ligand binding substantially reduce the transcriptional activity of this receptor. Importantly, both retinol and retinoic acid are able to promote TR4 to recruit coactivators and to activate a TR4-regulated reporter. These findings demonstrate that TR4 is a ligand-regulated nuclear receptor and suggest that retinoids might have a much wider regulatory role via activation of orphan receptors such as TR4.

  13. The insulin receptor activation process involves localized conformational changes.

    Science.gov (United States)

    Baron, V; Kaliman, P; Gautier, N; Van Obberghen, E

    1992-11-15

    The molecular process by which insulin binding to the receptor alpha-subunit induces activation of the receptor beta-subunit with ensuing substrate phosphorylation remains unclear. In this study, we aimed at approaching this molecular mechanism of signal transduction and at delineating the cytoplasmic domains implied in this process. To do this, we used antipeptide antibodies to the following sequences of the receptor beta-subunit: (i) positions 962-972 in the juxtamembrane domain, (ii) positions 1247-1261 at the end of the kinase domain, and (iii) positions 1294-1317 and (iv) positions 1309-1326, both in the receptor C terminus. We have previously shown that insulin binding to its receptor induces a conformational change in the beta-subunit C terminus. Here, we demonstrate that receptor autophosphorylation induces an additional conformational change. This process appears to be distinct from the one produced by ligand binding and can be detected in at least three different beta-subunit regions: the juxtamembrane domain, the kinase domain, and the C terminus. Hence, the cytoplasmic part of the receptor beta-subunit appears to undergo an extended conformational change upon autophosphorylation. By contrast, the insulin-induced change does not affect the juxtamembrane domain 962-972 nor the kinase domain 1247-1261 and may be limited to the receptor C terminus. Further, we show that the hormone-dependent conformational change is maintained in a kinase-deficient receptor due to a mutation at lysine 1018. Therefore, during receptor activation, the ligand-induced change could precede ATP binding and receptor autophosphorylation. We propose that insulin binding leads to a transient receptor form that may allow ATP binding and, subsequently, autophosphorylation. The second conformational change could unmask substrate-binding sites and stabilize the receptor in an active conformation. PMID:1331080

  14. RNAi-Mediated Simultaneous Downregulation of uPAR and Cathepsin B Induces Caspase 8-Mediated Apoptosis in SNB19 Human Glioma Cells

    OpenAIRE

    Christopher S Gondi; Kandhukuri, Neelima; Kondraganti, Shakuntala; Gujrati, Meena; Olivero, William C.; Dinh, Dzung H.; Rao, Jasti S.

    2006-01-01

    The invasive character of gliomas depends on proteolytic cleavage of the surrounding extracellular matrix. Cathepsin B and uPAR together are known to be overexpressed in gliomas, and as such, are attractive targets for gene therapy. In the present study, we used plasmid constructs to induce the RNAi-mediated downregulation of uPAR and Cathepsin B in SNB19 human glioma cells. We observed that the simultaneous downregulation of uPAR and Cathepsin B induces the up-regulation of pro-apoptotic gen...

  15. Activating Receptor Signals Drive Receptor Diversity in Developing Natural Killer Cells.

    Science.gov (United States)

    Freund, Jacquelyn; May, Rebecca M; Yang, Enjun; Li, Hongchuan; McCullen, Matthew; Zhang, Bin; Lenvik, Todd; Cichocki, Frank; Anderson, Stephen K; Kambayashi, Taku

    2016-08-01

    It has recently been appreciated that NK cells exhibit many features reminiscent of adaptive immune cells. Considerable heterogeneity exists with respect to the ligand specificity of individual NK cells and as such, a subset of NK cells can respond, expand, and differentiate into memory-like cells in a ligand-specific manner. MHC I-binding inhibitory receptors, including those belonging to the Ly49 and KIR families, are expressed in a variegated manner, which creates ligand-specific diversity within the NK cell pool. However, how NK cells determine which inhibitory receptors to express on their cell surface during a narrow window of development is largely unknown. In this manuscript, we demonstrate that signals from activating receptors are critical for induction of Ly49 and KIR receptors during NK cell development; activating receptor-derived signals increased the probability of the Ly49 bidirectional Pro1 promoter to transcribe in the forward versus the reverse direction, leading to stable expression of Ly49 receptors in mature NK cells. Our data support a model where the balance of activating and inhibitory receptor signaling in NK cells selects for the induction of appropriate inhibitory receptors during development, which NK cells use to create a diverse pool of ligand-specific NK cells.

  16. Interaction of chemokines with their receptors--from initial chemokine binding to receptor activating steps

    DEFF Research Database (Denmark)

    Thiele, Stefanie; Rosenkilde, Mette Marie

    2014-01-01

    interactions possibly occur, resulting in a multi-step process, as recently proposed for other 7TM receptors. Overall, the N-terminus of chemokine receptors is pivotal for binding of all chemokines. During receptor activation, differences between the two major chemokine subgroups occur, as CC-chemokines mainly......The human chemokine system comprises 19 seven-transmembrane helix (7TM) receptors and 45 endogenous chemokines that often interact with each other in a promiscuous manner. Due to the chemokine system's primary function in leukocyte migration, it has a central role in immune homeostasis...... and surveillance. Chemokines are a group of 8-12 kDa large peptides with a secondary structure consisting of a flexible N-terminus and a core-domain usually stabilized by two conserved disulfide bridges. They mainly interact with the extracellular domains of their cognate 7TM receptors. Affinityand activity...

  17. Human receptor activation by aroclor 1260, a polychlorinated biphenyl mixture.

    Science.gov (United States)

    Wahlang, Banrida; Falkner, K Cameron; Clair, Heather B; Al-Eryani, Laila; Prough, Russell A; States, J Christopher; Coslo, Denise M; Omiecinski, Curtis J; Cave, Matthew C

    2014-08-01

    Polychlorinated biphenyls (PCBs) are persistent environmental toxicants, present in 100% of U.S. adults and dose-dependently associated with obesity and non-alcoholic fatty liver disease (NAFLD). PCBs are predicted to interact with receptors previously implicated in xenobiotic/energy metabolism and NAFLD. These receptors include the aryl hydrocarbon receptor (AhR), pregnane xenobiotic receptor (PXR), constitutive androstane receptor (CAR), peroxisome proliferator-activated receptors (PPARs), liver-X-receptor (LXRα), and farnesoid-X-receptor (FXR). This study evaluates Aroclor 1260, a PCB mixture with congener composition mimicking that of human adipose tissue, and selected congeners, as potential ligands for these receptors utilizing human hepatoma-derived (HepG2) and primate-derived (COS-1) cell lines, and primary human hepatocytes. Aroclor 1260 (20 μg/ml) activated AhR, and PCB 126, a minor component, was a potent inducer. Aroclor 1260 activated PXR in a simple concentration-dependent manner at concentrations ≥10 μg/ml. Among the congeners tested, PCBs 138, 149, 151, 174, 183, 187, and 196 activated PXR. Aroclor 1260 activated CAR2 and CAR3 variants at lower concentrations and antagonize CAR2 activation by the CAR agonist, CITCO, at higher concentrations (≥20 μg/ml). Additionally, Aroclor 1260 induced CYP2B6 in primary hepatocytes. At subtoxic doses, Aroclor 1260 did not activate LXR or FXR and had no effect on LXR- or FXR-dependent induction by the agonists T0901317 or GW4064, respectively. Aroclor 1260 (20 μg/ml) suppressed PPARα activation by the agonist nafenopin, although none of the congeners tested demonstrated significant inhibition. The results suggest that Aroclor 1260 is a human AhR, PXR and CAR3 agonist, a mixed agonist/antagonist for CAR2, and an antagonist for human PPARα. PMID:24812009

  18. Influence of phasic and tonic dopamine release on receptor activation

    DEFF Research Database (Denmark)

    Dreyer, Jakob Kristoffer Kisbye; Herrik, Kjartan F; Berg, Rune W;

    2010-01-01

    Tonic and phasic dopamine release is implicated in learning, motivation, and motor functions. However, the relationship between spike patterns in dopaminergic neurons, the extracellular concentration of dopamine, and activation of dopamine receptors remains unresolved. In the present study, we...... of the boundaries of dopaminergic volume transmission. Bursts primarily increase occupancy of D(1) receptors, whereas pauses translate into low occupancy of D(1) and D(2) receptors. Phasic firing patterns, composed of bursts and pauses, reduce the average D(2) receptor occupancy and increase average D(1) receptor...... occupancy compared with equivalent tonic firing. Receptor occupancy is crucially dependent on synchrony and the balance between tonic and phasic firing modes. Our results provide quantitative insight in the dynamics of volume transmission and complement experimental data obtained with electrophysiology...

  19. Tonic activation of presynaptic GABAB receptors on rat pallidosubthalamic terminals

    Institute of Scientific and Technical Information of China (English)

    Lei CHEN; Wing-ho YUNG

    2005-01-01

    Aim: The subthalamic nucleus plays a critical role in the regulation of movement,and abnormal activity of its neurons is associated with some basal ganglia motor symptoms. We examined the presence of functional presynaptic GABAB receptors on pallidosubthalamic terminals and tested whether they were tonically active in the in vitro subthalamic slices. Methods: Whole-cell patch-clamp recordings were applied to acutely prepared rat subthalamic nucleus slices. The effects of specific GABAB agonist and antagonist on action potential-independent inhibitory postsynapfic currents (IPSCs), as well as holding current, were examined.Results: Superfusion of baclofen, a GABAB receptor agonist, significantly reduced the frequency of GABAA receptor-mediated miniature IPSCs (mIPSCs), in a Cd2+-sensitive manner, with no effect on the amplitude, indicating presynaptic inhibition on GABA release. In addition, baclofen induced a weak outward current only in a minority of subthalamic neurons. Both the pre- and post-synaptic effects of baclofen were prevented by the specific GABAB receptor antagonist,CGP55845. Furthermore, CGP55845 alone increased the frequency of mIPSCs,but had no effect on the holding current. Conclusion: These findings suggest the functional dominance of presynaptic GABAB receptors on the pallidosubthalamic terminals over the postsynaptic GABAB receptors on subthalamic neurons.Furthermore, the presynaptic, but not the postsynaptic, GABAB receptors are tonically active, suggesting that the presynaptic GABAB receptors in the subthalamic nucleus are potential therapeutic target for the treatment of Parkinson disease.

  20. 5-HT7 receptor activation promotes an increase in TrkB receptor expression and phosphorylation

    Directory of Open Access Journals (Sweden)

    Anshula eSamarajeewa

    2014-11-01

    Full Text Available The serotonin (5-HT type 7 receptor is expressed throughout the CNS including cortical neurons. We have previously demonstrated that the application of 5-HT7 receptor agonists to primary hippocampal neurons and SH-SY5Y cells increases platelet-derived growth factor (PDGF receptor expression and promotes neuroprotection against N-methyl-D-aspartate-(NMDA-induced toxicity. The tropomyosin-related kinase B (TrkB receptor is one of the receptors for brain-derived neurotrophic factor (BDNF and is associated with neurodevelopmental and neuroprotective effects. Application of LP 12 to primary cerebral cortical cultures, SH-SY5Y cells, as well as the retinal ganglion cell line, RGC-5, increased both the expression of full length TrkB as well as its basal phosphorylation state at tyrosine 816. The increase in TrkB expression and phosphorylation was observed as early as 30 min after 5-HT7 receptor activation. In addition to full-length TrkB, kinase domain-deficient forms may be expressed and act as dominant-negative proteins towards the full length receptor. We have identified distinct patterns of TrkB isoform expression across our cell lines and cortical cultures. Although TrkB receptor expression is regulated by cyclic AMP and Gαs-coupled GPCRs in several systems, we demonstrate that, depending on the model system, pathways downstream of both Gαs and Gα12 are involved in the regulation of TrkB expression by 5-HT7 receptors. Given the number of psychiatric and degenerative diseases associated with TrkB/BDNF deficiency and the current interest in developing 5-HT7 receptor ligands as pharmaceuticals, identifying signaling relationships between these two receptors will aid in our understanding of the potential therapeutic effects of 5-HT7 receptor ligands.

  1. 5-HT7 receptor activation promotes an increase in TrkB receptor expression and phosphorylation

    Science.gov (United States)

    Samarajeewa, Anshula; Goldemann, Lolita; Vasefi, Maryam S.; Ahmed, Nawaz; Gondora, Nyasha; Khanderia, Chandni; Mielke, John G.; Beazely, Michael A.

    2014-01-01

    The serotonin (5-HT) type 7 receptor is expressed throughout the CNS including the cortex and hippocampus. We have previously demonstrated that the application of 5-HT7 receptor agonists to primary hippocampal neurons and SH-SY5Y cells increases platelet-derived growth factor (PDGF) receptor expression and promotes neuroprotection against N-methyl-D-aspartate-(NMDA)-induced toxicity. The tropomyosin-related kinase B (TrkB) receptor is one of the receptors for brain-derived neurotrophic factor (BDNF) and is associated with neurodevelopmental and neuroprotective effects. Application of LP 12 to primary cerebral cortical cultures, SH-SY5Y cells, as well as the retinal ganglion cell line, RGC-5, increased both the expression of full length TrkB as well as its basal phosphorylation state at tyrosine 816. The increase in TrkB expression and phosphorylation was observed as early as 30 min after 5-HT7 receptor activation. In addition to full-length TrkB, kinase domain-deficient forms may be expressed and act as dominant-negative proteins toward the full length receptor. We have identified distinct patterns of TrkB isoform expression across our cell lines and cortical cultures. Although TrkB receptor expression is regulated by cyclic AMP and Gαs-coupled GPCRs in several systems, we demonstrate that, depending on the model system, pathways downstream of both Gαs and Gα12 are involved in the regulation of TrkB expression by 5-HT7 receptors. Given the number of psychiatric and degenerative diseases associated with TrkB/BDNF deficiency and the current interest in developing 5-HT7 receptor ligands as pharmaceuticals, identifying signaling relationships between these two receptors will aid in our understanding of the potential therapeutic effects of 5-HT7 receptor ligands. PMID:25426041

  2. Helix 11 Dynamics is Critical for Constitutive Androstane Receptor Activity

    OpenAIRE

    Wright, Edward; Busby, Scott A.; Wisecarver, Sarah; Vincent, Jeremy; Griffin, Patrick R.; Fernandez, Elias J.

    2011-01-01

    The constitutive androstane receptor (CAR) transactivation can occur in the absence of exogenous ligand and this activity is enhanced by agonists TCPOBOP and meclizine. We use biophysical and cell-based assays to show that increased activity of CAR(TCPOBOP) relative to CAR(meclizine) corresponds to a higher affinity of CAR(TCPOBOP) for the steroid receptor coactivator-1. Additionally, steady-state fluorescence spectra suggest conformational differences between CAR(TCPOBOP):RXR and CAR(meclizi...

  3. Differential trafficking of AMPA receptors following activation of NMDA receptors and mGluRs

    Directory of Open Access Journals (Sweden)

    Sanderson Thomas M

    2011-07-01

    Full Text Available Abstract The removal of AMPA receptors from synapses is a major component of long-term depression (LTD. How this occurs, however, is still only partially understood. To investigate the trafficking of AMPA receptors in real-time we previously tagged the GluA2 subunit of AMPA receptors with ecliptic pHluorin and studied the effects of NMDA receptor activation. In the present study we have compared the effect of NMDA receptor and group I mGluR activation, using GluA2 tagged with super ecliptic pHluorin (SEP-GluA2 expressed in cultured hippocampal neurons. Surprisingly, agonists of the two receptors, which are both able to induce chemical forms of LTD, had clearly distinct effects on AMPA receptor trafficking. In agreement with our previous work we found that transient NMDA receptor activation results in an initial decrease in surface GluA2 from extrasynaptic sites followed by a delayed reduction in GluA2 from puncta (putative synapses. In contrast, transient activation of group I mGluRs, using DHPG, led to a pronounced but more delayed decrease in GluA2 from the dendritic shafts. Surprisingly, there was no average change in the fluorescence of the puncta. Examination of fluorescence at individual puncta, however, indicated that alterations did take place, with some puncta showing an increase and others a decrease in fluorescence. The effects of DHPG were, like DHPG-induced LTD, prevented by treatment with a protein tyrosine phosphatase (PTP inhibitor. The electrophysiological correlate of the effects of DHPG in the SEP-GluA2 infected cultures was a reduction in mEPSC frequency with no change in amplitude. The implications of these findings for the initial mechanisms of expression of both NMDA receptor- and mGluR-induced LTD are discussed.

  4. Retinoic Acid-mediated Nuclear Receptor Activation and Hepatocyte Proliferation

    Science.gov (United States)

    Bushue, Nathan; Wan, Yu-Jui Yvonne

    2016-01-01

    Due to their well-known differentiation and apoptosis-inducing abilities, retinoic acid (RA) and its analogs have strong anti-cancer efficacy in human cancers. However, in vivo RA is a liver mitogen. While speculation has persisted that RA-mediated signaling is likely involved in hepatocyte proliferation during liver regeneration, direct evidence is still required. Findings in support of this proposition include observations that a release of retinyl palmitate (the precursor of RA) occurs in liver stellate cells following liver injury. Nevertheless, the biological action of this released vitamin A is virtually unknown. More likely is that the released vitamin A is converted to RA, the biological form, and then bound to a specific receptor (retinoid x receptor; RXRα), which is most abundantly expressed in the liver. Considering the mitogenic effects of RA, the RA-activated RXRα would likely then influence hepatocyte proliferation and liver tissue repair. At present, the mechanism by which RA stimulates hepatocyte proliferation is largely unknown. This review summarizes the activation of nuclear receptors (peroxisome proliferator activated receptor-α, pregnane x receptor, constitutive androstane receptor, and farnesoid x receptor) in an RXRα dependent manner to induce hepatocyte proliferation, providing a link between RA and its proliferative role.

  5. Environmental polycyclic aromatic hydrocarbons affect androgen receptor activation in vitro

    DEFF Research Database (Denmark)

    Vinggaard, Anne Marie; Hnida, Christina; Larsen, John Christian

    2000-01-01

    of certain PAHs to activate the Ah receptor was assessed in H4IIE liver cancer cells, stably transfected with a luciferase reporter gene system. The positive control 2, 3,7, 8-tetrachlorodibenzodioxin (TCDD) caused a 13-14-fold induction of luciferase activity reaching maximum activity at 0.1 nM. DB[a,h]A, B...

  6. Cloning, constitutive activity and expression profiling of two receptors related to relaxin receptors in Drosophila melanogaster.

    Science.gov (United States)

    Van Hiel, Matthias B; Vandersmissen, Hans Peter; Proost, Paul; Vanden Broeck, Jozef

    2015-06-01

    Leucine-rich repeat containing G protein-coupled receptors (LGRs) comprise a cluster of transmembrane proteins, characterized by the presence of a large N-terminal extracellular domain. This receptor group can be classified into three subtypes. Belonging to the subtype C LGRs are the mammalian relaxin receptors LGR7 (RXFP1) and LGR8 (RXFP2), which mediate important reproductive and other processes. We identified two related receptors in the genome of the fruit fly and cloned their open reading frames into an expression vector. Interestingly, dLGR3 demonstrated constitutive activity at very low doses of transfected plasmid, whereas dLGR4 did not show any basal activity. Both receptors exhibited a similar expression pattern during development, with relatively high transcript levels during the first larval stage. In addition, both receptors displayed higher expression in male adult flies as compared to female flies. Analysis of the tissue distribution of both receptor transcripts revealed a high expression of dLGR3 in the female fat body, while the expression of dLGR4 peaked in the midgut of both the wandering and adult stage. PMID:25064813

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

    Directory of Open Access Journals (Sweden)

    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.

  8. Implications of compound heterozygous insulin receptor mutations in congenital muscle fibre type disproportion myopathy for the receptor kinase activation

    DEFF Research Database (Denmark)

    Klein, H H; Müller, R; Vestergaard, H;

    1999-01-01

    We studied insulin receptor kinase activation in two brothers with congenital muscle fibre type disproportion myopathy and compound heterozygous mutations of the insulin receptor gene, their parents, and their unaffected brother. In the father who has a heterozygote Arg1174-->Gln mutation, in situ......% of the receptors to become insulin-dependently activated. The mother carries a point mutation at the last base pair in exon 17 which, due to abnormal alternative splicing, could lead to normally transcribed receptor or truncated receptor lacking the kinase region. Kinase activation was normal in the mother...... activation of the receptor kinase in skeletal muscle was reduced about 70%. Selection of only those receptors that bound to anti-phosphotyrosine antibody showed that these receptors had normal kinase activity and that the reduction in overall kinase activity was due to the inability of about 70...

  9. Nuclear receptor corepressor-dependent repression of peroxisome-proliferator-activated receptor delta-mediated transactivation

    DEFF Research Database (Denmark)

    Krogsdam, Anne-M; Nielsen, Curt A F; Neve, Søren;

    2002-01-01

    The nuclear receptor corepressor (NCoR) was isolated as a peroxisome-proliferator-activated receptor (PPAR) delta interacting protein using the yeast two-hybrid system. NCoR interacted strongly with the ligand-binding domain of PPAR delta, whereas interactions with the ligand-binding domains...... delta-RXR alpha heterodimer bound to an acyl-CoA oxidase (ACO)-type peroxisome-proliferator response element recruited a glutathione S-transferase-NCoR fusion protein in a ligand-independent manner. Contrasting with most other nuclear receptors, PPAR delta was found to interact equally well...

  10. Transcriptional activation of nuclear estrogen receptor and progesterone receptor and its regulation.

    Science.gov (United States)

    Xin, Qi-Liang; Qiu, Jing-Tao; Cui, Sheng; Xia, Guo-Liang; Wang, Hai-Bin

    2016-08-25

    Estrogen receptor (ER) and progesterone receptor (PR) are two important members of steroid receptors family, an evolutionarily conserved family of transcription factors. Upon binding to their ligands, ER and PR enter cell nucleus to interact with specific DNA element in the context of chromatin to initiate the transcription of diverse target genes, which largely depends on the timely recruitment of a wide range of cofactors. Moreover, the interactions between steroid hormones and their respective receptors also trigger post-translational modifications on these receptors to fine-tune their transcriptional activities. Besides the well-known phosphorylation modifications on tyrosine and serine/threonine residues, recent studies have identified several other covalent modifications, such as ubiquitylation and sumoylation. These post-translational modifications of steroid receptors affect its stability, subcellular localization, and/or cofactor recruitment; eventually influence the duration and extent of transcriptional activation. This review is to focus on the recent research progress on the transcriptional activation of nuclear ER and PR as well as their physiological functions in early pregnancy, which may help us to better understand related female reproductive diseases. PMID:27546504

  11. Identification of COUP-TFII Orphan Nuclear Receptor as a Retinoic Acid-Activated Receptor

    Energy Technology Data Exchange (ETDEWEB)

    Kruse, Schoen W; Suino-Powell, Kelly; Zhou, X Edward; Kretschman, Jennifer E; Reynolds, Ross; Vonrhein, Clemens; Xu, Yong; Wang, Liliang; Tsai, Sophia Y; Tsai, Ming-Jer; Xu, H Eric [Baylor; (Van Andel); (Globel Phasing); (Grand Valley)

    2010-01-12

    The chicken ovalbumin upstream promoter-transcription factors (COUP-TFI and II) make up the most conserved subfamily of nuclear receptors that play key roles in angiogenesis, neuronal development, organogenesis, cell fate determination, and metabolic homeostasis. Although the biological functions of COUP-TFs have been studied extensively, little is known of their structural features or aspects of ligand regulation. Here we report the ligand-free 1.48 {angstrom} crystal structure of the human COUP-TFII ligand-binding domain. The structure reveals an autorepressed conformation of the receptor, where helix {alpha}10 is bent into the ligand-binding pocket and the activation function-2 helix is folded into the cofactor binding site, thus preventing the recruitment of coactivators. In contrast, in multiple cell lines, COUP-TFII exhibits constitutive transcriptional activity, which can be further potentiated by nuclear receptor coactivators. Mutations designed to disrupt cofactor binding, dimerization, and ligand binding, substantially reduce the COUP-TFII transcriptional activity. Importantly, retinoid acids are able to promote COUP-TFII to recruit coactivators and activate a COUP-TF reporter construct. Although the concentration needed is higher than the physiological levels of retinoic acids, these findings demonstrate that COUP-TFII is a ligand-regulated nuclear receptor, in which ligands activate the receptor by releasing it from the autorepressed conformation.

  12. Monitoring leptin activity using the chicken leptin receptor.

    Science.gov (United States)

    Hen, Gideon; Yosefi, Sera; Ronin, Ana; Einat, Paz; Rosenblum, Charles I; Denver, Robert J; Friedman-Einat, Miriam

    2008-05-01

    We report on the construction of a leptin bioassay based on the activation of chicken leptin receptor in cultured cells. A human embryonic kidney (HEK)-293 cell line, stably transfected with the full-length cDNA of chicken leptin receptor together with a STAT3-responsive reporter gene specifically responded to recombinant human and Xenopus leptins. The observed higher sensitivity of chicken leptin receptor to the former is in agreement with the degree of sequence similarity among these species (about 60 and 38% identical amino acids between humans and chickens, and between humans and Xenopus respectively). The specific activation of signal transduction through the chicken leptin receptor, shown here for the first time, suggests that the transition of Gln269 (implicated in the Gln-to-Pro Zucker fatty mutation in rats) to Glu in chickens does not impair its activity. Analysis of leptin-like activity in human serum samples of obese and lean subjects coincided well with leptin levels determined by RIA. Serum samples of pre- and post partum cows showed a tight correlation with the degree of adiposity. However, specific activation of the chicken leptin receptor in this assay was not observed with serum samples from broiler or layer chickens (representing fat and lean phenotypes respectively) or with those from turkey. Similar leptin receptor activation profiles were observed with cells transfected with human leptin receptor. Further work is needed to determine whether the lack of leptin-like activity in the chicken serum samples is due to a lack of leptin in this species or simply to a serum level of leptin that is below the detection threshold.

  13. Monitoring leptin activity using the chicken leptin receptor.

    Science.gov (United States)

    Hen, Gideon; Yosefi, Sera; Ronin, Ana; Einat, Paz; Rosenblum, Charles I; Denver, Robert J; Friedman-Einat, Miriam

    2008-05-01

    We report on the construction of a leptin bioassay based on the activation of chicken leptin receptor in cultured cells. A human embryonic kidney (HEK)-293 cell line, stably transfected with the full-length cDNA of chicken leptin receptor together with a STAT3-responsive reporter gene specifically responded to recombinant human and Xenopus leptins. The observed higher sensitivity of chicken leptin receptor to the former is in agreement with the degree of sequence similarity among these species (about 60 and 38% identical amino acids between humans and chickens, and between humans and Xenopus respectively). The specific activation of signal transduction through the chicken leptin receptor, shown here for the first time, suggests that the transition of Gln269 (implicated in the Gln-to-Pro Zucker fatty mutation in rats) to Glu in chickens does not impair its activity. Analysis of leptin-like activity in human serum samples of obese and lean subjects coincided well with leptin levels determined by RIA. Serum samples of pre- and post partum cows showed a tight correlation with the degree of adiposity. However, specific activation of the chicken leptin receptor in this assay was not observed with serum samples from broiler or layer chickens (representing fat and lean phenotypes respectively) or with those from turkey. Similar leptin receptor activation profiles were observed with cells transfected with human leptin receptor. Further work is needed to determine whether the lack of leptin-like activity in the chicken serum samples is due to a lack of leptin in this species or simply to a serum level of leptin that is below the detection threshold. PMID:18434362

  14. Flavonoids with M1 Muscarinic Acetylcholine Receptor Binding Activity

    Directory of Open Access Journals (Sweden)

    Meyyammai Swaminathan

    2014-06-01

    Full Text Available Muscarinic acetylcholine receptor-active compounds have potential for the treatment of Alzheimer’s disease. In this study, a series of natural and synthetic flavones and flavonols was assayed in vitro for their ability to inhibit radioligand binding at human cloned M1 muscarinic receptors. Several compounds were found to possess competitive binding affinity (Ki = 40–110 µM, comparable to that of acetylcholine (Ki = 59 µM. Despite the fact that these compounds lack a positively-charged ammonium group under physiological conditions, molecular modelling studies suggested that they bind to the orthosteric site of the receptor, mainly through non-polar interactions.

  15. Structure-based engineering of species selectivity in the interaction between urokinase and its receptor: implication for preclinical cancer therapy

    DEFF Research Database (Denmark)

    Lin, Lin; Gårdsvoll, Henrik; Huai, Qing;

    2010-01-01

    The high affinity interaction between the urokinase-type plasminogen activator (uPA) and its glycolipid-anchored receptor (uPAR) is decisive for cell surface-associated plasminogen activation. Because plasmin activity controls fibrinolysis in a variety of pathological conditions, including cancer...... and wound healing, several intervention studies have focused on targeting the uPA.uPAR interaction in vivo. Evaluations of such studies in xenotransplanted tumor models are, however, complicated by the pronounced species selectivity in this interaction. We now report the molecular basis underlying...... the structural basis required for a successful rational design of the species selectivity in the uPA.uPAR interaction, which is highly relevant for functional studies in mouse models, but it also suggests the possible development of general inhibitors that will target the uPA.uPAR interaction across species...

  16. 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.

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

    International Nuclear Information System (INIS)

    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

  18. Extended Synaptotagmin Interaction with the Fibroblast Growth Factor Receptor Depends on Receptor Conformation, Not Catalytic Activity.

    Science.gov (United States)

    Tremblay, Michel G; Herdman, Chelsea; Guillou, François; Mishra, Prakash K; Baril, Joëlle; Bellenfant, Sabrina; Moss, Tom

    2015-06-26

    We previously demonstrated that ESyt2 interacts specifically with the activated FGF receptor and is required for a rapid phase of receptor internalization and for functional signaling via the ERK pathway in early Xenopus embryos. ESyt2 is one of the three-member family of Extended Synaptotagmins that were recently shown to be implicated in the formation of endoplasmic reticulum (ER)-plasma membrane (PM) junctions and in the Ca(2+) dependent regulation of these junctions. Here we show that ESyt2 is directed to the ER by its putative transmembrane domain, that the ESyts hetero- and homodimerize, and that ESyt2 homodimerization in vivo requires a TM adjacent sequence but not the SMP domain. ESyt2 and ESyt3, but not ESyt1, selectively interact in vivo with activated FGFR1. In the case of ESyt2, this interaction requires a short TM adjacent sequence and is independent of receptor autophosphorylation, but dependent on receptor conformation. The data show that ESyt2 recognizes a site in the upper kinase lobe of FGFR1 that is revealed by displacement of the kinase domain activation loop during receptor activation.

  19. Neurotrophin receptors expression and JNK pathway activation in human astrocytomas

    International Nuclear Information System (INIS)

    Neurotrophins are growth factors that regulate cell growth, differentiation and apoptosis in the nervous system. Their diverse actions are mediated through two different transmembrane – receptor signaling systems: Trk receptor tyrosine kinases (TrkA, TrkB, TrkC) and p75NTR neurotrophin receptor. Trk receptors promote cell survival and differentiation while p75NTR induces, in most cases, the activity of JNK-p53-Bax apoptosis pathway or suppresses intracellular survival signaling cascades. Robust Trk activation blocks p75NTR -induced apoptosis by suppressing the JNK-p53-Bax pathway. The aim of this exploratory study was to investigate the expression levels of neurotrophin receptors, Trks and p75NTR, and the activation of JNK pathway in human astrocytomas and in adjacent non-neoplastic brain tissue. Formalin-fixed paraffin-embedded serial sections from 33 supratentorial astrocytomas (5 diffuse fibrillary astrocytomas, WHO grade II; 6 anaplastic astrocytomas, WHO grade III; 22 glioblastomas multiforme, WHO grade IV) were immunostained following microwave pretreatment. Polyclonal antibodies against TrkA, TrkB, TrkC and monoclonal antibodies against p75NTR and phosphorylated forms of JNK (pJNK) and c-Jun (pc-Jun) were used. The labeling index (LI), defined as the percentage of positive (labeled) cells out of the total number of tumor cells counted, was determined. Moderate to strong, granular cytoplasmic immunoreactivity for TrkA, TrkB and TrkC receptors was detected in greater than or equal to 10% of tumor cells in the majority of tumors independently of grade; on the contrary, p75NTR receptor expression was found in a small percentage of tumor cells (~1%) in some tumors. The endothelium of tumor capillaries showed conspicuous immunoreactivity for TrkB receptor. Trk immunoreactivity seemed to be localized in some neurons and astrocytes in non-neoplastic tissue. Phosphorylated forms of JNK (pJNK) and c-Jun (pc-Jun) were significantly co-expressed in a tumor grade

  20. Structural mechanism of glutamate receptor activation and desensitization.

    Science.gov (United States)

    Meyerson, Joel R; Kumar, Janesh; Chittori, Sagar; Rao, Prashant; Pierson, Jason; Bartesaghi, Alberto; Mayer, Mark L; Subramaniam, Sriram

    2014-10-16

    Ionotropic glutamate receptors are ligand-gated ion channels that mediate excitatory synaptic transmission in the vertebrate brain. To gain a better understanding of how structural changes gate ion flux across the membrane, we trapped rat AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) and kainate receptor subtypes in their major functional states and analysed the resulting structures using cryo-electron microscopy. We show that transition to the active state involves a 'corkscrew' motion of the receptor assembly, driven by closure of the ligand-binding domain. Desensitization is accompanied by disruption of the amino-terminal domain tetramer in AMPA, but not kainate, receptors with a two-fold to four-fold symmetry transition in the ligand-binding domains in both subtypes. The 7.6 Å structure of a desensitized kainate receptor shows how these changes accommodate channel closing. These findings integrate previous physiological, biochemical and structural analyses of glutamate receptors and provide a molecular explanation for key steps in receptor gating. PMID:25119039

  1. PSD-95 regulates D1 dopamine receptor resensitization, but not receptor-mediated Gs-protein activation

    Institute of Scientific and Technical Information of China (English)

    Peihua Sun; Jingru Wang; Weihua Gu; Wei Cheng; Guo-zhang Jin; Eitan Friedman; Jie Zheng; Xuechu Zhen

    2009-01-01

    The present study aims to define the role of postsynaptic density (PSD)-95 in the regulation of dopamine (DA) receptor function. We found that PSD-95 physically associates with either D1 or D2 DA receptors in co-transfected HEK-293 cells. Stimulation of DA receptors altered the association between D1 receptor and PSD-95 in a time-depen-dent manner. Functional assays indicated that PSD-95 co-expression did not affect D1 receptor-stimulated cAMP pro-duction, Gs-protein activation or receptor desensitization. However, PSD-95 accelerated the recovery of internalized membrane receptors by promoting receptor recycling, thus resulting in enhanced resensitization of internalized D1 receptors. Our results provide a novel mechanism for regulating DA receptor recycling that may play an important role in postsynaptic DA functional modulation and synaptic neuroplasticity.

  2. UROKINASE-TYPE PLASMINOGEN ACTIVATOR, ITS RECEPTOR AND INHIBITOR EXPRESSION IN HEPATOCELLULAR CARCINOMA RELATION TO CANCER INVASIVENESS AND PROGNOSIS

    Institute of Scientific and Technical Information of China (English)

    Zheng Qi; Tang Zhaoyou; Wu Zhiquan; Shi Daren; Tang Huibin; Zhu Yunsong; Song Houyan

    1998-01-01

    Objective:To study the relevance of uPA, uPAR and PAI-1 to hepatocellular carcinoma (HCC). Methods:The expression at protein level of uPA, uPAR and PAI-1was determined in 48 cases of HCC and 12 cases of benign tumors of liver (as control) by immunohistochemistry.Results: When compared to cancer-adjacent liver tissue and the control, positive rate of immune staining for uPA,uPAR and PAI-1 on cell membrane were significantly higher in HCC cells (P<0.05). Positive staining of uPA and uPAR was seen in 16 of 22 and 19 of 22 cases of HCC with invasion, respectively (P<0.01 and P<0.001). In 8 of 8cases with cancer embolus, and in 6 of 6 cases with lymph node metastasis was the expression of positive uPAR.Compared with 2 of 17 cases without recurrence, uPAR was positive in 15 of 17 recurrent cases (P<0.01). In 36cases who survived, 17 was positive uPAR and 15 positive PAI-1, while in 12 cases who died 2 years after surgery, 12were positive for uPAR and 9 positive PAI-1, respectively (P<0.01 and P<0.05). In 15 positive cases for all three parameters, 11 had cancer invasion and 7 died within 2 years, while in negative cases, 2 had invasion and none died within 2 years (P<0.05). Conclusion: Expression of.uPA, uPAR and PAI-1 is increased in HCC, uPA and uPAR may contribute significantly to HCC invasion and metastasis. uPAR and PAI-1 are associated with poor prognosis of HCC.

  3. Abelson tyrosine kinase links PDGFbeta receptor activation to cytoskeletal regulation of NMDA receptors in CA1 hippocampal neurons

    Directory of Open Access Journals (Sweden)

    Beazely Michael A

    2008-12-01

    Full Text Available Abstract Background We have previously demonstrated that PDGF receptor activation indirectly inhibits N-methyl-D-aspartate (NMDA currents by modifying the cytoskeleton. PDGF receptor ligand is also neuroprotective in hippocampal slices and cultured neurons. PDGF receptors are tyrosine kinases that control a variety of signal transduction pathways including those mediated by PLCγ. In fibroblasts Src and another non-receptor tyrosine kinase, Abelson kinase (Abl, control PDGF receptor regulation of cytoskeletal dynamics. The mechanism whereby PDGF receptor regulates cytoskeletal dynamics in central neurons remains poorly understood. Results Intracellular applications of active Abl, but not heat-inactivated Abl, decreased NMDA-evoked currents in isolated hippocampal neurons. This mimics the effects of PDGF receptor activation in these neurons. The Abl kinase inhibitor, STI571, blocked the inhibition of NMDA currents by Abl. We demonstrate that PDGF receptors can activate Abl kinase in hippocampal neurons via mechanisms similar to those observed previously in fibroblasts. Furthermore, PDGFβ receptor activation alters the subcellular localization of Abl. Abl kinase is linked to actin cytoskeletal dynamics in many systems. We show that the inhibition of NMDA receptor currents by Abl kinase is blocked by the inclusion of the Rho kinase inhibitor, Y-27632, and that activation of Abl correlates with an increase in ROCK tyrosine phosphorylation. Conclusion This study demonstrates that PDGFβ receptors act via an interaction with Abl kinase and Rho kinase to regulated cytoskeletal regulation of NMDA receptor channels in CA1 pyramidal neurons.

  4. Glycine Potentiates AMPA Receptor Function through Metabotropic Activation of GluN2A-Containing NMDA Receptors

    Science.gov (United States)

    Li, Li-Jun; Hu, Rong; Lujan, Brendan; Chen, Juan; Zhang, Jian-Jian; Nakano, Yasuko; Cui, Tian-Yuan; Liao, Ming-Xia; Chen, Jin-Cao; Man, Heng-Ye; Feng, Hua; Wan, Qi

    2016-01-01

    NMDA receptors are Ca2+-permeable ion channels. The activation of NMDA receptors requires agonist glutamate and co-agonist glycine. Recent evidence indicates that NMDA receptor also has metabotropic function. Here we report that in cultured mouse hippocampal neurons, glycine increases AMPA receptor-mediated currents independent of the channel activity of NMDA receptors and the activation of glycine receptors. The potentiation of AMPA receptor function by glycine is antagonized by the inhibition of ERK1/2. In the hippocampal neurons and in the HEK293 cells transfected with different combinations of NMDA receptors, glycine preferentially acts on GluN2A-containing NMDA receptors (GluN2ARs), but not GluN2B-containing NMDA receptors (GluN2BRs), to enhance ERK1/2 phosphorylation independent of the channel activity of GluN2ARs. Without requiring the channel activity of GluN2ARs, glycine increases AMPA receptor-mediated currents through GluN2ARs. Thus, these results reveal a metabotropic function of GluN2ARs in mediating glycine-induced potentiation of AMPA receptor function via ERK1/2 activation.

  5. Activities of nicotinic acetylcholine receptors modulate neurotransmission and synaptic architecture

    Institute of Scientific and Technical Information of China (English)

    Akira Oda; Hidekazu Tanaka

    2014-01-01

    The cholinergic system is involved in a broad spectrum of brain function, and its failure has been implicated in Alzheimer’s disease. Acetylcholine transduces signals through muscarinic and nicotinic acetylcholine receptors, both of which inlfuence synaptic plasticity and cognition. However, the mechanisms that relate the rapid gating of nicotinic acetylcholine receptors to per-sistent changes in brain function have remained elusive. Recent evidence indicates that nicotinic acetylcholine receptors activities affect synaptic morphology and density, which result in per-sistent rearrangements of neural connectivity. Further investigations of the relationships between nicotinic acetylcholine receptors and rearrangements of neural circuitry in the central nervous system may help understand the pathogenesis of Alzheimer’s disease.

  6. Epidermis-type lipoxygenase 3 regulates adipocyte differentiation and peroxisome proliferator-activated receptor gamma activity

    DEFF Research Database (Denmark)

    Hallenborg, Philip; Jørgensen, Claus; Petersen, Rasmus K;

    2010-01-01

    The nuclear receptor peroxisome proliferator-activated receptor gamma (PPAR gamma) is essential for adipogenesis. Although several fatty acids and their derivatives are known to bind and activate PPAR gamma, the nature of the endogenous ligand(s) promoting the early stages of adipocyte differenti...

  7. New GABA amides activating GABA A-receptors

    OpenAIRE

    Peter Raster; Andreas Späth; Svetlana Bultakova; Pau Gorostiza; Burkhard König; Piotr Bregestovski

    2013-01-01

    We have prepared a series of new and some literature-reported GABA-amides and determined their effect on the activation of GABAA-receptors expressed in CHO cells. Special attention was paid to the purification of the target compounds to remove even traces of GABA contaminations, which may arise from deprotection steps in the synthesis. GABA-amides were previously reported to be partial, full or superagonists. In our hands these compounds were not able to activate GABAA-receptor channels in wh...

  8. Mechanisms of Activation of Receptor Tyrosine Kinases: Monomers or Dimers

    Directory of Open Access Journals (Sweden)

    Ichiro N. Maruyama

    2014-04-01

    Full Text Available Receptor tyrosine kinases (RTKs play essential roles in cellular processes, including metabolism, cell-cycle control, survival, proliferation, motility and differentiation. RTKs are all synthesized as single-pass transmembrane proteins and bind polypeptide ligands, mainly growth factors. It has long been thought that all RTKs, except for the insulin receptor (IR family, are activated by ligand-induced dimerization of the receptors. An increasing number of diverse studies, however, indicate that RTKs, previously thought to exist as monomers, are present as pre-formed, yet inactive, dimers prior to ligand binding. The non-covalently associated dimeric structures are reminiscent of those of the IR family, which has a disulfide-linked dimeric structure. Furthermore, recent progress in structural studies has provided insight into the underpinnings of conformational changes during the activation of RTKs. In this review, I discuss two mutually exclusive models for the mechanisms of activation of the epidermal growth factor receptor, the neurotrophin receptor and IR families, based on these new insights.

  9. Calcium-sensing receptor activation depresses synaptic transmission

    OpenAIRE

    Phillips, Cecilia G.; Harnett, Mark T.; Chen, Wenyan; Smith, Stephen M.

    2008-01-01

    At excitatory synapses, decreases in cleft [Ca] arising from activity-dependent transmembrane Ca flux reduce the probability of subsequent transmitter release. Intense neural activity, induced by physiological and pathological stimuli, disturb the external microenvironment reducing extracellular [Ca] ([Ca]o) and thus may impair neurotransmission. Increases in [Ca]o activate the extracellular calcium sensing receptor (CaSR) which in turn inhibits non-selective cation channels (NSCC) at the maj...

  10. Allosteric activation mechanism of the cys-loop receptors

    Institute of Scientific and Technical Information of China (English)

    Yong-chang CHANG; Wen WU; Jian-liang ZHANG; Yao HUANG

    2009-01-01

    Binding of a neurotransmitter to its ionotropic receptor opens a distantly located ion channel, a process termed allosteric activation. Here we review recent advances in the molecular mechanism by which the cys-loop receptors are activated with emphasis on the best studied nicotinic acetylcholine receptors (nAChRs). With a combination of affinity labeling, mutagenesis, electrophysiology, kinetic modeling, electron microscopy (EM), and crystal structure analysis, the allosteric activation mechanism is emerging. Specifically, the binding domain and gating domain are interconnected by an allosteric activation network. Agonist binding induces conformational changes, resulting in the rotation of a β sheet of amino-terminal domain and outward movement of loop 2, loop F, and cys-loop, which are coupled to the M2-M3 linker to pull the channel to open. However, there are still some controversies about the movement of the channel-lining domain M2. Nine angstrom resolution EM structure of a nAChR imaged in the open state suggests that channel opening is the result of rotation of the M2 domain. In contrast, recent crystal structures of bacterial homologues of the cys-loop receptor family in apparently open state have implied an M2 tilting model with pore dilation and quaternary twist of the whole pentameric receptor. An elegant study of the nAChR using protonation scanning of M2 domain supports a similar pore dilation activation mechanism with minimal rotation of M2. This remains to be validated with other approaches including high resolution structure determination of the mammalian cys-loop receptors in the open state.

  11. A bacterial tyrosine phosphatase inhibits plant pattern recognition receptor activation.

    Science.gov (United States)

    Macho, Alberto P; Schwessinger, Benjamin; Ntoukakis, Vardis; Brutus, Alexandre; Segonzac, Cécile; Roy, Sonali; Kadota, Yasuhiro; Oh, Man-Ho; Sklenar, Jan; Derbyshire, Paul; Lozano-Durán, Rosa; Malinovsky, Frederikke Gro; Monaghan, Jacqueline; Menke, Frank L; Huber, Steven C; He, Sheng Yang; Zipfel, Cyril

    2014-03-28

    Innate immunity relies on the perception of pathogen-associated molecular patterns (PAMPs) by pattern-recognition receptors (PRRs) located on the host cell's surface. Many plant PRRs are kinases. Here, we report that the Arabidopsis receptor kinase EF-TU RECEPTOR (EFR), which perceives the elf18 peptide derived from bacterial elongation factor Tu, is activated upon ligand binding by phosphorylation on its tyrosine residues. Phosphorylation of a single tyrosine residue, Y836, is required for activation of EFR and downstream immunity to the phytopathogenic bacterium Pseudomonas syringae. A tyrosine phosphatase, HopAO1, secreted by P. syringae, reduces EFR phosphorylation and prevents subsequent immune responses. Thus, host and pathogen compete to take control of PRR tyrosine phosphorylation used to initiate antibacterial immunity.

  12. Interaction of the C-terminal acidic domain of the insulin receptor with histone modulates the receptor kinase activity.

    Science.gov (United States)

    Baron, V; Kaliman, P; Alengrin, F; Van Obberghen, E

    1995-04-01

    In this study, we investigated the role of the insulin receptor domain 1270-1280, an acid-rich sequence located in the receptor C-terminus. Antipeptide IgG raised against this sequence were obtained and used to analyze their effect on receptor function. Antipeptide IgG inhibited receptor autophosphorylation at Tyr1146, Tyr1150 and Tyr1151. These sites are known to be key modulators of the receptor activity. Autophosphorylation at other sites may also have been inhibited. The antipeptide antibody decreased the receptor kinase activity measured with poly(Glu80Tyr20) and a synthetic peptide corresponding to the proreceptor sequence 1142-1158. We provide evidence that the effect of the antibody on substrate phosphorylation may result from the control of the phosphorylation level of the receptor. Concerning the action of the antipeptide IgG on the receptor kinase activity, histone did not behave similarly to poly(Glu80Tyr20). The antibody recognizing sequence 1270-1280 competed with histone for an overlapping binding site. Histone also modulated insulin receptor autophosphorylation, supporting the idea that interference with domain 1270-1280 alters the receptor kinase. Our data suggest that the acidic region including residues 1270-1280 of the insulin receptor C-terminus is involved in the following events: (a) receptor binding with histone, an exogenous substrate of the receptor kinase, and (b) the regulation of receptor autophosphorylation and kinase activity. Based on these observations, we would like to propose that this insulin receptor domain could interact with cellular proteins modulating the receptor kinase. PMID:7744039

  13. A composite role of vitronectin and urokinase in the modulation of cell morphology upon expression of the urokinase receptor

    DEFF Research Database (Denmark)

    Engelholm, L.H.; Ingvarsen, S.; Madsen, D.H.;

    2008-01-01

    a different single-site mutant, uPAR(Y57A), in the presence of a synthetic uPAR-binding peptide, as well as with wild-type uPAR, which underwent cytoskeletal rearrangements even when cultivated in uPA-deficient serum. Blocking of integrins with an Arg-Gly-Asp-containing peptide counteracted the matrix...... adhesion and cytoskeletal rearrangements, whereas a mutant uPAR, uPAR(W32A) with defective vitronectin binding, failed to induce both phenomena. However, upon saturation of uPAR(W32A) with the protease ligand, pro-uPA, or its receptor-binding domain, the ability to induce cytoskeletal rearrangements......, and this event is followed by downstream effects including changes in the cytoskeletal organization. However, it remains unclear whether the adhesion through uPAR-vitronectin is the only event capable of initiating these morphological rearrangements or whether lateral interactions between uPAR and integrins can...

  14. Ah receptor agonist activity in frequently consumed food items

    NARCIS (Netherlands)

    Waard, de W.J.; Aarts, J.M.M.J.G.; Peijnenburg, A.A.C.M.; Kok, de T.M.C.M.; Schooten, van F.J.; Hoogenboom, L.A.P.

    2008-01-01

    The aryl hydrocarbon receptor (AhR) receives much attention for its role in the toxicity of dioxins and dioxin-like polychlorinated biphenyls. However, many other compounds have also been reported to bind and activate AhR, of which natural food components are of special interest from a human health

  15. Sphingosine-1-phosphate receptor 1 reporter mice reveal receptor activation sites in vivo

    OpenAIRE

    Kono, Mari; Tucker, Ana E.; Tran, Jennifer; Bergner, Jennifer B.; Turner, Ewa M; Proia, Richard L.

    2014-01-01

    Activation of the GPCR sphingosine-1-phosphate receptor 1 (S1P1) by sphingosine-1-phosphate (S1P) regulates key physiological processes. S1P1 activation also has been implicated in pathologic processes, including autoimmunity and inflammation; however, the in vivo sites of S1P1 activation under normal and disease conditions are unclear. Here, we describe the development of a mouse model that allows in vivo evaluation of S1P1 activation. These mice, known as S1P1 GFP signaling mice, produce a ...

  16. The aryl hydrocarbon receptor and glucocorticoid receptor interact to activate human metallothionein 2A

    Energy Technology Data Exchange (ETDEWEB)

    Sato, Shoko, E-mail: satosho@rs.tus.ac.jp [Laboratory of Nutrition, Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555 (Japan); Shirakawa, Hitoshi, E-mail: shirakah@m.tohoku.ac.jp [Laboratory of Nutrition, Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555 (Japan); Tomita, Shuhei, E-mail: tomita@med.tottori-u.ac.jp [Division of Molecular Pharmacology, Department of Pathophysiological and Therapeutic Science, Yonago 683-8503 (Japan); Tohkin, Masahiro, E-mail: tohkin@phar.nagoya-cu.ac.jp [Department of Medical Safety Science, Graduate School of Pharmaceutical Science, Nagoya City University, Nagoya 267-8603 (Japan); Gonzalez, Frank J., E-mail: gonzalef@mail.nih.gov [Laboratory of Metabolism, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 (United States); Komai, Michio, E-mail: mkomai@m.tohoku.ac.jp [Laboratory of Nutrition, Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555 (Japan)

    2013-11-15

    Although the aryl hydrocarbon receptor (AHR) and glucocorticoid receptor (GR) play essential roles in mammalian development, stress responses, and other physiological events, crosstalk between these receptors has been the subject of much debate. Metallothioneins are classic glucocorticoid-inducible genes that were reported to increase upon treatment with AHR agonists in rodent tissues and cultured human cells. In this study, the mechanism of human metallothionein 2A (MT2A) gene transcription activation by AHR was investigated. Cotreatment with 3-methylcholanthrene and dexamethasone, agonists of AHR and GR respectively, synergistically increased MT2A mRNA levels in HepG2 cells. MT2A induction was suppressed by RNA interference against AHR or GR. Coimmunoprecipitation experiments revealed a physical interaction between AHR and GR proteins. Moreover, chromatin immunoprecipitation assays indicated that AHR was recruited to the glucocorticoid response element in the MT2A promoter. Thus, we provide a novel mechanism whereby AHR modulates expression of human MT2A via the glucocorticoid response element and protein–protein interactions with GR. - Highlights: • Aryl hydrocarbon receptor forms a complex with glucocorticoid receptor in cells. • Human metallothionein gene is regulated by the AHR and GR interaction. • AHR–GR complex binds to glucocorticoid response element in metallothionein gene. • We demonstrated a novel transcriptional mechanism via AHR and GR interaction.

  17. BioKnife, a uPA activity-dependent oncolytic Sendai virus, eliminates pleural spread of malignant mesothelioma via simultaneous stimulation of uPA expression.

    Science.gov (United States)

    Morodomi, Yosuke; Yano, Tokujiro; Kinoh, Hiroaki; Harada, Yui; Saito, Satoru; Kyuragi, Ryoichi; Yoshida, Kumi; Onimaru, Mitsuho; Shoji, Fumihiro; Yoshida, Tsukihisa; Ito, Kensaku; Shikada, Yasunori; Maruyama, Riichiroh; Hasegawa, Mamoru; Maehara, Yoshihiko; Yonemitsu, Yoshikazu

    2012-04-01

    Malignant pleural mesothelioma (MPM) is highly intractable and readily spreads throughout the surface of the pleural cavity, and these cells have been shown to express urokinase-type plasminogen activator receptor (uPAR). We here examined the potential of our new and powerful recombinant Sendai virus (rSeV), which shows uPAR-specific cell-to-cell fusion activity (rSeV/dMFct14 (uPA2), named "BioKnife"), for tumor cell killing in two independent orthotopic xenograft models of human. Multicycle treatment using BioKnife resulted in the efficient rescue of these models, in association with tumor-specific fusion and apoptosis. Such an effect was also seen on both MSTO-211H and H226 cells in vitro; however, we confirmed that the latter expressed uPAR but not uPA. Of interest, infection with BioKnife strongly facilitated the uPA release from H226 cells, and this effect was completely abolished by use of either pyrrolidine dithiocarbamate (PDTC) or BioKnife expressing the C-terminus-deleted dominant negative inhibitor for retinoic acid-inducible gene-I (RIG-IC), indicating that BioKnife-dependent expression of uPA was mediated by the RIG-I/nuclear factor-κB (NF-κB) axis, detecting RNA viral genome replication. Therefore, these results suggest a proof of concept that the tumor cell-killing mechanism via BioKnife may have significant potential to treat patients with MPM that is characterized by frequent uPAR expression in a clinical setting. PMID:22314292

  18. The angiotensin II type 1 receptor antagonist Losartan binds and activates bradykinin B2 receptor signaling

    DEFF Research Database (Denmark)

    Bonde, Marie Mi; Olsen, Kristine Boisen; Erikstrup, Niels;

    2011-01-01

    The angiotensin II type 1 receptor (AT1R) blocker (ARB) Losartan has cardioprotective effects during ischemia-reperfusion injury and inhibits reperfusion arrhythmias -effects that go beyond the benefits of lowering blood pressure. The renin-angiotensin and kallikrein-kinin systems are intricately...... connected and some of the cardioprotective effects of Losartan are abolished by blocking the bradykinin B2 receptor (B2R) signaling. In this study, we investigated the ability of six clinically available ARBs to specifically bind and activate the B2R. First, we investigated their ability to activate...... phosphoinositide (PI) hydrolysis in COS-7 cells transiently expressing the B2R. We found that only Losartan activated the B2R, working as a partial agonist compared to the endogenous ligand bradykinin. This effect was blocked by the B2R antagonist HOE 140. A competitive binding analysis revealed that Losartan does...

  19. Plasminogen Activator System and Breast Cancer: Potential Role in Therapy Decision Making and Precision Medicine.

    Science.gov (United States)

    Gouri, Adel; Dekaken, Aoulia; El Bairi, Khalid; Aissaoui, Arifa; Laabed, Nihad; Chefrour, Mohamed; Ciccolini, Joseph; Milano, Gérard; Benharkat, Sadek

    2016-01-01

    Shifting from the historical TNM paradigm to the determination of molecular and genetic subtypes of tumors has been a major improvement to better picture cancerous diseases. The sharper the picture is, the better will be the possibility to develop subsequent strategies, thus achieving higher efficacy and prolonged survival eventually. Recent studies suggest that urokinase-type plasminogen activator (uPA), uPA Receptor (uPAR), and plasmino-gen activator inhibitor-1 (PAI-1) may play a critical role in cancer invasion and metastasis. Consistent with their role in cancer dissemination, high levels of uPA, PAI-1, and uPAR in multiple cancer types correlate with dismal prognosis. In this respect, upfront determination of uPA and PAI-1 as invasion markers has further opened up the possibilities for individualized therapy of breast cancer. Indeed, uPA and PAI-1 could help to classify patients on their risk for metastatic spreading and subsequent relapse, thus helping clinicians in their decision-making process to propose, or not propose, adjuvant therapy. This review covers the implications for cancer diagnosis, prognosis, and therapy of uPA and PAI-1, and therefore how they could be major actors in the development of a precision medicine in breast cancer. PMID:27578963

  20. Peroxisome proliferator-activated receptors, metabolic syndrome and cardiovascular disease

    OpenAIRE

    Azhar, Salman

    2010-01-01

    Metabolic syndrome (MetS) is a constellation of risk factors including insulin resistance, central obesity, dyslipidemia and hypertension that markedly increase the risk of Type 2 diabetes (T2DM) and cardiovascular disease (CVD). The peroxisome proliferators-activated receptor (PPAR) isotypes, PPARα, PPARδ/β and PPARγ are ligand-activated nuclear transcription factors, which modulate the expression of an array of genes that play a central role in regulating glucose, lipid and cholesterol meta...

  1. Steroid signaling activation and intracellular localization of sex steroid receptors

    OpenAIRE

    Giraldi, Tiziana; Giovannelli, Pia; Di Donato, Marzia; Castoria, Gabriella; Migliaccio, Antimo; Auricchio, Ferdinando

    2010-01-01

    In addition to stimulating gene transcription, sex steroids trigger rapid, non-genomic responses in the extra-nuclear compartment of target cells. These events take place within seconds or minutes after hormone administration and do not require transcriptional activity of sex steroid receptors. Depending on cell systems, activation of extra-nuclear signaling pathways by sex steroids fosters cell cycle progression, prevents apoptosis, leads to epigenetic modifications and increases cell migrat...

  2. An improved ivermectin-activated chloride channel receptor for inhibiting electrical activity in defined neuronal populations

    DEFF Research Database (Denmark)

    Lynagh, Timothy Peter; Lynch, Joseph W

    2010-01-01

    for surgically implanted stimulus delivery methods and their use of nonhuman receptors. A third silencing method, an invertebrate glutamate-gated chloride channel receptor (GluClR) activated by ivermectin, solves the stimulus delivery problem as ivermectin is a safe, well tolerated drug that reaches the brain...

  3. Activation of serotonin receptors promotes microglial injury-induced motility but attenuates phagocytic activity

    NARCIS (Netherlands)

    Krabbe, Grietje; Matyash, Vitali; Pannasch, Ulrike; Mamer, Lauren; Boddeke, Hendrikus W. G. M.; Kettenmann, Helmut

    2012-01-01

    Microglia, the brain immune cell, express several neurotransmitter receptors which modulate microglial functions. In this project we studied the impact of serotonin receptor activation on distinct microglial properties as serotonin deficiency not only has been linked to a number of psychiatric disea

  4. Screening of selected pesticides for oestrogen receptor activation in vitro

    DEFF Research Database (Denmark)

    Vinggaard, Anne; Breinholt, Vibeke; Larsen, John Christian

    1999-01-01

    Twenty pesticides were tested for their ability to activate the oestrogen receptor in vitro using an,MCF7 cell proliferation assay and a Yeast Oestrogen Screen. The fungicides fenarimol, triadimefon, and triadimenol were identified as weak oestrogen receptor agonists, which at 10 mu M induces a 2.......0, 2.4, and 1.9-fold increase in proliferation of human MCF7 breast cancer cells (E3 clone). The relative proliferation efficiency (RPE) was 43-69%, indicating partial agonism at the oestrogen receptor. Several pesticides did not have any effect oil the proliferation response after 6 days of exposure......, including. chlorpyrifos, diuron, iprodion, linuron, pentachlorphenol, prochloraz, propioconazol, propyzamine, quintozen, tetrachorvinphos and tetradifon. Some pesticides resulted in a negligible proliferation response, which was nor statistically significant under the present experimental conditions...

  5. Urokinase-Type Plasminogen Activator Receptor as a Potential PET Biomarker in Glioblastoma

    DEFF Research Database (Denmark)

    Persson, Morten; Nedergaard, Mette K; Brandt-Larsen, Malene;

    2016-01-01

    an orthotopic xenograft model of glioblastoma. Tumor growth was monitored using bioluminescence imaging. Five to six weeks after inoculation, all mice were scanned with small-animal PET/CT using two new uPAR PET ligands ((64)Cu-NOTA-AE105 and (68)Ga-NOTA-AE105) and, for comparison, O-(2-(18)F...

  6. Glycine Receptor α2 Subunit Activation Promotes Cortical Interneuron Migration

    Directory of Open Access Journals (Sweden)

    Ariel Avila

    2013-08-01

    Full Text Available Glycine receptors (GlyRs are detected in the developing CNS before synaptogenesis, but their function remains elusive. This study demonstrates that functional GlyRs are expressed by embryonic cortical interneurons in vivo. Furthermore, genetic disruption of these receptors leads to interneuron migration defects. We discovered that extrasynaptic activation of GlyRs containing the α2 subunit in cortical interneurons by endogenous glycine activates voltage-gated calcium channels and promotes calcium influx, which further modulates actomyosin contractility to fine-tune nuclear translocation during migration. Taken together, our data highlight the molecular events triggered by GlyR α2 activation that control cortical tangential migration during embryogenesis.

  7. Conserved phosphorylation sites in the activation loop of the Arabidopsis phytosulfokine receptor PSKR1 differentially affect kinase and receptor activity

    OpenAIRE

    Hartmann, Jens; Linke, Dennis; Bönniger, Christine; Tholey, Andreas; Sauter, Margret

    2015-01-01

    PSK (phytosulfokine) is a plant peptide hormone perceived by a leucine-rich repeat receptor kinase. Phosphosite mapping of epitope-tagged PSKR1 (phytosulfokine receptor 1) from Arabidopsis thaliana plants identified Ser696 and Ser698 in the JM (juxtamembrane) region and probably Ser886 and/or Ser893 in the AL (activation loop) as in planta phosphorylation sites. In vitro-expressed kinase was autophosphorylated at Ser717 in the JM, and at Ser733, Thr752, Ser783, Ser864, Ser911, Ser958 and Thr9...

  8. Conserved phosphorylation sites in the activation loop of the Arabidopsis phytosulfokine receptor PSKR1 differentially affect kinase and receptor activity.

    Science.gov (United States)

    Hartmann, Jens; Linke, Dennis; Bönniger, Christine; Tholey, Andreas; Sauter, Margret

    2015-12-15

    PSK (phytosulfokine) is a plant peptide hormone perceived by a leucine-rich repeat receptor kinase. Phosphosite mapping of epitope-tagged PSKR1 (phytosulfokine receptor 1) from Arabidopsis thaliana plants identified Ser(696) and Ser(698) in the JM (juxtamembrane) region and probably Ser(886) and/or Ser(893) in the AL (activation loop) as in planta phosphorylation sites. In vitro-expressed kinase was autophosphorylated at Ser(717) in the JM, and at Ser(733), Thr(752), Ser(783), Ser(864), Ser(911), Ser(958) and Thr(998) in the kinase domain. The LC-ESI-MS/MS spectra provided support that up to three sites (Thr(890), Ser(893) and Thr(894)) in the AL were likely to be phosphorylated in vitro. These sites are evolutionarily highly conserved in PSK receptors, indicative of a conserved function. Site-directed mutagenesis of the four conserved residues in the activation segment, Thr(890), Ser(893), Thr(894) and Thr(899), differentially altered kinase activity in vitro and growth-promoting activity in planta. The T899A and the quadruple-mutated TSTT-A (T890A/S893A/T894A/T899A) mutants were both kinase-inactive, but PSKR1(T899A) retained growth-promoting activity. The T890A and S893A/T894A substitutions diminished kinase activity and growth promotion. We hypothesize that phosphorylation within the AL activates kinase activity and receptor function in a gradual and distinctive manner that may be a means to modulate the PSK response. PMID:26472115

  9. Hyaluronic acid induces activation of the κ-opioid receptor.

    Directory of Open Access Journals (Sweden)

    Barbara Zavan

    Full Text Available INTRODUCTION: Nociceptive pain is one of the most common types of pain that originates from an injury involving nociceptors. Approximately 60% of the knee joint innervations are classified as nociceptive. The specific biological mechanism underlying the regulation of nociceptors is relevant for the treatment of symptoms affecting the knee joint. Intra-articular administration of exogenous hyaluronic acid (HA in patients with osteoarthritis (OA appears to be particularly effective in reducing pain and improving patient function. METHODS: We performed an in vitro study conducted in CHO cells that expressed a panel of opioid receptors and in primary rat dorsal root ganglion (DRG neurons to determine if HA induces the activation of opioid peptide receptors (OPr using both aequorin and the fluorescent dye Fura-2/AM. RESULTS: Selective agonists and antagonists for each OPr expressed on CHO cells were used to test the efficacy of our in vitro model followed by stimulation with HA. The results showed that HA induces stimulatory effects on the κ receptor (KOP. These effects of HA were also confirmed in rat DRG neurons, which express endogenously the OPr. CONCLUSIONS: HA activates the KOP receptor in a concentration dependent manner, with a pEC(50 value of 7.57.

  10. Allosteric receptor activation by the plant peptide hormone phytosulfokine.

    Science.gov (United States)

    Wang, Jizong; Li, Hongju; Han, Zhifu; Zhang, Heqiao; Wang, Tong; Lin, Guangzhong; Chang, Junbiao; Yang, Weicai; Chai, Jijie

    2015-09-10

    Phytosulfokine (PSK) is a disulfated pentapeptide that has a ubiquitous role in plant growth and development. PSK is perceived by its receptor PSKR, a leucine-rich repeat receptor kinase (LRR-RK). The mechanisms underlying the recognition of PSK, the activation of PSKR and the identity of the components downstream of the initial binding remain elusive. Here we report the crystal structures of the extracellular LRR domain of PSKR in free, PSK- and co-receptor-bound forms. The structures reveal that PSK interacts mainly with a β-strand from the island domain of PSKR, forming an anti-β-sheet. The two sulfate moieties of PSK interact directly with PSKR, sensitizing PSKR recognition of PSK. Supported by biochemical, structural and genetic evidence, PSK binding enhances PSKR heterodimerization with the somatic embryogenesis receptor-like kinases (SERKs). However, PSK is not directly involved in PSKR-SERK interaction but stabilizes PSKR island domain for recruitment of a SERK. Our data reveal the structural basis for PSKR recognition of PSK and allosteric activation of PSKR by PSK, opening up new avenues for the design of PSKR-specific small molecules. PMID:26308901

  11. Neurohumoral activation in heart failure: the role of adrenergic receptors

    Directory of Open Access Journals (Sweden)

    Patricia C. Brum

    2006-09-01

    Full Text Available Heart failure (HF is a common endpoint for many forms of cardiovascular disease and a significant cause of morbidity and mortality. The development of end-stage HF often involves an initial insult to the myocardium that reduces cardiac output and leads to a compensatory increase in sympathetic nervous system activity. Acutely, the sympathetic hyperactivity through the activation of beta-adrenergic receptors increases heart rate and cardiac contractility, which compensate for decreased cardiac output. However, chronic exposure of the heart to elevated levels of catecholamines released from sympathetic nerve terminals and the adrenal gland may lead to further pathologic changes in the heart, resulting in continued elevation of sympathetic tone and a progressive deterioration in cardiac function. On a molecular level, altered beta-adrenergic receptor signaling plays a pivotal role in the genesis and progression of HF. beta-adrenergic receptor number and function are decreased, and downstream mechanisms are altered. In this review we will present an overview of the normal beta-adrenergic receptor pathway in the heart and the consequences of sustained adrenergic activation in HF. The myopathic potential of individual components of the adrenergic signaling will be discussed through the results of research performed in genetic modified animals. Finally, we will discuss the potential clinical impact of beta-adrenergic receptor gene polymorphisms for better understanding the progression of HF.A insuficiência cardíaca (IC é a via final comum da maioria das doenças cardiovasculares e uma das maiores causas de morbi-mortalidade. O desenvolvimento do estágio final da IC freqüentemente envolve um insulto inicial do miocárdio, reduzindo o débito cardíaco e levando ao aumento compensatório da atividade do sistema nervoso simpático (SNS. Existem evidências de que apesar da exposição aguda ser benéfica, exposições crônicas a elevadas concentra

  12. Activation of Penile Proadipogenic Peroxisome Proliferator-Activated Receptor with an Estrogen: Interaction with Estrogen Receptor Alpha during Postnatal Development

    Directory of Open Access Journals (Sweden)

    Mahmoud M. Mansour

    2008-01-01

    Full Text Available Exposure to the estrogen receptor alpha (ER ligand diethylstilbesterol (DES between neonatal days 2 to 12 induces penile adipogenesis and adult infertility in rats. The objective of this study was to investigate the in vivo interaction between DES-activated ER and the proadipogenic transcription factor peroxisome proliferator-activated receptor gamma (PPAR. Transcripts for PPARs , , and and 1a splice variant were detected in Sprague-Dawley normal rat penis with PPAR predominating. In addition, PPAR1b and PPAR2 were newly induced by DES. The PPAR transcripts were significantly upregulated with DES and reduced by antiestrogen ICI 182, 780. At the cellular level, PPAR protein was detected in urethral transitional epithelium and stromal, endothelial, neuronal, and smooth muscular cells. Treatment with DES activated ER and induced adipocyte differentiation in corpus cavernosum penis. Those adipocytes exhibited strong nuclear PPAR expression. These results suggest a biological overlap between PPAR and ER and highlight a mechanism for endocrine disruption.

  13. Monocyte Signal Transduction Receptors in Active and Latent Tuberculosis

    Directory of Open Access Journals (Sweden)

    Magdalena Druszczynska

    2013-01-01

    Full Text Available The mechanisms that promote either resistance or susceptibility to TB disease remain insufficiently understood. Our aim was to compare the expression of cell signaling transduction receptors, CD14, TLR2, CD206, and β2 integrin LFA-1 on monocytes from patients with active TB or nonmycobacterial lung disease and healthy individuals with M.tb latency and uninfected controls to explain the background of the differences between clinical and subclinical forms of M.tb infection. A simultaneous increase in the expression of the membrane bound mCD14 receptor and LFA-1 integrin in patients with active TB may be considered a prodrome of breaking immune control by M.tb bacilli in subjects with the latent TB and absence of clinical symptoms.

  14. Allosterism and Structure in Thermally Activated Transient Receptor Potential Channels.

    Science.gov (United States)

    Diaz-Franulic, Ignacio; Poblete, Horacio; Miño-Galaz, Germán; González, Carlos; Latorre, Ramón

    2016-07-01

    The molecular sensors that mediate temperature changes in living organisms are a large family of proteins known as thermosensitive transient receptor potential (TRP) ion channels. These membrane proteins are polymodal receptors that can be activated by cold or hot temperatures, depending on the channel subtype, voltage, and ligands. The stimuli sensors are allosterically coupled to a pore domain, increasing the probability of finding the channel in its ion conductive conformation. In this review we first discuss the allosteric coupling between the temperature and voltage sensor modules and the pore domain, and then discuss the thermodynamic foundations of thermo-TRP channel activation. We provide a structural overview of the molecular determinants of temperature sensing. We also posit an anisotropic thermal diffusion model that may explain the large temperature sensitivity of TRP channels. Additionally, we examine the effect of several ligands on TRP channel function and the evidence regarding their mechanisms of action. PMID:27297398

  15. Manipulation of P2X Receptor Activities by Light Stimulation

    Directory of Open Access Journals (Sweden)

    Sang Seong Kim

    2016-01-01

    Full Text Available P2X receptors are involved in amplification of inflammatory responses in peripheral nociceptive fibers and in mediating pain-related signals to the CNS. Control of P2X activation has significant importance in managing unwanted hypersensitive neuron responses. To overcome the limitations of chemical ligand treatment, optical stimulation methods of optogenetics and photoswitching achieve efficient control of P2X activation while allowing specificity at the target site and convenient stimulation by light illumination. There are many potential applications for photosensitive elements, such as improved uncaging methods, photoisomerizable ligands, photoswitches, and gold nanoparticles. Each technique has both advantages and downsides, and techniques are selected according to the purpose of the application. Technical advances not only provide novel approaches to manage inflammation or pain mediated by P2X receptors but also suggest a similar approach for controlling other ion channels.

  16. Activation of the chicken gonadotropin-inhibitory hormone receptor reduces gonadotropin releasing hormone receptor signaling.

    Science.gov (United States)

    Shimizu, Mamiko; Bédécarrats, Grégoy Y

    2010-06-01

    Gonadotropin-inhibitory hormone (GnIH) is a hypothalamic peptide from the RFamide peptide family that has been identified in multiple avian species. Although GnIH has clearly been shown to reduce LH release from the anterior pituitary gland, its mechanism of action remains to be determined. The overall objectives of this study were (1) to characterize the GnIH receptor (GnIH-R) signaling pathway, (2) to evaluate potential interactions with gonadotropin releasing hormone type III receptor (GnRH-R-III) signaling, and (3) to determine the molecular mechanisms by which GnIH and GnRH regulate pituitary gonadotrope function during a reproductive cycle in the chicken. Using real-time PCR, we showed that in the chicken pituitary gland, GnIH-R mRNA levels fluctuate in an opposite manner to GnRH-R-III, with higher and lower levels observed during inactive and active reproductive stages, respectively. We demonstrated that the chicken GnIH-R signals by inhibiting adenylyl cyclase cAMP production, most likely by coupling to G(alphai). We also showed that this inhibition is sufficient to significantly reduce GnRH-induced cAMP responsive element (CRE) activation in a dose-dependent manner, and that the ratio of GnRH/GnIH receptors is a significant factor. We propose that in avian species, sexual maturation is characterized by a change in GnIH/GnRH receptor ratio, resulting in a switch in pituitary sensitivity from inhibitory (involving GnIH) to stimulatory (involving GnRH). In turn, decreasing GnIH-R signaling, combined with increasing GnRH-R-III signaling, results in significant increases in CRE activation, possibly initiating gonadotropin synthesis. PMID:20350548

  17. uPAR Targeted Radionuclide Therapy with 177Lu-DOTA-AE105 Inhibits Dissemination of Metastatic Prostate Cancer

    DEFF Research Database (Denmark)

    Persson, Morten; Juhl, Karina; Rasmussen, Palle;

    2014-01-01

    value of 100 nM in a competitive binding experiment. In vivo, uPAR targeted radionuclide therapy significantly reduced the number of metastatic lesions in the disseminated metastatic prostate cancer model, when compared to vehicle and nontargeted 177Lu groups (p bioluminescence imaging...... with bioluminescence imaging in a cohort of animals during the treatment study. In conclusion, uPAR targeted radiotherapy resulted in a significant reduction in the number of metastatic lesions in a human metastatic prostate cancer model. Furthermore, we have provided the first evidence of the potential...

  18. Presence of urokinase plasminogen activator, its inhibitor and receptor in small cell lung cancer and non-small cell lung cancer

    DEFF Research Database (Denmark)

    Pappot, H.; Pfeiffer, P.; Grøndahl Hansen, J.;

    1997-01-01

    Spreading of cancer cells is dependent on the combined action of several proteolytic enzymes, such as serine proteases, comprising the urokinase pathway of plasminogen activation. Previous studies of lung cancer indicate that expression, localization and prognostic impact of the components of the...... plasminogen activation system differ in the different non-small cell lung cancer (NSCLC) types, whereas the expression of the components in small cell lung cancer (SCLC) has only sparingly been investigated. In the present study we investigate the presence of the components of the plasminogen activation...... the clinical parameters. This is the first report of a study using a quantitative method to compare levels of the components of the plasminogen activation system in tissue extracts from the two major lung cancer groups. The study shows that uPA, PAI-1 and uPAR are present in SCLC-tissue, suggesting...

  19. A novel hydroxyfuroic acid compound as an insulin receptor activator – structure and activity relationship of a prenylindole moiety to insulin receptor activation

    Directory of Open Access Journals (Sweden)

    Tsai Henry J

    2009-07-01

    Full Text Available Abstract Background Diabetes Mellitus is a chronic disease and many patients of which require frequent subcutaneous insulin injection to maintain proper blood glucose levels. Due to the inconvenience of insulin administration, an orally active insulin replacement has long been a prime target for many pharmaceutical companies. Demethylasterriquinone (DMAQ B1, extracted from tropical fungus, Pseudomassaria sp., has been reported to be an orally effective agent at lowering circulating glucose levels in diabetic (db/db mice; however, the cytotoxicity associated with the quinone moiety has not been addressed thus far. Methods A series of hydroxyfuroic acid compounds were synthesized and tested for their efficacies at activating human insulin receptor. Cytotoxicity to Chinese hamster ovary cells, selectivities over insulin-like growth factor-1 (IGF-1, epidermal growth factor (EGF, and fibroblast growth factor (FGF receptors were examined in this study. Result and Conclusion This study reports a new non-quinone DMAQ B1 derivative, a hydroxyfuroic acid compound (D-410639, which is 128 fold less cytotoxic as DMAQ B1 and as potent as compound 2, a DMAQ B1 synthetic derivative from Merck, at activating human insulin receptor. D-410639 has little activation potential on IGF-1 receptor but is a moderate inhibitor to EGF receptor. Structure and activity relationship of the prenylindole moiety to insulin receptor activation is discussed.

  20. Dosimetry of 64Cu-DOTA-AE105, a PET tracer for uPAR imaging

    DEFF Research Database (Denmark)

    Persson, Morten; El Ali, Henrik H.; Binderup, Tina;

    2014-01-01

    the heart wall to receive the highest dose (0.0918mSv/MBq) followed by the liver (0.0815mSv/MBq), All other organs/tissue were estimated to receive doses in the range of 0.02–0.04mSv/MBq. The mean effective whole-body dose of 64Cu-DOTA-AE105 was estimated to be 0.0317mSv/MBq. Relatively good....... ConclusionFavorable dosimetry estimates together with previously reported uPAR PET data fully support human testing of 64Cu-DOTA-AE105....

  1. Liver X Receptor β and Peroxisome Proliferator-Activated Receptor δ regulate cholesterol transport in cholangiocytes

    Science.gov (United States)

    Xia, Xuefeng; Jung, Dongju; Webb, Paul; Zhang, Aijun; Zhang, Bin; Li, Lifei; Ayers, Stephen D.; Gabbi, Chiara; Ueno, Yoshiyuki; Gustafsson, Jan-Åke; Alpini, Gianfranco; Moore, David D.; LeSage, Gene D.

    2012-01-01

    Nuclear receptors (NRs) play crucial roles in regulation of hepatic cholesterol synthesis, metabolism and conversion to bile acids, but their actions in cholangiocytes have not been examined. In this study, we investigated the roles of NRs in cholangiocyte physiology and cholesterol metabolism and flux. We examined the expression of NRs and other genes involved in cholesterol homeostasis in freshly isolated and cultured rodent cholangiocytes and found that these cells express a specific subset of NRs which includes Liver X Receptor β (LXRβ) and Peroxisome Proliferator-Activated Receptor δ (PPARδ). Activation of LXRβ and/or PPARδ in cholangiocytes induces ATP-binding cassette cholesterol transporter A1 (ABCA1) and increases cholesterol export at the basolateral compartment in polarized cultured cholangiocytes. In addition, PPARδ induces Niemann Pick C1 Like L1 (NPC1L1), which imports cholesterol into cholangiocytes and is expressed on the apical cholangiocyte membrane, via specific interaction with a PPRE within the NPC1L1 promoter. Based on these studies, we propose that (i) LXRβ and PPARδ coordinate NPC1L1/ABCA1 dependent vectorial cholesterol flux from bile through cholangiocytes and (ii) manipulation of these processes may influence bile composition with important applications in cholestatic liver disease and gallstone disease, serious health concerns for humans. PMID:22729460

  2. DMPD: Receptor tyrosine kinases and the regulation of macrophage activation. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 14726496 Receptor tyrosine kinases and the regulation of macrophage activation. Cor...(.csml) Show Receptor tyrosine kinases and the regulation of macrophage activation. PubmedID 14726496 Title ...Receptor tyrosine kinases and the regulation of macrophage activation. Authors Co

  3. Detecting constitutive activity and protean agonism at cannabinoid-2 receptor.

    Science.gov (United States)

    Beltramo, Massimiliano; Brusa, Rossella; Mancini, Isabella; Scandroglio, Paola

    2010-01-01

    Since the cannabinoid system is involved in regulating several physiological functions such as locomotor activity, cognition, nociception, food intake, and inflammatory reaction, it has been the subject of intense study. Research on the pharmacology of this system has enormously progressed in the last 20years. One intriguing aspect that emerged from this research is that cannabinoid receptors (CBs) express a high level of constitutive activity. Investigation on this particular aspect of receptor pharmacology has largely focused on CB1, the CB subtype highly expressed in several brain regions. More recently, research on constitutive activity on the other CB subtype, CB2, was stimulated by the increasing interest on its potential as target for the treatment of various pathologies (e.g., pain and inflammation). There are several possible implications of constitutive activity on the therapeutic action of both agonists and antagonists, and consequently, it is important to have valuable methods to study this aspect of CB2 pharmacology. In the present chapter, we describe three methods to study constitutive activity at CB2: two classical methods relying on the detection of changes in cAMP level and GTPγS binding and a new one based on cell impedance measurement. In addition, we also included a section on detection of protean agonism, which is an interesting pharmacological phenomenon strictly linked to constitutive activity. PMID:21036225

  4. Bioluminescence imaging of estrogen receptor activity during breast cancer progression.

    Science.gov (United States)

    Vantaggiato, Cristina; Dell'Omo, Giulia; Ramachandran, Balaji; Manni, Isabella; Radaelli, Enrico; Scanziani, Eugenio; Piaggio, Giulia; Maggi, Adriana; Ciana, Paolo

    2016-01-01

    Estrogen receptors (ER) are known to play an important regulatory role in mammary gland development as well as in its neoplastic transformation. Although several studies highlighted the contribution of ER signaling in the breast transformation, little is known about the dynamics of ER state of activity during carcinogenesis due to the lack of appropriate models for measuring the extent of receptor signaling in time, in the same animal. To this aim, we have developed a reporter mouse model for the non-invasive in vivo imaging of ER activity: the ERE-Luc reporter mouse. ERE-Luc is a transgenic mouse generated with a firefly luciferase (Luc) reporter gene driven by a minimal promoter containing an estrogen responsive element (ERE). This model allows to measure receptor signaling in longitudinal studies by bioluminescence imaging (BLI). Here, we have induced sporadic mammary cancers by treating systemically ERE-Luc reporter mice with DMBA (9,10-dimethyl 1,2-benzanthracene) and measured receptor signaling by in vivo imaging in individual animals from early stage until a clinically palpable tumor appeared in the mouse breast. We showed that DMBA administration induces an increase of bioluminescence in the whole abdominal area 6 h after treatment, the signal rapidly disappears. Several weeks later, strong bioluminescence is observed in the area corresponding to the mammary glands. In vivo and ex vivo imaging analysis demonstrated that this bioluminescent signal is localized in the breast area undergoing neoplastic transformation. We conclude that this non-invasive assay is a novel relevant tool to identify the activation of the ER signaling prior the morphological detection of the neoplastic transformation.

  5. D1 dopamine receptor activity of anti-parkinsonian drugs.

    Science.gov (United States)

    Fici, G J; Wu, H; VonVoigtlander, P F; Sethy, V H

    1997-01-01

    Clinical and preclinical investigations suggest that stimulation of D1 dopamine receptors may be responsible for dyskinesias induced by dopamine agonist treatment of Parkinson's Disease (PD), and that these dyskinesias may be decreased by treatment with a D1 antagonist (clozapine). Therefore, the effects of dopamine agonists and antagonists have been investigated in a primary cerebellar granule cell model of cAMP formation that seems to be highly responsive to the D1 receptors. SKF 38393, lisuride, apomorphine, pergolide, dopamine, bromocriptine and 7-OH-DPAT showed concentration-dependent increases in cAMP formation, with EC50s (in microM) of 0.013, 0.053, 0.25, 1.04, 2.18, 50.9 and 54.4, respectively. SKF 38393, apomorphine, dopamine and pergolide had similar intrinsic activity (100%), while the intrinsic activities of 7-OH-DPAT, bromocriptine and lisuride were 28.0%, 20.7% and 17.2%, respectively. SCH 23390, a selective D1 dopamine receptor antagonist, blocked an increase in cAMP formation produced by EC50 concentrations of all of the dopamine agonists investigated in this study. Clozapine concentration-dependently blocked pergolide-induced increases in cAMP and was approximately 1700-fold less potent than SCH 23390 (IC50: 0.97 microM and 0.56 nM, respectively). U-95666A (1-1000 microM), selective for the D2 receptors, showed no significant effect on cAMP, while pramipexole (0.1-100 microM), a D3 preferring agonist, did not elevate cAMP. These data suggest that primary cerebellar granule cell cultures are an excellent model for measuring D1 dopamine receptor-mediated changes in cellular cAMP. The results are discussed with reference to the relationship between the D1 receptor-stimulated increase in cAMP formation and the induction of dyskinesia in humans by these anti-parkinsonian drugs. PMID:9126882

  6. Identification of Modulators of the Nuclear Receptor Peroxisome Proliferator-Activated Receptor α (PPARα) in a Mouse Liver Gene Expression Compendium

    Science.gov (United States)

    The nuclear receptor family member peroxisome proliferator-activated receptor α (PPARα) is activated by therapeutic hypolipidemic drugs and environmentally-relevant chemicals to regulate genes involved in lipid transport and catabolism. Chronic activation of PPARα in rodents inc...

  7. Phagocytic receptors activate and immune inhibitory receptor SIRPα inhibits phagocytosis through paxillin and cofilin.

    Science.gov (United States)

    Gitik, Miri; Kleinhaus, Rachel; Hadas, Smadar; Reichert, Fanny; Rotshenker, Shlomo

    2014-01-01

    The innate immune function of phagocytosis of apoptotic cells, tissue debris, pathogens, and cancer cells is essential for homeostasis, tissue repair, fighting infection, and combating malignancy. Phagocytosis is carried out in the central nervous system (CNS) by resident microglia and in both CNS and peripheral nervous system by recruited macrophages. While phagocytosis proceeds, bystander healthy cells protect themselves by sending a "do not eat me" message to phagocytes as CD47 on their surface ligates immune inhibitory receptor SIRPα on the surface of phagocytes and SIRPα then produces the signaling which inhibits phagocytosis. This helpful mechanism becomes harmful when tissue debris and unhealthy cells inhibit their own phagocytosis by employing the same mechanism. However, the inhibitory signaling that SIRPα produces has not been fully revealed. We focus here on how SIRPα inhibits the phagocytosis of the tissue debris "degenerated myelin" which hinders repair in axonal injury and neurodegenerative diseases. We tested whether SIRPα inhibits phagocytosis by regulating cytoskeleton function through paxillin and cofilin since (a) the cytoskeleton generates the mechanical forces that drive phagocytosis and (b) both paxillin and cofilin control cytoskeleton function. Paxillin and cofilin were transiently activated in microglia as phagocytosis was activated. In contrast, paxillin and cofilin were continuously activated and phagocytosis augmented in microglia in which SIRPα expression was knocked-down by SIRPα-shRNA. Further, levels of phagocytosis, paxillin activation, and cofilin activation positively correlated with one another. Taken together, these observations suggest a novel mechanism whereby paxillin and cofilin are targeted to control phagocytosis by both the activating signaling that phagocytic receptors produce by promoting the activation of paxillin and cofilin and the inhibiting signaling that immune inhibitory SIRPα produces by promoting the

  8. Phagocytic receptors activate and immune inhibitory receptor SIRPalpha inhibits phagocytosis through paxillin and cofilin

    Directory of Open Access Journals (Sweden)

    Miri eGitik

    2014-04-01

    Full Text Available The innate-immune function of phagocytosis of apoptotic cells, tissue-debris, pathogens and cancer cells is essential for homeostasis, tissue repair, fighting infection and combating malignancy. Phagocytosis is carried out in the CNS by resident microglia and in both CNS and PNS by recruited macrophages. While phagocytosis proceeds, bystander healthy cells protect themselves by sending a do not eat me message to phagocytes as CD47 on their surface ligates immune inhibitory receptor SIRPα on the surface of phagocytes and SIRPα then produces the signaling which inhibits phagocytosis. This helpful mechanism becomes harmful when tissue-debris and unhealthy cells inhibit their own phagocytosis by employing the same mechanism. However, the inhibitory signaling that SIRPα produces has not been fully revealed. We focus here on how SIRPα inhibits the phagocytosis of the tissue-debris degenerated-myelin which hinders repair in axonal injury and neurodegenerative diseases. We tested whether SIRPα inhibits phagocytosis by regulating cytoskeleton function through paxillin and cofilin since (a the cytoskeleton generates the mechanical forces that drive phagocytosis and (b both paxillin and cofilin control cytoskeleton function. Paxillin and cofilin were transiently activated in microglia as phagocytosis was activated. In contrast, paxillin and cofilin were continuously activated and phagocytosis augmented in microglia in which SIRPα expression was knocked-down by SIRPα-shRNA. Further, levels of phagocytosis, paxillin activation and cofilin activation positively correlated with one another. Taken together, these observations suggest a novel mechanism whereby paxillin and cofilin are targeted to control phagocytosis by both the activating signaling that phagocytic receptors produce by promoting the activation of paxillin and cofilin and the inhibiting signaling that immune inhibitory SIRPα produces by promoting the inactivation of paxillin and cofilin.

  9. UV ACTIVATION OF RECEPTOR TYROSINE KINASE-ACTIVITY

    NARCIS (Netherlands)

    COFFER, PJ; BURGERING, BMT; PEPPELENBOSCH, MP; BOS, JL; KRUIJER, W

    1995-01-01

    The exposure of mammalian cells to ultraviolet radiation (UV) may lead to DNA damage resulting in mutation and thus possibly cancer, while irradiation can further act as a potent tumour promoter. In addition UV induces p21ras-mediated signalling leading to activation of transcription factors such as

  10. FATTY ACIDS MODULATE TOLL-LIKE RECEPTOR 4 ACTIVATION THROUGH REGULATION OF RECEPTOR DIMERIZATION AND RECRUITMENT INTO LIPID RAFTS

    Science.gov (United States)

    The saturated fatty acids acylated on Lipid A of lipopolysaccharide (LPS) or bacterial lipoproteins play critical roles in ligand recognition and receptor activation for Toll-like Receptor 4 (TLR4) and TLR2. The results from our previous studies (J Biol Chem 2003, 2004) demonstrated that saturated ...

  11. Exercise reduces adipose tissue via cannabinoid receptor type 1 which is regulated by peroxisome proliferator-activated receptor-delta

    DEFF Research Database (Denmark)

    Yan, Zhen Cheng; Liu, Dao Yan; Zhang, Li Li;

    2007-01-01

    Obesity is one major cardiovascular risk factor. We tested effects of endurance exercise on cannabinoid receptor type 1 (CB1) and peroxisome proliferator-activated receptor-delta (PPAR-delta)-dependent pathways in adipose tissue. Male Wistar rats were randomly assigned to standard laboratory chow...

  12. Stimulation of cannabinoid receptor 2 (CB2 suppresses microglial activation

    Directory of Open Access Journals (Sweden)

    Fernandez Francisco

    2005-12-01

    Full Text Available Abstract Background Activated microglial cells have been implicated in a number of neurodegenerative disorders, including Alzheimer's disease (AD, multiple sclerosis (MS, and HIV dementia. It is well known that inflammatory mediators such as nitric oxide (NO, cytokines, and chemokines play an important role in microglial cell-associated neuron cell damage. Our previous studies have shown that CD40 signaling is involved in pathological activation of microglial cells. Many data reveal that cannabinoids mediate suppression of inflammation in vitro and in vivo through stimulation of cannabinoid receptor 2 (CB2. Methods In this study, we investigated the effects of a cannabinoid agonist on CD40 expression and function by cultured microglial cells activated by IFN-γ using RT-PCR, Western immunoblotting, flow cytometry, and anti-CB2 small interfering RNA (siRNA analyses. Furthermore, we examined if the stimulation of CB2 could modulate the capacity of microglial cells to phagocytise Aβ1–42 peptide using a phagocytosis assay. Results We found that the selective stimulation of cannabinoid receptor CB2 by JWH-015 suppressed IFN-γ-induced CD40 expression. In addition, this CB2 agonist markedly inhibited IFN-γ-induced phosphorylation of JAK/STAT1. Further, this stimulation was also able to suppress microglial TNF-α and nitric oxide production induced either by IFN-γ or Aβ peptide challenge in the presence of CD40 ligation. Finally, we showed that CB2 activation by JWH-015 markedly attenuated CD40-mediated inhibition of microglial phagocytosis of Aβ1–42 peptide. Taken together, these results provide mechanistic insight into beneficial effects provided by cannabinoid receptor CB2 modulation in neurodegenerative diseases, particularly AD.

  13. Methamphetamine Increases Locomotion and Dopamine Transporter Activity in Dopamine D5 Receptor-Deficient Mice

    OpenAIRE

    Seiji Hayashizaki; Shinobu Hirai; Yumi Ito; Yoshiko Honda; Yosefu Arime; Ichiro Sora; Haruo Okado; Tohru Kodama; Masahiko Takada

    2013-01-01

    Dopamine regulates the psychomotor stimulant activities of amphetamine-like substances in the brain. The effects of dopamine are mediated through five known dopamine receptor subtypes in mammals. The functional relevance of D5 dopamine receptors in the central nervous system is not well understood. To determine the functional relevance of D5 dopamine receptors, we created D5 dopamine receptor-deficient mice and then used these mice to assess the roles of D5 dopamine receptors in the behaviora...

  14. Behavioral meaningful opioidergic stimulation activates kappa receptor gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Teodorov, E. [Centro de Matemática, Computação e Cognição, Universidade Federal do ABC, São Paulo, SP (Brazil); Ferrari, M.F.R. [Departamento de Genética e Biologia Evolutiva, Instituto de Biociências, Universidade de São Paulo, São Paulo, SP (Brazil); Fior-Chadi, D.R. [Departamento de Fisiologia, Instituto de Biociências, Universidade de São Paulo, São Paulo, SP (Brazil); Camarini, R. [Departamento de Farmacologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, SP (Brazil); Felício, L.F. [Departamento de Patologia, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, São Paulo, SP (Brazil)

    2012-06-01

    The periaqueductal gray (PAG) has been reported to be a location for opioid regulation of pain and a potential site for behavioral selection in females. Opioid-mediated behavioral and physiological responses differ according to the activity of opioid receptor subtypes. The present study investigated the effects of the peripheral injection of the kappa-opioid receptor agonist U69593 into the dorsal subcutaneous region of animals on maternal behavior and on Oprk1 gene activity in the PAG of female rats. Female Wistar rats weighing 200-250 g at the beginning of the study were randomly divided into 2 groups for maternal behavior and gene expression experiments. On day 5, pups were removed at 7:00 am and placed in another home cage that was distant from their mother. Thirty minutes after removing the pups, the dams were treated with U69593 (0.15 mg/kg, sc) or 0.9% saline (up to 1 mL/kg) and after 30 min were evaluated in the maternal behavior test. Latencies in seconds for pup retrieval, grouping, crouching, and full maternal behavior were scored. The results showed that U69593 administration inhibited maternal behavior (P < 0.05) because a lower percentage of U69593 group dams showed retrieval of first pup, retrieving all pups, grouping, crouching and displaying full maternal behavior compared to the saline group. Opioid gene expression was evaluated using real-time reverse-transcription polymerase chain reaction (RT-PCR). A single injection of U69593 increased Oprk1 PAG expression in both virgin (P < 0.05) and lactating female rats (P < 0.01), with no significant effect on Oprm1 or Oprd1 gene activity. Thus, the expression of kappa-opioid receptors in the PAG may be modulated by single opioid receptor stimulation and behavioral meaningful opioidergic transmission in the adult female might occur simultaneously to specific changes in gene expression of kappa-opioid receptor subtype. This is yet another alert for the complex role of the opioid system in female

  15. Behavioral meaningful opioidergic stimulation activates kappa receptor gene expression

    International Nuclear Information System (INIS)

    The periaqueductal gray (PAG) has been reported to be a location for opioid regulation of pain and a potential site for behavioral selection in females. Opioid-mediated behavioral and physiological responses differ according to the activity of opioid receptor subtypes. The present study investigated the effects of the peripheral injection of the kappa-opioid receptor agonist U69593 into the dorsal subcutaneous region of animals on maternal behavior and on Oprk1 gene activity in the PAG of female rats. Female Wistar rats weighing 200-250 g at the beginning of the study were randomly divided into 2 groups for maternal behavior and gene expression experiments. On day 5, pups were removed at 7:00 am and placed in another home cage that was distant from their mother. Thirty minutes after removing the pups, the dams were treated with U69593 (0.15 mg/kg, sc) or 0.9% saline (up to 1 mL/kg) and after 30 min were evaluated in the maternal behavior test. Latencies in seconds for pup retrieval, grouping, crouching, and full maternal behavior were scored. The results showed that U69593 administration inhibited maternal behavior (P < 0.05) because a lower percentage of U69593 group dams showed retrieval of first pup, retrieving all pups, grouping, crouching and displaying full maternal behavior compared to the saline group. Opioid gene expression was evaluated using real-time reverse-transcription polymerase chain reaction (RT-PCR). A single injection of U69593 increased Oprk1 PAG expression in both virgin (P < 0.05) and lactating female rats (P < 0.01), with no significant effect on Oprm1 or Oprd1 gene activity. Thus, the expression of kappa-opioid receptors in the PAG may be modulated by single opioid receptor stimulation and behavioral meaningful opioidergic transmission in the adult female might occur simultaneously to specific changes in gene expression of kappa-opioid receptor subtype. This is yet another alert for the complex role of the opioid system in female

  16. Facilitation of neocortical presynaptic terminal development by NMDA receptor activation

    Directory of Open Access Journals (Sweden)

    Sceniak Michael P

    2012-02-01

    Full Text Available Abstract Background Neocortical circuits are established through the formation of synapses between cortical neurons, but the molecular mechanisms of synapse formation are only beginning to be understood. The mechanisms that control synaptic vesicle (SV and active zone (AZ protein assembly at developing presynaptic terminals have not yet been defined. Similarly, the role of glutamate receptor activation in control of presynaptic development remains unclear. Results Here, we use confocal imaging to demonstrate that NMDA receptor (NMDAR activation regulates accumulation of multiple SV and AZ proteins at nascent presynaptic terminals of visual cortical neurons. NMDAR-dependent regulation of presynaptic assembly occurs even at synapses that lack postsynaptic NMDARs. We also provide evidence that this control of presynaptic terminal development is independent of glia. Conclusions Based on these data, we propose a novel NMDAR-dependent mechanism for control of presynaptic terminal development in excitatory neocortical neurons. Control of presynaptic development by NMDARs could ultimately contribute to activity-dependent development of cortical receptive fields.

  17. The adipogenic acetyltransferase Tip60 targets activation function 1 of peroxisome proliferator-activated receptor gamma

    DEFF Research Database (Denmark)

    van Beekum, Olivier; Brenkman, Arjan B; Grøntved, Lars;

    2008-01-01

    The transcription factor peroxisome proliferator-activated receptor gamma (PPARgamma) plays a key role in the regulation of lipid and glucose metabolism in adipocytes, by regulating their differentiation, maintenance, and function. The transcriptional activity of PPARgamma is dictated by the set...... of proteins with which this nuclear receptor interacts under specific conditions. Here we identify the HIV-1 Tat-interacting protein 60 (Tip60) as a novel positive regulator of PPARgamma transcriptional activity. Using tandem mass spectrometry, we found that PPARgamma and the acetyltransferase Tip60 interact...

  18. Characterization of human endothelial cell urokinase-type plasminogen activator receptor protein and messenger RNA

    DEFF Research Database (Denmark)

    Barnathan, E S; Kuo, A; Karikó, K;

    1990-01-01

    Human umbilical vein endothelial cells in culture (HUVEC) express receptors for urokinase-type plasminogen activators (u-PA). The immunochemical nature of this receptor and its relationship to u-PA receptors expressed by other cell types is unknown. Cross-linking active site-blocked u-PA to HUVEC...

  19. Dopamine receptor activation increases HIV entry into primary human macrophages.

    Directory of Open Access Journals (Sweden)

    Peter J Gaskill

    Full Text Available Macrophages are the primary cell type infected with HIV in the central nervous system, and infection of these cells is a major component in the development of neuropathogenesis and HIV-associated neurocognitive disorders. Within the brains of drug abusers, macrophages are exposed to increased levels of dopamine, a neurotransmitter that mediates the addictive and reinforcing effects of drugs of abuse such as cocaine and methamphetamine. In this study we examined the effects of dopamine on HIV entry into primary human macrophages. Exposure to dopamine during infection increased the entry of R5 tropic HIV into macrophages, irrespective of the concentration of the viral inoculum. The entry pathway affected was CCR5 dependent, as antagonizing CCR5 with the small molecule inhibitor TAK779 completely blocked entry. The effect was dose-dependent and had a steep threshold, only occurring above 108 M dopamine. The dopamine-mediated increase in entry required dopamine receptor activation, as it was abrogated by the pan-dopamine receptor antagonist flupenthixol, and could be mediated through both subtypes of dopamine receptors. These findings indicate that the effects of dopamine on macrophages may have a significant impact on HIV pathogenesis. They also suggest that drug-induced increases in CNS dopamine may be a common mechanism by which drugs of abuse with distinct modes of action exacerbate neuroinflammation and contribute to HIV-associated neurocognitive disorders in infected drug abusers.

  20. NMDA receptor subunit expression and PAR2 receptor activation in colospinal afferent neurons (CANs during inflammation induced visceral hypersensitivity

    Directory of Open Access Journals (Sweden)

    Caudle Robert M

    2009-09-01

    Full Text Available Abstract Background Visceral hypersensitivity is a clinical observation made when diagnosing patients with functional bowel disorders. The cause of visceral hypersensitivity is unknown but is thought to be attributed to inflammation. Previously we demonstrated that a unique set of enteric neurons, colospinal afferent neurons (CANs, co-localize with the NR1 and NR2D subunits of the NMDA receptor as well as with the PAR2 receptor. The aim of this study was to determine if NMDA and PAR2 receptors expressed on CANs contribute to visceral hypersensitivity following inflammation. Recently, work has suggested that dorsal root ganglion (DRG neurons expressing the transient receptor potential vanilloid-1 (TRPV1 receptor mediate inflammation induced visceral hypersensitivity. Therefore, in order to study CAN involvement in visceral hypersensitivity, DRG neurons expressing the TRPV1 receptor were lesioned with resiniferatoxin (RTX prior to inflammation and behavioural testing. Results CANs do not express the TRPV1 receptor; therefore, they survive following RTX injection. RTX treatment resulted in a significant decrease in TRPV1 expressing neurons in the colon and immunohistochemical analysis revealed no change in peptide or receptor expression in CANs following RTX lesioning as compared to control data. Behavioral studies determined that both inflamed non-RTX and RTX animals showed a decrease in balloon pressure threshold as compared to controls. Immunohistochemical analysis demonstrated that the NR1 cassettes, N1 and C1, of the NMDA receptor on CANs were up-regulated following inflammation. Furthermore, inflammation resulted in the activation of the PAR2 receptors expressed on CANs. Conclusion Our data show that inflammation causes an up-regulation of the NMDA receptor and the activation of the PAR2 receptor expressed on CANs. These changes are associated with a decrease in balloon pressure in response to colorectal distension in non-RTX and RTX lesioned

  1. Genome-Wide Profiling of Liver X Receptor, Retinoid X Receptor, and Peroxisome Proliferator-Activated Receptor α in Mouse Liver Reveals Extensive Sharing of Binding Sites

    DEFF Research Database (Denmark)

    Boergesen, Michael; Pedersen, Thomas Åskov; Gross, Barbara;

    2012-01-01

    The liver X receptors (LXRs) are nuclear receptors that form permissive heterodimers with retinoid X receptor (RXR) and are important regulators of lipid metabolism in the liver. We have recently shown that RXR agonist-induced hypertriglyceridemia and hepatic steatosis in mice are dependent on LXRs...... increases the genomic binding of RXR, whereas the LXR agonist T0901317 greatly increases both LXR and RXR binding. Functional annotation of putative direct LXR target genes revealed a significant association with classical LXR-regulated pathways as well as peroxisome proliferator-activated receptor (PPAR...

  2. "Mirror image" antagonists of thrombin-induced platelet activation based on thrombin receptor structure.

    OpenAIRE

    Hung, D. T.; Vu, T K; Wheaton, V I; Charo, I F; Nelken, N A; Esmon, N; Esmon, C T; Coughlin, S R

    1992-01-01

    Platelet activation by thrombin plays a critical role in hemostasis and thrombosis. Based on structure-activity studies of a cloned platelet thrombin receptor, we designed two "mirror image" antagonists of thrombin and thrombin receptor function. First, "uncleavable" peptides mimicking the receptor domain postulated to interact with thrombin were found to be potent thrombin inhibitors. Second, proteolytically inactive mutant thrombins designed to bind but not cleave the thrombin receptor were...

  3. The glucagon-like peptide 1 receptor agonist enhances intrinsic peroxisome proliferator-activated receptor γ activity in endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Onuma, Hirohisa; Inukai, Kouichi, E-mail: kinukai@ks.kyorin-u.ac.jp; Kitahara, Atsuko; Moriya, Rie; Nishida, Susumu; Tanaka, Toshiaki; Katsuta, Hidenori; Takahashi, Kazuto; Sumitani, Yoshikazu; Hosaka, Toshio; Ishida, Hitoshi

    2014-08-22

    Highlights: • PPARγ activation was involved in the GLP-1-mediated anti-inflammatory action. • Exendin-4 enhanced endogenous PPARγ transcriptional activity in HUVECs. • H89, a PKA inhibitor, abolished GLP-1-induced PPARγ enhancement. • The anti-inflammatory effects of GLP-1 may be explained by PPARγ activation. - Abstract: Recent studies have suggested glucagon-like peptide-1 (GLP-1) signaling to exert anti-inflammatory effects on endothelial cells, although the precise underlying mechanism remains to be elucidated. In the present study, we investigated whether PPARγ activation is involved in the GLP-1-mediated anti-inflammatory action on endothelial cells. When we treated HUVEC cells with 0.2 ng/ml exendin-4, a GLP-1 receptor agonist, endogenous PPARγ transcriptional activity was significantly elevated, by approximately 20%, as compared with control cells. The maximum PPARγ activity enhancing effect of exendin-4 was observed 12 h after the initiation of incubation with exendin-4. As H89, a PKA inhibitor, abolished GLP-1-induced PPARγ enhancement, the signaling downstream from GLP-1 cross-talk must have been involved in PPARγ activation. In conclusion, our results suggest that GLP-1 has the potential to induce PPARγ activity, partially explaining the anti-inflammatory effects of GLP-1 on endothelial cells. Cross-talk between GLP-1 signaling and PPARγ activation would have major impacts on treatments for patients at high risk for cardiovascular disease.

  4. Influence of metabotropic glutamate receptor agonists on the inhibitory effects of adenosine A1 receptor activation in the rat hippocampus

    OpenAIRE

    de Mendonça, Alexandre; Ribeiro, J. A.

    1997-01-01

    Glutamate and other amino acids are the main excitatory neurotransmitters in many brain regions, including the hippocampus, by activating ion channel-coupled glutamate receptors, as well as metabotropic receptors linked to G proteins and second messenger systems. Several conditions which promote the release of glutamate, like frequency stimulation and hypoxia, also lead to an increase in the extracellular levels of the important neuromodulator, adenosine. We studied whether the activation of ...

  5. Role of Peroxisome Proliferator-Activated Receptors in Inflammation Control

    Directory of Open Access Journals (Sweden)

    Jihan Youssef

    2004-01-01

    Full Text Available Peroxisome proliferator-activated receptors (PPARs were discovered over a decade ago, and were classified as orphan members of the nuclear receptor superfamily. To date, three PPAR subtypes have been discovered and characterized (PPARα, β/δ, γ. Different PPAR subtypes have been shown to play crucial roles in important diseases and conditions such as obesity, diabetes, atherosclerosis, cancer, and fertility. Among the most studied roles of PPARs is their involvement in inflammatory processes. Numerous studies have revealed that agonists of PPARα and PPARγ exert anti-inflammatory effects both in vitro and in vivo. Using the carrageenan-induced paw edema model of inflammation, a recent study in our laboratories showed that these agonists hinder the initiation phase, but not the late phase of the inflammatory process. Furthermore, in the same experimental model, we recently also observed that activation of PPARδ exerted an anti-inflammatory effect. Despite the fact that exclusive dependence of these effects on PPARs has been questioned, the bulk of evidence suggests that all three PPAR subtypes, PPARα,δ,γ, play a significant role in controlling inflammatory responses. Whether these subtypes act via a common mechanism or are independent of each other remains to be elucidated. However, due to the intensity of research efforts in this area, it is anticipated that these efforts will result in the development of PPAR ligands as therapeutic agents for the treatment of inflammatory diseases.

  6. Antitussive activity of Withania somnifera and opioid receptors.

    Science.gov (United States)

    Nosálová, Gabriela; Sivová, Veronika; Ray, Bimalendu; Fraňová, Soňa; Ondrejka, Igor; Flešková, Dana

    2015-01-01

    Arabinogalactan is a polysaccharide isolated from the roots of the medicinal plant Withania somnifera L. It contains 65% arabinose and 18% galactose. The aim of the present study was to evaluate the antitussive activity of arabinogalactan in conscious, healthy adult guinea pigs and the role of the opioid pathway in the antitussive action. A polysaccharide extract was given orally in a dose of 50 mg/kg. Cough was induced by an aerosol of citric acid in a concentration 0.3 mol/L, generated by a jet nebulizer into a plethysmographic chamber. The intensity of cough response was defined as the number of cough efforts counted during a 3-min exposure to the aerosol. The major finding was that arabinogalactan clearly suppressed the cough reflex; the suppression was comparable with that of codeine that was taken as a reference drug. The involvement of the opioid system was tested with the use of a blood-brain barrier penetrable, naloxone hydrochloride, and non-penetrable, naloxone methiodide, to distinguish between the central and peripheral mu-opioid receptor pathways. Both opioid antagonists acted to reverse the arabinogalactan-induced cough suppression; the reversion was total over time with the latter antagonist. We failed to confirm the presence of a bronchodilating effect of the polysaccharide, which could be involved in its antitussive action. We conclude that the polysaccharide arabinogalactan from Withania somnifera has a distinct antitussive activity consisting of cough suppression and that this action involves the mu-opioid receptor pathways.

  7. Involvement of Activating NK Cell Receptors and Their Modulation in Pathogen Immunity

    Directory of Open Access Journals (Sweden)

    Francesco Marras

    2011-01-01

    Full Text Available Natural Killer (NK cells are endowed with cell-structure-sensing receptors providing inhibitory protection from self-destruction (inhibitory NK receptors, iNKRs, including killer inhibitory receptors and other molecules and rapid triggering potential leading to functional cell activation by Toll-like receptors (TLRs, cytokine receptors, and activating NK cell receptors including natural cytotoxicity receptors (NCRs, i.e., NKp46, NKp46, and NKp44. NCR and NKG2D recognize ligands on infected cells which may be endogenous or may directly bind to some structures derived from invading pathogens. In this paper, we address the known direct or indirect interactions between activating receptors and pathogens and their expression during chronic HIV and HCV infections.

  8. ERK5 activation by Gq-coupled muscarinic receptors is independent of receptor internalization and β-arrestin recruitment.

    Directory of Open Access Journals (Sweden)

    Guzmán Sánchez-Fernández

    Full Text Available G-protein-coupled receptors (GPCRs are known to activate both G protein- and β-arrestin-dependent signalling cascades. The initiation of mitogen-activated protein kinase (MAPK pathways is a key downstream event in the control of cellular functions including proliferation, differentiation, migration and apoptosis. Both G proteins and β-arrestins have been reported to mediate context-specific activation of ERK1/2, p38 and JNK MAPKs. Recently, the activation of ERK5 MAPK by Gq-coupled receptors has been described to involve a direct interaction between Gαq and two novel effectors, PKCζ and MEK5. However, the possible contribution of β-arrestin towards this pathway has not yet been addressed. In the present work we sought to investigate the role of receptor internalization processes and β-arrestin recruitment in the activation of ERK5 by Gq-coupled GPCRs. Our results show that ERK5 activation is independent of M1 or M3 muscarinic receptor internalization. Furthermore, we demonstrate that phosphorylation-deficient muscarinic M1 and M3 receptors are still able to fully activate the ERK5 pathway, despite their reported inability to recruit β-arrestins. Indeed, the overexpression of Gαq, but not that of β-arrestin1 or β-arrestin2, was found to potently enhance ERK5 activation by GPCRs, whereas silencing of β-arrestin2 expression did not affect the activation of this pathway. Finally, we show that a β-arrestin-biased mutant form of angiotensin II (SII; Sar1-Ile4-Ile8 AngII failed to promote ERK5 phosphorylation in primary cardiac fibroblasts, as compared to the natural ligand. Overall, this study shows that the activation of ERK5 MAPK by model Gq-coupled GPCRs does not depend on receptor internalization, β-arrestin recruitment or receptor phosphorylation but rather is dependent on Gαq-signalling.

  9. Relationship between somatostatin receptors and activation of hepatic stellate cells

    Institute of Scientific and Technical Information of China (English)

    潘勤; 李定国; 陆汉明; 陆良勇; 尤汉宁; 徐芹芳

    2004-01-01

    Background Somafostatin receptors (SSTRs) have been suggested to involve in mediating the effect of somatostatin on hepatic stellate cells (HSCs) in an activation-dependent way. We, therefore, try to investigate the relationship between expression of SSTRs and activation of rat HSCs.Methods HSCs were isolated from rats by in situ perfusion and single-step density gradient centrifugation.SSTR1-5 mRNA levels in the differentiated first passage HSCs were detected by means of a reverse transcription polymerase chain reaction. On the other hand, hepatic fibrosis was induced in adult male Sprague-Dawley rats by carbon tetrachloride intoxication, and the expression of SSTR1-5 in normal as well as fibrotic livers was measured by immunohistochemical staining.Results SSTR mRNA and SSTR could not be found in freshly isolated rat HSCs or normal rat liver. However, SSTR1-3 mRNA appeared as HSCs became wholly activated, and could also be identified on the membrane of activated HSCs in the perisinusoid space, fibrous septa, etc.Conclusion The expression of SSTR1-3 in the rat HSC is closely related to its activation. This may reflect one of the main negative regulation mechanisms in the course of HSC activation.

  10. The novel plasminogen receptor, plasminogen receptor(KT) (Plg-R(KT)), regulates catecholamine release.

    Science.gov (United States)

    Bai, Hongdong; Baik, Nagyung; Kiosses, William B; Krajewski, Stan; Miles, Lindsey A; Parmer, Robert J

    2011-09-23

    Neurotransmitter release by catecholaminergic cells is negatively regulated by prohormone cleavage products formed from plasmin-mediated proteolysis. Here, we investigated the expression and subcellular localization of Plg-R(KT), a novel plasminogen receptor, and its role in catecholaminergic cell plasminogen activation and regulation of catecholamine release. Prominent staining with anti-Plg-R(KT) mAb was observed in adrenal medullary chromaffin cells in murine and human tissue. In Western blotting, Plg-R(KT) was highly expressed in bovine adrenomedullary chromaffin cells, human pheochromocytoma tissue, PC12 pheochromocytoma cells, and murine hippocampus. Expression of Plg-R(KT) fused in-frame to GFP resulted in targeting of the GFP signal to the cell membrane. Phase partitioning, co-immunoprecipitation with urokinase-type plasminogen activator receptor (uPAR), and FACS analysis with antibody directed against the C terminus of Plg-R(KT) were consistent with Plg-R(KT) being an integral plasma membrane protein on the surface of catecholaminergic cells. Cells stably overexpressing Plg-R(KT) exhibited substantial enhancement of plasminogen activation, and antibody blockade of non-transfected PC12 cells suppressed plasminogen activation. In functional secretion assays, nicotine-evoked [(3)H]norepinephrine release from cells overexpressing Plg-R(KT) was markedly decreased (by 51 ± 2%, p < 0.001) when compared with control transfected cells, and antibody blockade increased [(3)H]norepinephrine release from non-transfected PC12 cells. In summary, Plg-R(KT) is present on the surface of catecholaminergic cells and functions to stimulate plasminogen activation and modulate catecholamine release. Plg-R(KT) thus represents a new mechanism and novel control point for regulating the interface between plasminogen activation and neurosecretory cell function.

  11. Identification of key residues involved in the activation and signaling properties of dopamine D3 receptor.

    Science.gov (United States)

    Kota, Kokila; Kuzhikandathil, Eldo V; Afrasiabi, Milad; Lacy, Brett; Kontoyianni, Maria; Crider, A Michael; Song, Daniel

    2015-09-01

    The dopamine D3 receptor exhibits agonist-dependent tolerance and slow response termination (SRT) signaling properties that distinguish it from the closely-related D2 receptors. While amino acid residues important for D3 receptor ligand binding have been identified, the residues involved in activation of D3 receptor signaling and induction of signaling properties have not been determined. In this paper, we used cis and trans isomers of a novel D3 receptor agonist, 8-OH-PBZI, and site-directed mutagenesis to identify key residues involved in D3 receptor signaling function. Our results show that trans-8-OH-PBZI, but not cis-8-OH-PBZI, elicit the D3 receptor tolerance and SRT properties. We show that while both agonists require a subset of residues in the orthosteric binding site of D3 receptors for activation of the receptor, the ability of the two isomers to differentially induce tolerance and SRT is mediated by interactions with specific residues in the sixth transmembrane helix and third extracellular loop of the D3 receptor. We also show that unlike cis-8-OH-PBZI, which is a partial agonist at the dopamine D2S receptor and full agonist at dopamine D2L receptor, trans-8-OH-PBZI is a full agonist at both D2S and D2L receptors. The different effect of the two isomers on D3 receptor signaling properties and D2S receptor activation correlated with differential effects of the isomers on agonist-induced mouse locomotor activity. The two isomers of 8-OH-PBZI represent novel pharmacological tools for in silico D3 and D2 receptor homology modeling and for determining the role of D3 receptor tolerance and SRT properties in signaling and behavior. PMID:26116441

  12. Activation of glucocorticoid receptors increases 5-HT2A receptor levels

    DEFF Research Database (Denmark)

    Trajkovska, Viktorija; Kirkegaard, Lisbeth; Krey, Gesa;

    2009-01-01

    of depression is unknown. In mice with altered glucocorticoid receptor (GR) expression we investigated 5-HT2A receptor levels by Western blot and 3H-MDL100907 receptor binding. Serotonin fibre density was analyzed by stereological quantification of serotonin transporter immunopositive fibers. To establish...... in dorsal hippocampus (77 +/- 35%, p

  13. Monoclonal antibodies to the human insulin receptor that activate glucose transport but not insulin receptor kinase activity.

    OpenAIRE

    Forsayeth, J R; Caro, J F; Sinha, M K; Maddux, B A; Goldfine, I D

    1987-01-01

    Three mouse monoclonal antibodies were produced that reacted with the alpha subunit of the human insulin receptor. All three both immunoprecipitated 125I-labeled insulin receptors from IM-9 lymphocytes and competitively inhibited 125I-labeled insulin binding to its receptor. Unlike insulin, the antibodies failed to stimulate receptor autophosphorylation in both intact IM-9 lymphocytes and purified human placental insulin receptors. Moreover, unlike insulin, the antibodies failed to stimulate ...

  14. RELATIONSHIP BETWEEN SOMATOSTATIN RECEPTORS AND ACTIVATION OF HEPATIC STELLATE CELL

    Institute of Scientific and Technical Information of China (English)

    潘勤; 李定国; 陆汉明; 尤汉宁; 徐芹芳; 陆良勇

    2004-01-01

    Objective To investigate the relationship between expression of somatostatin receptors (SSTRs) and activation of rat hepatic stellate cell (HSC). Methods HSCs were isolated from rats by in situ perfusion and single-step density gradient centrifugation, and then SSTR1 ~5 mRNA levels in the differentiated first passage HSCs were detected by means of reverse transcription polymerase chain reaction. On the other hand, hepatic fibrosis was induced in adult male Sprague-Dawley rats by carbon tetrachloride intoxication, and the expression of SSTR1 ~5 in normal as well as fibrotic liver was measured by immunohistochemical staining. Results SSTR mRNA and SSTR could not be found in freshly isolated rat HSCs and normal rat liver. But SSTR1~3 mRNA appeared as HSCs became wholly activated, and SSTR1 ~3 could also be identified on the membrane of activated HSCs in the perisinusoid space, fibrous septa, etc Conclusion The expression of SSTR1~3 in the rat HSC is closely related to its activation. This may reflect one of the main negative regulation mechanisms in the course of HSC activation.

  15. New Target Genes for the Peroxisome Proliferator-Activated Receptor-γ (PPARγ Antitumour Activity: Perspectives from the Insulin Receptor

    Directory of Open Access Journals (Sweden)

    Daniela P. Foti

    2009-01-01

    Full Text Available The insulin receptor (IR plays a crucial role in mediating the metabolic and proliferative functions triggered by the peptide hormone insulin. There is considerable evidence that abnormalities in both IR expression and function may account for malignant transformation and tumour progression in some human neoplasias, including breast cancer. PPARγ is a ligand-activated, nuclear hormone receptor implicated in many pleiotropic biological functions related to cell survival and proliferation. In the last decade, PPARγ agonists—besides their known action and clinical use as insulin sensitizers—have proved to display a wide range of antineoplastic effects in cells and tissues expressing PPARγ, leading to intensive preclinical research in oncology. PPARγ and activators affect tumours by different mechanisms, involving cell proliferation and differentiation, apoptosis, antiinflammatory, and antiangiogenic effects. We recently provided evidence that PPARγ and agonists inhibit IR by non canonical, DNA-independent mechanisms affecting IR gene transcription. We conclude that IR may be considered a new PPARγ “target” gene, supporting a potential use of PPARγ agonists as antiproliferative agents in selected neoplastic tissues that overexpress the IR.

  16. Diabetes or peroxisome proliferator-activated receptor alpha agonist increases mitochondrial thioesterase I activity in heart

    Science.gov (United States)

    Peroxisome proliferator-activated receptor alpha (PPAR alpha) is a transcriptional regulator of the expression of mitochondrial thioesterase I (MTE-I) and uncoupling protein 3 (UCP3), which are induced in the heart at the mRNA level in response to diabetes. Little is known about the regulation of pr...

  17. Pharmacological activation of mGlu4 metabotropic glutamate receptors inhibits the growth of medulloblastomas

    NARCIS (Netherlands)

    L. Iacovelli; A. Arcella; G. Battaglia; S. Pazzaglia; E. Aronica; P. Spinsanti; A. Caruso; E. de Smaele; A. Saran; A. Gulino; M. D'Onofrio; F. Giangaspero; F. Nicoletti

    2006-01-01

    Moving from the evidence that activation of type 4 metabotropic glutamate (mGlu4) receptors inhibits proliferation and promotes differentiation of cerebellar granule cell neuroprogenitors, we examined the expression and function of mGlu4 receptors in medulloblastoma cells. mGlu4 receptors were expre

  18. Guidance Receptors in the Nervous and Cardiovascular Systems.

    Science.gov (United States)

    Rubina, K A; Tkachuk, V A

    2015-10-01

    Blood vessels and nervous fibers grow in parallel, for they express similar receptors for chemokine substances. Recently, much attention is being given to studying guidance receptors and their ligands besides the growth factors, cytokines, and chemokines necessary to form structures in the nervous and vascular systems. Such guidance molecules determine trajectory for growing axons and vessels. Guidance molecules include Ephrins and their receptors, Neuropilins and Plexins as receptors for Semaphorins, Robos as receptors for Slit-proteins, and UNC5B receptors binding Netrins. Apart from these receptors and their ligands, urokinase and its receptor (uPAR) and T-cadherin are also classified as guidance molecules. The urokinase system mediates local proteolysis at the leading edge of cells, thereby providing directed migration. T-cadherin is a repellent molecule that regulates the direction of growing axons and blood vessels. Guidance receptors also play an important role in the diseases of the nervous and cardiovascular systems.

  19. Developmental stability of taurine's activation on glycine receptors in cultured neurons of rat auditory cortex.

    Science.gov (United States)

    Tang, Zheng-Quan; Lu, Yun-Gang; Chen, Lin

    2008-01-01

    Taurine is an endogenous amino acid that can activate glycine and/or gamma-aminobutyric acid type A (GABA(A)) receptors in the central nervous system. During natural development, taurine's receptor target undergoes a shift from glycine receptors to GABA(A) receptors in cortical neurons. Here, we demonstrate that taurine's receptor target in cortical neurons remains stable during in vitro development. With whole-cell patch-clamp recordings, we found that taurine always activated glycine receptors, rather than GABA(A) receptors, in neurons of rat auditory cortex cultured for 5-22 days. Our results suggest that the functional sensitivity of glycine and GABA(A) receptors to taurine is critically regulated by their developmental environments.

  20. Peroxisome Proliferator-Activated Receptor-γ in Thyroid Autoimmunity

    Directory of Open Access Journals (Sweden)

    Silvia Martina Ferrari

    2015-01-01

    Full Text Available Peroxisome proliferator-activated receptor- (PPAR- γ expression has been shown in thyroid tissue from patients with thyroiditis or Graves’ disease and furthermore in the orbital tissue of patients with Graves’ ophthalmopathy (GO, such as in extraocular muscle cells. An increasing body of evidence shows the importance of the (C-X-C motif receptor 3 (CXCR3 and cognate chemokines (C-X-C motif ligand (CXCL9, CXCL10, and CXCL11, in the T helper 1 immune response and in inflammatory diseases such as thyroid autoimmune disorders. PPAR-γ agonists show a strong inhibitory effect on the expression and release of CXCR3 chemokines, in vitro, in various kinds of cells, such as thyrocytes, and in orbital fibroblasts, preadipocytes, and myoblasts from patients with GO. Recently, it has been demonstrated that rosiglitazone is involved in a higher risk of heart failure, stroke, and all-cause mortality in old patients. On the contrary, pioglitazone has not shown these effects until now; this favors pioglitazone for a possible use in patients with thyroid autoimmunity. However, further studies are ongoing to explore the use of new PPAR-γ agonists in the treatment of thyroid autoimmune disorders.

  1. Full and partial agonists of thromboxane prostanoid receptor unveil fine tuning of receptor superactive conformation and G protein activation.

    Directory of Open Access Journals (Sweden)

    Valérie Capra

    Full Text Available The intrahelical salt bridge between E/D(3.49 and R(3.50 within the E/DRY motif on helix 3 (H3 and the interhelical hydrogen bonding between the E/DRY and residues on H6 are thought to be critical in stabilizing the class A G protein-coupled receptors in their inactive state. Removal of these interactions is expected to generate constitutively active receptors. This study examines how neutralization of E(3.49/6.30 in the thromboxane prostanoid (TP receptor alters ligand binding, basal, and agonist-induced activity and investigates the molecular mechanisms of G protein activation. We demonstrate here that a panel of full and partial agonists showed an increase in affinity and potency for E129V and E240V mutants. Yet, even augmenting the sensitivity to detect constitutive activity (CA with overexpression of the receptor or the G protein revealed resistance to an increase in basal activity, while retaining fully the ability to cause agonist-induced signaling. However, direct G protein activation measured through bioluminescence resonance energy transfer (BRET indicates that these mutants more efficiently communicate and/or activate their cognate G proteins. These results suggest the existence of additional constrains governing the shift of TP receptor to its active state, together with an increase propensity of these mutants to agonist-induced signaling, corroborating their definition as superactive mutants. The particular nature of the TP receptor as somehow "resistant" to CA should be examined in the context of its pathophysiological role in the cardiovascular system. Evolutionary forces may have favored regulation mechanisms leading to low basal activity and selected against more highly active phenotypes.

  2. Structural motifs of importance for the constitutive activity of the orphan 7TM receptor EBI2: analysis of receptor activation in the absence of an agonist

    DEFF Research Database (Denmark)

    Benned-Jensen, Tau; Rosenkilde, Mette M

    2008-01-01

    The Epstein-Barr induced receptor 2 (EBI2) is a lymphocyte-expressed orphan seven transmembrane-spanning (7TM) receptor that signals constitutively through Galphai, as shown, for instance by guanosine 5'-O-(3-thio)triphosphate incorporation. Two regions of importance for the constitutive activity...

  3. Complement Component C3 Binds to Activated Normal Platelets without Preceding Proteolytic Activation and Promotes Binding to Complement Receptor 1

    OpenAIRE

    Osama A Hamad; Nilsson, Per H.; Wouters, Diana; Lambris, John D.; Ekdahl, Kristina N.; Nilsson, Bo

    2010-01-01

    It has been reported that complement is activated on the surface of activated platelets, despite the presence of multiple regulators of complement activation. To reinvestigate the mechanisms by which activated platelets bind to complement components, the presence of complement proteins on the surfaces of nonactivated and thrombin receptor-activating peptide-activated platelets was analyzed by flow cytometry and Western blot analyses. C1q, C4, C3, and C9 were found to bind to thrombin receptor...

  4. Signaling Mechanism of Cannabinoid Receptor-2 Activation-Induced β-Endorphin Release.

    Science.gov (United States)

    Gao, Fang; Zhang, Ling-Hong; Su, Tang-Feng; Li, Lin; Zhou, Rui; Peng, Miao; Wu, Cai-Hua; Yuan, Xiao-Cui; Sun, Ning; Meng, Xian-Fang; Tian, Bo; Shi, Jing; Pan, Hui-Lin; Li, Man

    2016-08-01

    Activation of cannabinoid receptor-2 (CB2) results in β-endorphin release from keratinocytes, which then acts on primary afferent neurons to inhibit nociception. However, the underlying mechanism is still unknown. The CB2 receptor is generally thought to couple to Gi/o to inhibit cAMP production, which cannot explain the peripheral stimulatory effects of CB2 receptor activation. In this study, we found that in a keratinocyte cell line, the Gβγ subunits from Gi/o, but not Gαs, were involved in CB2 receptor activation-induced β-endorphin release. Inhibition of MAPK kinase, but not PLC, abolished CB2 receptor activation-induced β-endorphin release. Also, CB2 receptor activation significantly increased intracellular Ca(2+). Treatment with BAPTA-AM or thapsigargin blocked CB2 receptor activation-induced β-endorphin release. Using a rat model of inflammatory pain, we showed that the MAPK kinase inhibitor PD98059 abolished the peripheral effect of the CB2 receptor agonist on nociception. We thus present a novel mechanism of CB2 receptor activation-induced β-endorphin release through Gi/o-Gβγ-MAPK-Ca(2+) signaling pathway. Our data also suggest that stimulation of MAPK contributes to the peripheral analgesic effect of CB2 receptor agonists. PMID:26108183

  5. Peroxisome Proliferator-Activated Receptors in Female Reproduction and Fertility.

    Science.gov (United States)

    Vitti, Maurizio; Di Emidio, Giovanna; Di Carlo, Michela; Carta, Gaspare; Antonosante, Andrea; Artini, Paolo Giovanni; Cimini, Annamaria; Tatone, Carla; Benedetti, Elisabetta

    2016-01-01

    Reproductive functions may be altered by the exposure to a multitude of endogenous and exogenous agents, drug or environmental pollutants, which are known to affect gene transcription through the peroxisome proliferator-activated receptors (PPARs) activation. PPARs act as ligand activated transcription factors and regulate metabolic processes such as lipid and glucose metabolism, energy homeostasis, inflammation, and cell proliferation and differentiation. All PPARs isotypes are expressed along the hypothalamic-pituitary-gonadal axis and are strictly involved in reproductive functions. Since female fertility and energy metabolism are tightly interconnected, the research on female infertility points towards the exploration of potential PPARs activating/antagonizing compounds, mainly belonging to the class of thiazolidinediones (TZDs) and fibrates, as useful agents for the maintenance of metabolic homeostasis in women with ovarian dysfunctions. In the present review, we discuss the recent evidence about PPARs expression in the hypothalamic-pituitary-gonadal axis and their involvement in female reproduction. Finally, the therapeutic potential of their manipulation through several drugs is also discussed. PMID:27559343

  6. Peroxisome Proliferator-Activated Receptors in Female Reproduction and Fertility

    Science.gov (United States)

    Carta, Gaspare; Artini, Paolo Giovanni

    2016-01-01

    Reproductive functions may be altered by the exposure to a multitude of endogenous and exogenous agents, drug or environmental pollutants, which are known to affect gene transcription through the peroxisome proliferator-activated receptors (PPARs) activation. PPARs act as ligand activated transcription factors and regulate metabolic processes such as lipid and glucose metabolism, energy homeostasis, inflammation, and cell proliferation and differentiation. All PPARs isotypes are expressed along the hypothalamic-pituitary-gonadal axis and are strictly involved in reproductive functions. Since female fertility and energy metabolism are tightly interconnected, the research on female infertility points towards the exploration of potential PPARs activating/antagonizing compounds, mainly belonging to the class of thiazolidinediones (TZDs) and fibrates, as useful agents for the maintenance of metabolic homeostasis in women with ovarian dysfunctions. In the present review, we discuss the recent evidence about PPARs expression in the hypothalamic-pituitary-gonadal axis and their involvement in female reproduction. Finally, the therapeutic potential of their manipulation through several drugs is also discussed.

  7. Peroxisome Proliferator-Activated Receptors in Female Reproduction and Fertility

    Directory of Open Access Journals (Sweden)

    Maurizio Vitti

    2016-01-01

    Full Text Available Reproductive functions may be altered by the exposure to a multitude of endogenous and exogenous agents, drug or environmental pollutants, which are known to affect gene transcription through the peroxisome proliferator-activated receptors (PPARs activation. PPARs act as ligand activated transcription factors and regulate metabolic processes such as lipid and glucose metabolism, energy homeostasis, inflammation, and cell proliferation and differentiation. All PPARs isotypes are expressed along the hypothalamic-pituitary-gonadal axis and are strictly involved in reproductive functions. Since female fertility and energy metabolism are tightly interconnected, the research on female infertility points towards the exploration of potential PPARs activating/antagonizing compounds, mainly belonging to the class of thiazolidinediones (TZDs and fibrates, as useful agents for the maintenance of metabolic homeostasis in women with ovarian dysfunctions. In the present review, we discuss the recent evidence about PPARs expression in the hypothalamic-pituitary-gonadal axis and their involvement in female reproduction. Finally, the therapeutic potential of their manipulation through several drugs is also discussed.

  8. Peroxisome Proliferator-Activated Receptors in Female Reproduction and Fertility

    Science.gov (United States)

    Carta, Gaspare; Artini, Paolo Giovanni

    2016-01-01

    Reproductive functions may be altered by the exposure to a multitude of endogenous and exogenous agents, drug or environmental pollutants, which are known to affect gene transcription through the peroxisome proliferator-activated receptors (PPARs) activation. PPARs act as ligand activated transcription factors and regulate metabolic processes such as lipid and glucose metabolism, energy homeostasis, inflammation, and cell proliferation and differentiation. All PPARs isotypes are expressed along the hypothalamic-pituitary-gonadal axis and are strictly involved in reproductive functions. Since female fertility and energy metabolism are tightly interconnected, the research on female infertility points towards the exploration of potential PPARs activating/antagonizing compounds, mainly belonging to the class of thiazolidinediones (TZDs) and fibrates, as useful agents for the maintenance of metabolic homeostasis in women with ovarian dysfunctions. In the present review, we discuss the recent evidence about PPARs expression in the hypothalamic-pituitary-gonadal axis and their involvement in female reproduction. Finally, the therapeutic potential of their manipulation through several drugs is also discussed. PMID:27559343

  9. Activation of EphA receptors mediates the recruitment of the adaptor protein Slap, contributing to the downregulation of N-methyl-D-aspartate receptors.

    Science.gov (United States)

    Semerdjieva, Sophia; Abdul-Razak, Hayder H; Salim, Sharifah S; Yáñez-Muñoz, Rafael J; Chen, Philip E; Tarabykin, Victor; Alifragis, Pavlos

    2013-04-01

    Regulation of the activity of N-methyl-d-aspartate receptors (NMDARs) at glutamatergic synapses is essential for certain forms of synaptic plasticity underlying learning and memory and is also associated with neurotoxicity and neurodegenerative diseases. In this report, we investigate the role of Src-like adaptor protein (Slap) in NMDA receptor signaling. We present data showing that in dissociated neuronal cultures, activation of ephrin (Eph) receptors by chimeric preclustered eph-Fc ligands leads to recruitment of Slap and NMDA receptors at the sites of Eph receptor activation. Interestingly, our data suggest that prolonged activation of EphA receptors is as efficient in recruiting Slap and NMDA receptors as prolonged activation of EphB receptors. Using established heterologous systems, we examined whether Slap is an integral part of NMDA receptor signaling. Our results showed that Slap does not alter baseline activity of NMDA receptors and does not affect Src-dependent potentiation of NMDA receptor currents in Xenopus oocytes. We also demonstrate that Slap reduces excitotoxic cell death triggered by activation of NMDARs in HEK293 cells. Finally, we present evidence showing reduced levels of NMDA receptors in the presence of Slap occurring in an activity-dependent manner, suggesting that Slap is part of a mechanism that homeostatically modulates the levels of NMDA receptors.

  10. Activation of protease-activated receptor 2 induces VEGF independently of HIF-1

    DEFF Research Database (Denmark)

    Rasmussen, Jeppe Grøndahl; Riis, Simone Elkjær; Frøbert, Ole;

    2012-01-01

    Human adipose stem cells (hASCs) can promote angiogenesis through secretion of proangiogenic factors such as vascular endothelial growth factor (VEGF). In other cell types, it has been shown that induction of VEGF is mediated by both protease activated receptor 2 (PAR2) and hypoxia inducible fact...... 1(HIF-1). The present study hypothesized that PAR2 stimulation through activation of kinase signaling cascades lead to induction of HIF-1 and secretion of VEGF....

  11. Mu-opioid receptor knockout mice show diminished food-anticipatory activity

    NARCIS (Netherlands)

    Kas, Martien J H; van den Bos, Ruud; Baars, Annemarie M; Lubbers, Marianne; Lesscher, Heidi M B; Hillebrand, Jacquelien J G; Schuller, Alwin G; Pintar, John E; Spruijt, Berry M

    2004-01-01

    We have previously suggested that during or prior to activation of anticipatory behaviour to a coming reward, mu-opioid receptors are activated. To test this hypothesis schedule induced food-anticipatory activity in mu-opioid receptor knockout mice was measured using running wheels. We hypothesized

  12. Calcium is the switch in the moonlighting dual function of the ligand-activated receptor kinase phytosulfokine receptor 1

    KAUST Repository

    Muleya, Victor

    2014-09-23

    Background: A number of receptor kinases contain guanylate cyclase (GC) catalytic centres encapsulated in the cytosolic kinase domain. A prototypical example is the phytosulfokine receptor 1 (PSKR1) that is involved in regulating growth responses in plants. PSKR1 contains both kinase and GC activities however the underlying mechanisms regulating the dual functions have remained elusive. Findings: Here, we confirm the dual activity of the cytoplasmic domain of the PSKR1 receptor. We show that mutations within the guanylate cyclase centre modulate the GC activity while not affecting the kinase catalytic activity. Using physiologically relevant Ca2+ levels, we demonstrate that its GC activity is enhanced over two-fold by Ca2+ in a concentration-dependent manner. Conversely, increasing Ca2+ levels inhibits kinase activity up to 500-fold at 100 nM Ca2+. Conclusions: Changes in calcium at physiological levels can regulate the kinase and GC activities of PSKR1. We therefore propose a functional model of how calcium acts as a bimodal switch between kinase and GC activity in PSKR1 that could be relevant to other members of this novel class of ligand-activated receptor kinases.

  13. Thrombin-Mediated Direct Activation of Proteinase-Activated Receptor-2: Another Target for Thrombin Signaling.

    Science.gov (United States)

    Mihara, Koichiro; Ramachandran, Rithwik; Saifeddine, Mahmoud; Hansen, Kristina K; Renaux, Bernard; Polley, Danny; Gibson, Stacy; Vanderboor, Christina; Hollenberg, Morley D

    2016-05-01

    Thrombin is known to signal to cells by cleaving/activating a G-protein-coupled family of proteinase-activated receptors (PARs). The signaling mechanism involves the proteolytic unmasking of an N-terminal receptor sequence that acts as a tethered receptor-activating ligand. To date, the recognized targets of thrombin cleavage and activation for signaling are PAR1 and PAR4, in which thrombin cleaves at a conserved target arginine to reveal a tethered ligand. PAR2, which like PAR1 is also cleaved at an N-terminal arginine to unmask its tethered ligand, is generally regarded as a target for trypsin but not for thrombin signaling. We now show that thrombin, at concentrations that can be achieved at sites of acute injury or in a tumor microenvironment, can directly activate PAR2 vasorelaxation and signaling, stimulating calcium and mitogen-activated protein kinase responses along with triggeringβ-arrestin recruitment. Thus, PAR2 can be added alongside PAR1 and PAR4 to the targets, whereby thrombin can affect tissue function.

  14. Repressive effects of resveratrol on androgen receptor transcriptional activity.

    Directory of Open Access Journals (Sweden)

    Wen-feng Shi

    Full Text Available BACKGROUND: The chemopreventive effects of resveratrol (RSV on prostate cancer have been well established; the androgen receptor (AR plays pivotal roles in prostatic tumorigenesis. However, the exact underlying molecular mechanisms about the effects of RSV on AR have not been fully elucidated. A model system is needed to determine whether and how RSV represses AR transcriptional activity. METHODOLOGY: The AR cDNA was first cloned into the retroviral vector pOZ-N and then integrated into the genome of AR-negative HeLa cells to generate the AR(+ cells. The constitutively expressed AR was characterized by monitoring hormone-stimulated nuclear translocation, DNA binding, and transcriptional activation, with the AR(- cells serving as controls. AR(+ cells were treated with RSV, and both AR protein levels and AR transcriptional activity were measured simultaneously. Chromatin immunoprecipitation (ChIP assays were used to detect the effects of RSV on the recruitment of AR to its cognate element (ARE. RESULTS: AR in the AR (+ stable cell line functions in a manner similar to that of endogenously expressed AR. Using this model system we clearly demonstrated that RSV represses AR transcriptional activity independently of any effects on AR protein levels. However, neither the hormone-mediated nucleus translocation nor the AR/ARE interaction was affected by RSV treatment. CONCLUSION: We demonstrated unambiguously that RSV regulates AR target gene expression, at least in part, by repressing AR transcriptional activity. Repressive effects of RSV on AR activity result from mechanisms other than the affects of AR nuclear translocation or DNA binding.

  15. Contribution of regional brain melanocortin receptor subtypes to elevated activity energy expenditure in lean, active rats.

    Science.gov (United States)

    Shukla, C; Koch, L G; Britton, S L; Cai, M; Hruby, V J; Bednarek, M; Novak, C M

    2015-12-01

    Physical activity and non-exercise activity thermogenesis (NEAT) are crucial factors accounting for individual differences in body weight, interacting with genetic predisposition. In the brain, a number of neuroendocrine intermediates regulate food intake and energy expenditure (EE); this includes the brain melanocortin (MC) system, consisting of MC peptides as well as their receptors (MCR). MC3R and MC4R have emerged as critical modulators of EE and food intake. To determine how variance in MC signaling may underlie individual differences in physical activity levels, we examined behavioral response to MC receptor agonists and antagonists in rats that show high and low levels of physical activity and NEAT, that is, high- and low-capacity runners (HCR, LCR), developed by artificial selection for differential intrinsic aerobic running capacity. Focusing on the hypothalamus, we identified brain region-specific elevations in expression of MCR 3, 4, and also MC5R, in the highly active, lean HCR relative to the less active and obesity-prone LCR. Further, the differences in activity and associated EE as a result of MCR activation or suppression using specific agonists and antagonists were similarly region-specific and directly corresponded to the differential MCR expression patterns. The agonists and antagonists investigated here did not significantly impact food intake at the doses used, suggesting that the differential pattern of receptor expression may by more meaningful to physical activity than to other aspects of energy balance regulation. Thus, MCR-mediated physical activity may be a key neural mechanism in distinguishing the lean phenotype and a target for enhancing physical activity and NEAT.

  16. Activation of transient receptor potential ankyrin 1 by eugenol.

    Science.gov (United States)

    Chung, G; Im, S T; Kim, Y H; Jung, S J; Rhyu, M-R; Oh, S B

    2014-03-01

    Eugenol is a bioactive plant extract used as an analgesic agent in dentistry. The structural similarity of eugenol to cinnamaldehyde, an active ligand for transient receptor potential ankyrin 1 (TRPA1), suggests that eugenol might produce its effect via TRPA1, in addition to TRPV1 as we reported previously. In this study, we investigated the effect of eugenol on TRPA1, by fura-2-based calcium imaging and patch clamp recording in trigeminal ganglion neurons and in a heterologous expression system. As the result, eugenol induced robust calcium responses in rat trigeminal ganglion neurons that responded to a specific TRPA1 agonist, allyl isothiocyanate (AITC), and not to capsaicin. Capsazepine, a TRPV1 antagonist failed to inhibit eugenol-induced calcium responses in AITC-responding neurons. In addition, eugenol response was observed in trigeminal ganglion neurons from TRPV1 knockout mice and human embryonic kidney 293 cell lines that express human TRPA1, which was inhibited by TRPA1-specific antagonist HC-030031. Eugenol-evoked TRPA1 single channel activity and eugenol-induced TRPA1 currents were dose-dependent with EC50 of 261.5μM. In summary, these results demonstrate that the activation of TRPA1 might account for another molecular mechanism underlying the pharmacological action of eugenol.

  17. Modulation of Opioid Receptor Ligand Affinity and Efficacy Using Active and Inactive State Receptor Models

    OpenAIRE

    Anand, Jessica P.; Purington, Lauren C.; Pogozheva, Irina D.; Traynor, John R.; Mosberg, Henry I.

    2012-01-01

    Mu opioid receptor (MOR) agonists are widely used for the treatment of pain; however chronic use results in the development of tolerance and dependence. It has been demonstrated that co-administration of a MOR agonist with a delta opioid receptor (DOR) antagonist maintains the analgesia associated with MOR agonists, but with reduced negative side effects. Using our newly refined opioid receptor models for structure-based ligand design, we have synthesized several pentapeptides with tailored a...

  18. GabaB receptors activation in the NTS blocks the glycemic responses induced by carotid body receptor stimulation.

    Science.gov (United States)

    Lemus, Mónica; Montero, Sergio; Cadenas, José Luis; Lara, José Jesús; Tejeda-Chávez, Héctor Rafael; Alvarez-Buylla, Ramón; de Alvarez-Buylla, Elena Roces

    2008-08-18

    The carotid body receptors participate in glucose regulation sensing glucose levels in blood entering the cephalic circulation. The carotid body receptors information, is initially processed within the nucleus tractus solitarius (NTS) and elicits changes in circulating glucose and brain glucose uptake. Previous work has shown that gamma-aminobutyric acid (GABA) in NTS modulates respiratory reflexes, but the role of GABA within NTS in glucose regulation remains unknown. Here we show that GABA(B) receptor agonist (baclofen) or antagonists (phaclofen and CGP55845A) locally injected into NTS modified arterial glucose levels and brain glucose retention. Control injections outside NTS did not elicit these responses. In contrast, GABA(A) agonist and antagonist (muscimol or bicuculline) produced no significant changes in blood glucose levels. When these GABAergic drugs were applied before carotid body receptors stimulation, again, only GABA(B) agonist or antagonist significantly affected glycemic responses; baclofen microinjection significantly reduced the hyperglycemic response and brain glucose retention observed after carotid body receptors stimulation, while phaclofen produced the opposite effect, increasing significantly hyperglycemia and brain glucose retention. These results indicate that activation of GABA(B), but not GABA(A), receptors in the NTS modulates the glycemic responses after anoxic stimulation of the carotid body receptors, and suggest the presence of a tonic inhibitory mechanism in the NTS to avoid hyperglycemia.

  19. Triclocarban mediates induction of xenobiotic metabolism through activation of the constitutive androstane receptor and the estrogen receptor alpha.

    Directory of Open Access Journals (Sweden)

    Mei-Fei Yueh

    Full Text Available Triclocarban (3,4,4'-trichlorocarbanilide, TCC is used as a broad-based antimicrobial agent that is commonly added to personal hygiene products. Because of its extensive use in the health care industry and resistance to degradation in sewage treatment processes, TCC has become a significant waste product that is found in numerous environmental compartments where humans and wildlife can be exposed. While TCC has been linked to a range of health and environmental effects, few studies have been conducted linking exposure to TCC and induction of xenobiotic metabolism through regulation by environmental sensors such as the nuclear xenobiotic receptors (XenoRs. To identify the ability of TCC to activate xenobiotic sensors, we monitored XenoR activities in response to TCC treatment using luciferase-based reporter assays. Among the XenoRs in the reporter screening assay, TCC promotes both constitutive androstane receptor (CAR and estrogen receptor alpha (ERα activities. TCC treatment to hUGT1 mice resulted in induction of the UGT1A genes in liver. This induction was dependent upon the constitutive active/androstane receptor (CAR because no induction occurred in hUGT1Car(-/- mice. Induction of the UGT1A genes by TCC corresponded with induction of Cyp2b10, another CAR target gene. TCC was demonstrated to be a phenobarbital-like activator of CAR in receptor-based assays. While it has been suggested that TCC be classified as an endocrine disruptor, it activates ERα leading to induction of Cyp1b1 in female ovaries as well as in promoter activity. Activation of ERα by TCC in receptor-based assays also promotes induction of human CYP2B6. These observations demonstrate that TCC activates nuclear xenobiotic receptors CAR and ERα both in vivo and in vitro and might have the potential to alter normal physiological homeostasis. Activation of these xenobiotic-sensing receptors amplifies gene expression profiles that might represent a mechanistic base for

  20. Oleamide activates peroxisome proliferator-activated receptor gamma (PPARγ in vitro

    Directory of Open Access Journals (Sweden)

    Dionisi Mauro

    2012-05-01

    Full Text Available Abstract Background Oleamide (ODA is a fatty acid primary amide first identified in the cerebrospinal fluid of sleep-deprived cats, which exerts effects on vascular and neuronal tissues, with a variety of molecular targets including cannabinoid receptors and gap junctions. It has recently been reported to exert a hypolipidemic effect in hamsters. Here, we have investigated the nuclear receptor family of peroxisome proliferator-activated receptors (PPARs as potential targets for ODA action. Results Activation of PPARα, PPARβ and PPARγ was assessed using recombinant expression in Chinese hamster ovary cells with a luciferase reporter gene assay. Direct binding of ODA to the ligand binding domain of each of the three PPARs was monitored in a cell-free fluorescent ligand competition assay. A well-established assay of PPARγ activity, the differentiation of 3T3-L1 murine fibroblasts into adipocytes, was assessed using an Oil Red O uptake-based assay. ODA, at 10 and 50 μM, was able to transactivate PPARα, PPARβ and PPARγ receptors. ODA bound to the ligand binding domain of all three PPARs, although complete displacement of fluorescent ligand was only evident for PPARγ, at which an IC50 value of 38 μM was estimated. In 3T3-L1 cells, ODA, at 10 and 20 μM, induced adipogenesis. Conclusions We have, therefore, identified a novel site of action of ODA through PPAR nuclear receptors and shown how ODA should be considered as a weak PPARγ ligand in vitro.

  1. Role of receptors in Bacillus thuringiensis crystal toxin activity.

    Science.gov (United States)

    Pigott, Craig R; Ellar, David J

    2007-06-01

    Bacillus thuringiensis produces crystalline protein inclusions with insecticidal or nematocidal properties. These crystal (Cry) proteins determine a particular strain's toxicity profile. Transgenic crops expressing one or more recombinant Cry toxins have become agriculturally important. Individual Cry toxins are usually toxic to only a few species within an order, and receptors on midgut epithelial cells have been shown to be critical determinants of Cry specificity. The best characterized of these receptors have been identified for lepidopterans, and two major receptor classes have emerged: the aminopeptidase N (APN) receptors and the cadherin-like receptors. Currently, 38 different APNs have been reported for 12 different lepidopterans. Each APN belongs to one of five groups that have unique structural features and Cry-binding properties. While 17 different APNs have been reported to bind to Cry toxins, only 2 have been shown to mediate toxin susceptibly in vivo. In contrast, several cadherin-like proteins bind to Cry toxins and confer toxin susceptibility in vitro, and disruption of the cadherin gene has been associated with toxin resistance. Nonetheless, only a small subset of the lepidopteran-specific Cry toxins has been shown to interact with cadherin-like proteins. This review analyzes the interactions between Cry toxins and their receptors, focusing on the identification and validation of receptors, the molecular basis for receptor recognition, the role of the receptor in resistant insects, and proposed models to explain the sequence of events at the cell surface by which receptor binding leads to cell death. PMID:17554045

  2. Investigating real-time activation of adenosine receptors by bioluminescence resonance energy transfer technique

    Science.gov (United States)

    Huang, Yimei; Yang, Hongqin; Zheng, Liqin; Chen, Jiangxu; Wang, Yuhua; Li, Hui; Xie, Shusen

    2013-02-01

    Adenosine receptors play important roles in many physiological and pathological processes, for example regulating myocardial oxygen consumption and the release of neurotransmitters. The activations of adenosine receptors have been studied by some kinds of techniques, such as western blot, immunohistochemistry, etc. However, these techniques cannot reveal the dynamical response of adenosine receptors under stimulation. In this paper, bioluminescence resonance energy transfer technique was introduced to study the real-time activation of adenosine receptors by monitoring the dynamics of cyclic adenosine monophosphate (cAMP) level. The results showed that there were significant differences between adenosine receptors on real-time responses under stimulation. Moreover, the dynamics of cAMP level demonstrated that competition between adenosine receptors existed. Taken together, our study indicates that monitoring the dynamics of cAMP level using bioluminescence resonance energy transfer technique could be one potential approach to investigate the mechanism of competitions between adenosine receptors.

  3. The role of ECL2 in CGRP receptor activation: a combined modelling and experimental approach

    Science.gov (United States)

    Woolley, Michael. J.; Watkins, Harriet A.; Taddese, Bruck; Karakullukcu, Z. Gamze; Barwell, James; Smith, Kevin J.; Hay, Debbie L.; Poyner, David R.; Reynolds, Christopher A.; Conner, Alex C.

    2013-01-01

    The calcitonin gene-related peptide (CGRP) receptor is a complex of a calcitonin receptor-like receptor (CLR), which is a family B G-protein-coupled receptor (GPCR) and receptor activity modifying protein 1. The role of the second extracellular loop (ECL2) of CLR in binding CGRP and coupling to Gs was investigated using a combination of mutagenesis and modelling. An alanine scan of residues 271–294 of CLR showed that the ability of CGRP to produce cAMP was impaired by point mutations at 13 residues; most of these also impaired the response to adrenomedullin (AM). These data were used to select probable ECL2-modelled conformations that are involved in agonist binding, allowing the identification of the likely contacts between the peptide and receptor. The implications of the most likely structures for receptor activation are discussed. PMID:24047872

  4. Adipocyte insulin receptor activity maintains adipose tissue mass and lifespan.

    Science.gov (United States)

    Friesen, Max; Hudak, Carolyn S; Warren, Curtis R; Xia, Fang; Cowan, Chad A

    2016-08-01

    Type 2 diabetes follows a well-defined progressive pathogenesis, beginning with insulin resistance in metabolic tissues such as the adipose. Intracellular signaling downstream of insulin receptor activation regulates critical metabolic functions of adipose tissue, including glucose uptake, lipogenesis, lipolysis and adipokine secretion. Previous studies have used the aP2 promoter to drive Cre recombinase expression in adipose tissue. Insulin receptor (IR) knockout mice created using this aP2-Cre strategy (FIRKO mice) were protected from obesity and glucose intolerance. Later studies demonstrated the promiscuity of the aP2 promoter, casting doubts upon the tissue specificity of aP2-Cre models. It is our goal to use the increased precision of the Adipoq promoter to investigate adipocyte-specific IR function. Towards this end we generated an adipocyte-specific IR knockout (AIRKO) mouse using an Adipoq-driven Cre recombinase. Here we report AIRKO mice are less insulin sensitive throughout life, and less glucose tolerant than wild-type (WT) littermates at the age of 16 weeks. In contrast to WT littermates, the insulin sensitivity of AIRKO mice is unaffected by age or dietary regimen. At any age, AIRKO mice are comparably insulin resistant to old or obese WT mice and have a significantly reduced lifespan. Similar results were obtained when these phenotypes were re-examined in FIRKO mice. We also found that the AIRKO mouse is protected from high-fat diet-induced weight gain, corresponding with a 90% reduction in tissue weight of major adipose depots compared to WT littermates. Adipose tissue mass reduction is accompanied by hepatomegaly and increased hepatic steatosis. These data indicate that adipocyte IR function is crucial to systemic energy metabolism and has profound effects on adiposity, hepatic homeostasis and lifespan. PMID:27246738

  5. Cannabinoid receptor activation shifts temporally engendered patterns of dopamine release.

    Science.gov (United States)

    Oleson, Erik B; Cachope, Roger; Fitoussi, Aurelie; Tsutsui, Kimberly; Wu, Sharon; Gallegos, Jacqueline A; Cheer, Joseph F

    2014-05-01

    The ability to discern temporally pertinent environmental events is essential for the generation of adaptive behavior in conventional tasks, and our overall survival. Cannabinoids are thought to disrupt temporally controlled behaviors by interfering with dedicated brain timing networks. Cannabinoids also increase dopamine release within the mesolimbic system, a neural pathway generally implicated in timing behavior. Timing can be assessed using fixed-interval (FI) schedules, which reinforce behavior on the basis of time. To date, it remains unknown how cannabinoids modulate dopamine release when responding under FI conditions, and for that matter, how subsecond dopamine release is related to time in these tasks. In the present study, we hypothesized that cannabinoids would accelerate timing behavior in an FI task while concurrently augmenting a temporally relevant pattern of dopamine release. To assess this possibility, we measured subsecond dopamine concentrations in the nucleus accumbens while mice responded for food under the influence of the cannabinoid agonist WIN 55,212-2 in an FI task. Our data reveal that accumbal dopamine concentrations decrease proportionally to interval duration--suggesting that dopamine encodes time in FI tasks. We further demonstrate that WIN 55,212-2 dose-dependently increases dopamine release and accelerates a temporal behavioral response pattern in a CB1 receptor-dependent manner--suggesting that cannabinoid receptor activation modifies timing behavior, in part, by augmenting time-engendered patterns of dopamine release. Additional investigation uncovered a specific role for endogenous cannabinoid tone in timing behavior, as elevations in 2-arachidonoylglycerol, but not anandamide, significantly accelerated the temporal response pattern in a manner akin to WIN 55,212-2. PMID:24345819

  6. Nuclear factor RIP140 modulates transcriptional activation by the estrogen receptor.

    OpenAIRE

    Cavaillès, V; Dauvois, S; L'Horset, F; Lopez, G; Hoare, S.; Kushner, P J; Parker, M G

    1995-01-01

    A conserved region in the hormone-dependent activation domain AF2 of nuclear receptors plays an important role in transcriptional activation. We have characterized a novel nuclear protein, RIP140, that specifically interacts in vitro with this domain of the estrogen receptor. This interaction was increased by estrogen, but not by anti-estrogens and the in vitro binding capacity of mutant receptors correlates with their ability to stimulate transcription. RIP140 also interacts with estrogen re...

  7. Endocytosis of activated receptors and clathrin-coated pit formation: deciphering the chicken or egg relationship

    OpenAIRE

    1996-01-01

    The fundamental mechanisms by which receptors once targeted for endocytosis are found in coated pits is an important yet unresolved question. Specifically, are activated receptors simply trapped on encountering preexisting coated pits, subsequently being rapidly internalized? Or do the receptors themselves, by active recruitment, gather soluble coat and cytosolic components and initiate the rapid assembly of new coated pits that then mediate their internalization? To explore this question, we...

  8. Analysis of the Heat Shock Response in Mouse Liver Reveals Transcriptional Dependence on the Nuclear Receptor Peroxisome Proliferator-Activated Receptor alpha (PPARα)

    Science.gov (United States)

    BACKGROUND: The nuclear receptor peroxisome proliferator-activated receptor alpha (PPARalpha) regulates responses to chemical or physical stress in part by altering expression of genes involved in proteome maintenance. Many of these genes are also transcriptionally regulated by h...

  9. Endothelial Cells Promote Pigmentation through Endothelin Receptor B Activation.

    Science.gov (United States)

    Regazzetti, Claire; De Donatis, Gian Marco; Ghorbel, Houda Hammami; Cardot-Leccia, Nathalie; Ambrosetti, Damien; Bahadoran, Philippe; Chignon-Sicard, Bérengère; Lacour, Jean-Philippe; Ballotti, Robert; Mahns, Andre; Passeron, Thierry

    2015-12-01

    Findings of increased vascularization in melasma lesions and hyperpigmentation in acquired bilateral telangiectatic macules suggested a link between pigmentation and vascularization. Using high-magnification digital epiluminescence dermatoscopy, laser confocal microscopy, and histological examination, we showed that benign vascular lesions of the skin have restricted but significant hyperpigmentation compared with the surrounding skin. We then studied the role of microvascular endothelial cells in regulating skin pigmentation using an in vitro co-culture model using endothelial cells and melanocytes. These experiments showed that endothelin 1 released by microvascular endothelial cells induces increased melanogenesis signaling, characterized by microphthalmia-associated transcription factor phosphorylation, and increased tyrosinase and dopachrome tautomerase levels. Immunostaining for endothelin 1 in vascular lesions confirmed the increased expression on the basal layer of the epidermis above small vessels compared with perilesional skin. Endothelin acts through the activation of endothelin receptor B and the mitogen-activated protein kinase, extracellular signal-regulated kinase (ERK)1/2, and p38, to induce melanogenesis. Finally, culturing of reconstructed skin with microvascular endothelial cells led to increased skin pigmentation that could be prevented by inhibiting EDNRB. Taken together these results demonstrated the role of underlying microvascularization in skin pigmentation, a finding that could open new fields of research for regulating physiological pigmentation and for treating pigmentation disorders such as melasma. PMID:26308584

  10. Stereostructure-activity studies on agonists at the AMPA and kainate subtypes of ionotropic glutamate receptors

    DEFF Research Database (Denmark)

    Johansen, Tommy N; Greenwood, Jeremy R; Frydenvang, Karla Andrea;

    2003-01-01

    -methyl-4-isoxazolyl)propionic acid (AMPA) receptor subtype of ionotropic Glu receptors in the presence or absence of an agonist has provided important information about ligand-receptor interaction mechanisms. The availability of these binding domain crystal structures has formed the basis for rational...... design of ligands, especially for the AMPA and kainate subtypes of ionotropic Glu receptors. This mini-review will focus on structure-activity relationships on AMPA and kainate receptor agonists with special emphasis on stereochemical and three-dimensional aspects....

  11. Tyrosine kinase JAK1 is associated with the granulocyte-colony-stimulating factor receptor and both become tyrosine-phosphorylated after receptor activation.

    OpenAIRE

    Nicholson, S. E.; Oates, A. C.; Harpur, A G; Ziemiecki, A; Wilks, A F; Layton, J E

    1994-01-01

    Granulocyte-colony-stimulating factor (G-CSF) stimulates the proliferation and differentiation of cells of the neutrophil lineage by interaction with a specific receptor. Early signal transduction events following G-CSF receptor activation were studied. We detected tyrosine phosphorylation of both the G-CSF receptor and the protein tyrosine kinase JAK1 following G-CSF binding to the human G-CSF receptor. In vitro, the kinase activity of JAK1 was increased by G-CSF stimulation. Coimmunoprecipi...

  12. Plasma soluble urokinase plasminogen activator receptor in children with urinary tract infection

    DEFF Research Database (Denmark)

    Wittenhagen, Per; Andersen, Jesper Brandt; Hansen, Anita;

    2011-01-01

    In this prospective study we investigated the role of plasma levels of soluble urokinase plasminogen activator receptor (suPAR) in children with urinary tract infection.......In this prospective study we investigated the role of plasma levels of soluble urokinase plasminogen activator receptor (suPAR) in children with urinary tract infection....

  13. Activation of toll-like receptors and dendritic cells by a broad range of bacterial molecules

    NARCIS (Netherlands)

    Boele, L.C.L.; Bajramovic, J.J.; Vries, A.M.M.B.C. de; Voskamp-Visser, I.A.I.; Kaman, W.E.; Kleij, D. van der

    2009-01-01

    Activation of pattern recognition receptors such as Toll-like receptors (TLRs) by pathogens leads to activation and maturation of dendritic cells (DC), which orchestrate the development of the adaptive immune response. To create an overview of the effects of a broad range of pathogenic bacteria, the

  14. Activation of intracellular angiotensin AT2 receptors induces rapid cell death in human uterine leiomyosarcoma cells

    DEFF Research Database (Denmark)

    Zhao, Yi; Lützen, Ulf; Fritsch, Jürgen;

    2015-01-01

    densities in mitochondria. Activation of the cell membrane AT2 receptors by a concomitant treatment with angiotensin II and the AT1 receptor antagonist, losartan, induces apoptosis but does not affect the rate of cell death. We demonstrate for the first time that the high-affinity, non-peptide AT2 receptor...... of apoptosis and cell death in cultured human uterine leiomyosarcoma (SK-UT-1) cells and control human uterine smooth muscle cells (HutSMC). The intracellular levels of the AT2 receptor are low in proliferating SK-UT-1 cells but the receptor is substantially up-regulated in quiescent SK-UT-1 cells with high...... agonist, Compound 21 (C21) penetrates the cell membrane of quiescent SK-UT-1 cells, activates intracellular AT2 receptors and induces rapid cell death; approximately 70% of cells died within 24 h. The cells, which escaped from the cell death, displayed activation of the mitochondrial apoptotic pathway, i...

  15. NMDA receptor activation regulates sociability by its effect on mTOR signaling activity.

    Science.gov (United States)

    Burket, Jessica A; Benson, Andrew D; Tang, Amy H; Deutsch, Stephen I

    2015-07-01

    Tuberous Sclerosis Complex is one example of a syndromic form of autism spectrum disorder associated with disinhibited activity of mTORC1 in neurons (e.g., cerebellar Purkinje cells). mTORC1 is a complex protein possessing serine/threonine kinase activity and a key downstream molecule in a signaling cascade beginning at the cell surface with the transduction of neurotransmitters (e.g., glutamate and acetylcholine) and nerve growth factors (e.g., Brain-Derived Neurotrophic Factor). Interestingly, the severity of the intellectual disability in Tuberous Sclerosis Complex may relate more to this metabolic disturbance (i.e., overactivity of mTOR signaling) than the density of cortical tubers. Several recent reports showed that rapamycin, an inhibitor of mTORC1, improved sociability and other symptoms in mouse models of Tuberous Sclerosis Complex and autism spectrum disorder, consistent with mTORC1 overactivity playing an important pathogenic role. NMDA receptor activation may also dampen mTORC1 activity by at least two possible mechanisms: regulating intraneuronal accumulation of arginine and the phosphorylation status of a specific extracellular signal regulating kinase (i.e., ERK1/2), both of which are "drivers" of mTORC1 activity. Conceivably, the prosocial effects of targeting the NMDA receptor with agonists in mouse models of autism spectrum disorders result from their ability to dampen mTORC1 activity in neurons. Strategies for dampening mTORC1 overactivity by NMDA receptor activation may be preferred to its direct inhibition in chronic neurodevelopmental disorders, such as autism spectrum disorders.

  16. Antileishmanial activity of the estrogen receptor modulator raloxifene.

    Directory of Open Access Journals (Sweden)

    Juliana Q Reimão

    2014-05-01

    Full Text Available BACKGROUND: The treatment of leishmaniasis relies mostly on parenteral drugs with potentially serious adverse effects. Additionally, parasite resistance in the treatment of leishmaniasis has been demonstrated for the majority of drugs available, making the search for more effective and less toxic drugs and treatment regimens a priority for the control of leishmaniasis. The aims of this study were to evaluate the antileishmanial activity of raloxifene in vitro and in vivo and to investigate its mechanism of action against Leishmania amazonensis. METHODOLOGY/PRINCIPAL FINDINGS: Raloxifene was shown to possess antileishmanial activity in vitro against several species with EC50 values ranging from 30.2 to 38.0 µM against promastigotes and from 8.8 to 16.2 µM against intracellular amastigotes. Raloxifene's mechanism of action was investigated through transmission electron microscopy and labeling with propidium iodide, DiSBAC2(3, rhodamine 123 and monodansylcadaverine. Microscopic examinations showed that raloxifene treated parasites displayed autophagosomes and mitochondrial damage while the plasma membrane remained continuous. Nonetheless, plasma membrane potential was rapidly altered upon raloxifene treatment with initial hyperpolarization followed by depolarization. Loss of mitochondrial membrane potential was also verified. Treatment of L. amazonensis-infected BALB/c mice with raloxifene led to significant decrease in lesion size and parasite burden. CONCLUSIONS/SIGNIFICANCE: The results of this work extend the investigation of selective estrogen receptor modulators as potential candidates for leishmaniasis treatment. The antileishmanial activity of raloxifene was demonstrated in vitro and in vivo. Raloxifene produces functional disorder on the plasma membrane of L. amazonensis promastigotes and leads to functional and morphological disruption of mitochondria, which culminate in cell death.

  17. Modulation of receptors and adenylate cyclase activity during sucrose feeding, food deprivation, and cold exposure

    International Nuclear Information System (INIS)

    Thermogenesis in brown adipose tissue (BAT) serves as a regulator of body temperature and weight maintenance. Thermogenesis can be stimulated by catecholamine activation of adenylate cyclase through the β-adrenergic receptor. To investigate the effects of sucrose feeding, food deprivation, and cold exposure on the β-adrenergic pathway, adenylate cyclase activity and β-adrenergic receptors were assessed in rat BAT after 2 wk of sucrose feeding, 2 days of food deprivation, or 2 days of cold exposure. β-Adrenergic receptors were identified in BAT using [125I]iodocyanopindolol. Binding sites had the characteristics of mixed β1- and β2-type adrenergic receptors at a ratio of 60/40. After sucrose feeding or cold exposure, there was the expected increase in BAT mitochondrial mass as measured by total cytochrome-c oxidase activity but a decrease in β-adrenergic receptor density due to a loss of the β1-adrenergic subtype. This BAT β-adrenergic receptor downregulation was tissue specific, since myocardial β-adrenergic receptors were unchanged with either sucrose feeding or cold exposure. Forskolin-stimulated adenylate cyclase activity increased in BAT after sucrose feeding or cold exposure but not after food deprivation. These data suggest that in BAT, sucrose feeding or cold exposure result in downregulation of β-adrenergic receptors and that isoproterenol-stimulated adenylate cyclase activity was limited by receptor availability

  18. A bacterial tyrosine phosphatase inhibits plant pattern recognition receptor activation

    Science.gov (United States)

    Perception of pathogen-associated molecular patterns (PAMPs) by surface-localised pattern-recognition receptors (PRRs) is a key component of plant innate immunity. Most known plant PRRs are receptor kinases and initiation of PAMP-triggered immunity (PTI) signalling requires phosphorylation of the PR...

  19. Cloning and expression of the receptor for human urokinase plasminogen activator, a central molecule in cell surface, plasmin dependent proteolysis

    DEFF Research Database (Denmark)

    Roldan, A L; Cubellis, M V; Masucci, M T;

    1990-01-01

    , and therefore the capacity of cells to migrate and invade neighboring tissues. We have isolated a 1.4 kb cDNA clone coding for the entire human uPAR. An oligonucleotide synthesized on the basis of the N-terminal sequence of the purified protein was used to screen a cDNA library made from SV40 transformed human...

  20. Estrogen Receptor β Activation Rapidly Modulates Male Sexual Motivation through the Transactivation of Metabotropic Glutamate Receptor 1a.

    Science.gov (United States)

    Seredynski, Aurore L; Balthazart, Jacques; Ball, Gregory F; Cornil, Charlotte A

    2015-09-23

    In addition to the transcriptional activity of their liganded nuclear receptors, estrogens, such as estradiol (E2), modulate cell functions, and consequently physiology and behavior, within minutes through membrane-initiated events. The membrane-associated receptors (mERs) underlying the acute effects of estrogens on behavior have mostly been documented in females where active estrogens are thought to be of ovarian origin. We determined here, by acute intracerebroventricular injections of specific agonists and antagonists, the type(s) of mERs that modulate rapid effects of brain-derived estrogens on sexual motivation in male Japanese quail. Brain aromatase blockade acutely inhibited sexual motivation. Diarylpropionitrile (DPN), an estrogen receptor β (ERβ)-specific agonist, and to a lesser extent 17α-estradiol, possibly acting through ER-X, prevented this effect. In contrast, drugs targeting ERα (PPT and MPP), GPR30 (G1 and G15), and the Gq-mER (STX) did not affect sexual motivation. The mGluR1a antagonist LY367385 significantly inhibited sexual motivation but mGluR2/3 and mGluR5 antagonists were ineffective. LY367385 also blocked the behavioral restoration induced by E2 or DPN, providing functional evidence that ERβ interacts with metabotropic glutamate receptor 1a (mGluR1a) signaling to acutely regulate male sexual motivation. Together these results show that ERβ plays a key role in sexual behavior regulation and the recently uncovered cooperation between mERs and mGluRs is functional in males where it mediates the acute effects of estrogens produced centrally in response to social stimuli. The presence of an ER-mGluR interaction in birds suggests that this mechanism emerged relatively early in vertebrate history and is well conserved. Significance statement: The membrane-associated receptors underlying the acute effects of estrogens on behavior have mostly been documented in females, where active estrogens are thought to be of ovarian origin. Using acute

  1. Regulatory network of inflammation downstream of proteinase-activated receptors

    Directory of Open Access Journals (Sweden)

    Hurst Robert E

    2007-03-01

    Full Text Available Abstract Background Protease-activated receptors (PAR are present in the urinary bladder, and their expression is altered in response to inflammation. PARs are a unique class of G protein-coupled that carry their own ligands, which remain cryptic until unmasked by proteolytic cleavage. Although the canonical signal transduction pathway downstream of PAR activation and coupling with various G proteins is known and leads to the rapid transcription of genes involved in inflammation, the effect of PAR activation on the downstream transcriptome is unknown. We have shown that intravesical administration of PAR-activating peptides leads to an inflammatory reaction characterized by edema and granulocyte infiltration. Moreover, the inflammatory response to intravesical instillation of known pro-inflammatory stimuli such as E. coli lipopolysaccharide (LPS, substance P (SP, and antigen was strongly attenuated by PAR1- and to a lesser extent by PAR2-deficiency. Results Here, cDNA array experiments determined inflammatory genes whose expression is dependent on PAR1 activation. For this purpose, we compared the alteration in gene expression in wild type and PAR1-/- mice induced by classical pro-inflammatory stimuli (LPS, SP, and antigen. 75 transcripts were considered to be dependent on PAR-1 activation and further annotated in silico by Ingenuity Pathways Analysis (IPA and gene ontology (GO. Selected transcripts were target validated by quantitative PCR (Q-PCR. Among PAR1-dependent transcripts, the following have been implicated in the inflammatory process: b2m, ccl7, cd200, cd63, cdbpd, cfl1, dusp1, fkbp1a, fth1, hspb1, marcksl1, mmp2, myo5a, nfkbia, pax1, plaur, ppia, ptpn1, ptprcap, s100a10, sim2, and tnfaip2. However, a balanced response to signals of injury requires a transient cellular activation of a panel of genes together with inhibitory systems that temper the overwhelming inflammation. In this context, the activation of genes such as dusp1 and

  2. Group III metabotropic glutamate receptors and D1-like and D2-like dopamine receptors interact in the rat nucleus accumbens to influence locomotor activity.

    Science.gov (United States)

    David, Hélène N; Abraini, Jacques H

    2002-03-01

    Evidence for functional interactions between metabotropic glutamate (mGlu) receptors and dopamine (DA) neurotransmission is now clearly established. In the present study, we investigated interactions between group III mGlu receptors and D1- and D2-like receptors in the nucleus accumbens (NAcc). Administration, into the NAcc, of the selective group III mGlu receptor agonist, AP4, resulted in an increase in locomotor activity, which was blocked by pretreatment with the group III mGlu receptor antagonist, MPPG. In addition, pretreatment with AP4 further blocked the increase in motor activity induced by the D1-like receptor agonist, SKF 38393, but potentiated the locomotor responses induced by either the D2-like receptor agonist, quinpirole, or coinfusion of SKF 38393 and quinpirole. MPPG reversed the effects of AP4 on the motor responses induced by D1-like and/or D2-like receptor activation. These results confirm that glutamate transmission may control DA-dependent locomotor function through mGlu receptors and further indicate that group III mGlu receptors oppose the behavioural response produced by D1-like receptor activation and favour those produced by D2-like receptor activation. PMID:11906529

  3. Activation of transforming potential of the human insulin receptor gene

    International Nuclear Information System (INIS)

    A retrovirus containing part of the human insulin receptor (hIR) gene was constructed by replacing ros sequences in the avian sarcoma virus UR2 with hIR cDNA sequences coding for 46 amino acids of the extracellular domain and the entire transmembrane and cytoplasmic domains of the β subunit of hIR. The resulting virus, named UIR, contains the hIR sequence fused to the 5' portion of the UR2 gag gene coding for p19. UIR is capable of transforming chicken embryo fibroblasts and promoting formation of colonies in soft agar; however, it does not form tumors in vivo. A variant that arose from the parental UIR is capable of efficiently inducing sarcomas in vivo. UIR-transformed cells exhibit higher rates of glucose uptake and growth than normal cells. The 4-kilobase UIR genome codes for a membrane-associated, glycosylated gag-hIR fusion protein of 75 kDa designated P75/sup gag-hir/. P75/sup gag-hir/ contains a protein tyrosine kinase activity that is capable of undergoing autophosphorylation and of phosphorylating foreign substrates in vitro; it is phosphorylated at both serine and tyrosine residues in vivo

  4. Activation of transforming potential of the human insulin receptor gene

    Energy Technology Data Exchange (ETDEWEB)

    Wang, L.H.; Lin, B.; Jong, S.M.J.; Dixon, D.; Ellis, L.; Roth, R.A.; Rutter, W.J.

    1987-08-01

    A retrovirus containing part of the human insulin receptor (hIR) gene was constructed by replacing ros sequences in the avian sarcoma virus UR2 with hIR cDNA sequences coding for 46 amino acids of the extracellular domain and the entire transmembrane and cytoplasmic domains of the ..beta.. subunit of hIR. The resulting virus, named UIR, contains the hIR sequence fused to the 5' portion of the UR2 gag gene coding for p19. UIR is capable of transforming chicken embryo fibroblasts and promoting formation of colonies in soft agar; however, it does not form tumors in vivo. A variant that arose from the parental UIR is capable of efficiently inducing sarcomas in vivo. UIR-transformed cells exhibit higher rates of glucose uptake and growth than normal cells. The 4-kilobase UIR genome codes for a membrane-associated, glycosylated gag-hIR fusion protein of 75 kDa designated P75/sup gag-hir/. P75/sup gag-hir/ contains a protein tyrosine kinase activity that is capable of undergoing autophosphorylation and of phosphorylating foreign substrates in vitro; it is phosphorylated at both serine and tyrosine residues in vivo

  5. Selective glucocorticoid receptor-activating adjuvant therapy in cancer treatments

    Science.gov (United States)

    Sundahl, Nora; Clarisse, Dorien; Bracke, Marc; Offner, Fritz; Berghe, Wim Vanden; Beck, Ilse M.

    2016-01-01

    Although adverse effects and glucocorticoid resistance cripple their chronic use, glucocorticoids form the mainstay therapy for acute and chronic inflammatory disorders, and play an important role in treatment protocols of both lymphoid malignancies and as adjuvant to stimulate therapy tolerability in various solid tumors. Glucocorticoid binding to their designate glucocorticoid receptor (GR), sets off a plethora of cell-specific events including therapeutically desirable effects, such as cell death, as well as undesirable effects, including chemotherapy resistance, systemic side effects and glucocorticoid resistance. In this context, selective GR agonists and modulators (SEGRAMs) with a more restricted GR activity profile have been developed, holding promise for further clinical development in anti-inflammatory and potentially in cancer therapies. Thus far, the research into the prospective benefits of selective GR modulators in cancer therapy limped behind. Our review discusses how selective GR agonists and modulators could improve the therapy regimens for lymphoid malignancies, prostate or breast cancer. We summarize our current knowledge and look forward to where the field should move to in the future. Altogether, our review clarifies novel therapeutic perspectives in cancer modulation via selective GR targeting.

  6. Phytoceramide and sphingoid bases derived from brewer's yeast Saccharomyces pastorianus activate peroxisome proliferator-activated receptors

    Directory of Open Access Journals (Sweden)

    Mitsutake Susumu

    2011-08-01

    Full Text Available Abstract Background Peroxisome proliferator-activated receptors (PPARs are ligand-activated transcription factors that regulate lipid and glucose metabolism. PPARα is highly expressed in the liver and controls genes involved in lipid catabolism. We previously reported that synthetic sphingolipid analogs, part of which contains shorter-length fatty acid chains than natural sphingolipids, stimulated the transcriptional activities of PPARs. Sphingosine and dihydrosphingosine (DHS are abundant sphingoid bases, and ceramide and dihydroceramide are major ceramide species in mammals. In contrast, phytosphingosine (PHS and DHS are the main sphingoid bases in fungi. PHS and phytoceramide exist in particular tissues such as the epidermis in mammals, and involvement of ceramide species in PPARβ activation in cultured keratinocytes has been reported. The purpose of the present study is to investigate whether natural sphingolipids with C18 fatty acid and yeast-derived sphingoid bases activate PPARs as PPAR agonists. Method Lipids of brewer's yeast contain PHS- and DHS-based sphingolipids. To obtain the sphingoid bases, lipids were extracted from brewer's yeast and acid-hydrolyzed. The sphingoid base fraction was purified and quantified. To assess the effects of sphingolipids on PPAR activation, luciferase reporter assay was carried out. NIH/3T3 and human hepatoma (HepG2 cells were transfected with expression vectors for PPARs and retinoid × receptors, and PPAR responsive element reporter vector. When indicated, the PPAR/Gal4 chimera system was performed to enhance the credibility of experiments. Sphingolipids were added to the cells and the dual luciferase reporter assay was performed to determine the transcriptional activity of PPARs. Results We observed that phytoceramide increased the transcriptional activities of PPARs significantly, whereas ceramide and dihydroceramide did not change PPAR activities. Phytoceramide also increased transactivation of

  7. Molecular cloning and characterization of a nuclear androgen receptor activated by 11-ketotestosterone

    Directory of Open Access Journals (Sweden)

    Karlsson Johnny

    2005-08-01

    Full Text Available Abstract Although 11-ketotestosterone is a potent androgen and induces male secondary sex characteristics in many teleosts, androgen receptors with high binding affinity for 11-ketotestosterone or preferential activation by 11-ketotestosterone have not been identified. So, the mechanism by which 11-ketotestosterone exhibits such high potency remains unclear. Recently we cloned the cDNA of an 11-ketotestosterone regulated protein, spiggin, from three-spined stickleback renal tissue. As spiggin is the only identified gene product regulated by 11-ketotestosterone, the stickleback kidney is ideal for determination of the mechanism of 11-ketotestosterone gene regulation. A single androgen receptor gene with two splicing variants, belonging to the androgen receptor-β subfamily was cloned from stickleback kidney. A high affinity, saturable, single class of androgen specific binding sites, with the characteristics of an androgen receptor, was identified in renal cytosolic and nuclear fractions. Measurement of ligand binding moieties in the cytosolic and nuclear fractions as well as to the recombinant receptor revealed lower affinity for 11-ketotestosterone than for dihydrotestosterone. Treatment with different androgens did not up-regulate androgen receptor mRNA level or increase receptor abundance, suggesting that auto-regulation is not involved in differential ligand activation. However, comparison of the trans-activation potential of the stickleback androgen receptor with the human androgen receptor, in both human HepG2 cells and zebrafish ZFL cells, revealed preferential activation by 11-ketotestosterone of the stickleback receptor, but not of the human receptor. These findings demonstrate the presence of a receptor preferentially activated by 11-ketotestosterone in the three-spined stickleback, so far the only one known in any animal.

  8. Antipeptide antibody that specifically inhibits insulin receptor autophosphorylation and protein kinase activity

    International Nuclear Information System (INIS)

    Two site-specific antibodies that immunoprecipitate the human insulin receptor have been prepared by immunizing rabbits with chemically synthesized peptides derived from the cDNA-predicted amino acid sequence of the β subunit of the proreceptor. Antibodies to the carboxyl terminus (AbP5) and to a domain around tyrosine-960 (AbP4) specifically recognize the β subunit of the receptor on immunoblots. Both antibodies immunoprecipitated 125I-labeled insulin-receptor complexes and the autophosphorylated receptor. Although neither antibody inhibited insulin binding to the receptor, both insulin-dependent autophosphorylation and exogenous substrate phosphorylation were inhibited by AbP4. Inhibition by AbP4 was dependent upon the phosphorylation state of the receptor; it was not detected when the receptor was autophosphorylated prior to addition of AbP4. AbP4 did not inhibit activity of the related epidermal growth factor (EGF)-receptor tyrosine protein kinase nor did it inhibit the activity of cAMP-dependent kinase or protein kinase C. The observation that an antibody directed to residues 952-967 of the proreceptor neutralizes the protein kinase activity of the β subunit suggest that this region may play a critical role in the function of the hormone-dependent, protein tyrosine-specific kinase activity of the insulin receptor

  9. Activation profiles of opioid ligands in HEK cells expressing δ opioid receptors

    Directory of Open Access Journals (Sweden)

    Clark J

    2002-11-01

    Full Text Available Abstract Background The aim of the present study was to characterize the activation profiles of 15 opioid ligands in transfected human embryonic kidney cells expressing only δ opioid receptors. Activation profiles of most of these ligands at δ opioid receptors had not been previously characterized in vitro. Receptor activation was assessed by measuring the inhibition of forskolin-stimulated cAMP production. Results Naltrexone and nalorphine were classified as antagonists at δ opioid receptor. The other ligands studied were agonists at δ opioid receptors and demonstrated IC50 values of 0.1 nM to 2 μM, maximal inhibition of 39–77% and receptor binding affinities of 0.5 to 243 nM. The rank order of efficacy of the ligands tested was metazocine = xorphanol ≥ fentanyl = SKF 10047 = etorphine = hydromorphone = butorphanol = lofentanil > WIN 44,441 = Nalbuphine = cyclazocine ≥ met-enkephalin >> morphine = dezocine. For the first time these data describe and compare the function and relative efficacy of several ligands at δ opioid receptors. Conclusions The data produced from this study can lead to elucidation of the complete activation profiles of several opioid ligands, leading to clarification of the mechanisms involved in physiological effects of these ligands at δ opioid receptors. Furthermore, these data can be used as a basis for novel use of existing opioid ligands based on their pharmacology at δ opioid receptors.

  10. Function of the cytoplasmic tail of human calcitonin receptor-like receptor in complex with receptor activity-modifying protein 2

    International Nuclear Information System (INIS)

    Receptor activity-modifying protein 2 (RAMP2) enables calcitonin receptor-like receptor (CRLR) to form an adrenomedullin (AM)-specific receptor. Here we investigated the function of the cytoplasmic C-terminal tail (C-tail) of human (h)CRLR by co-transfecting its C-terminal mutants into HEK-293 cells stably expressing hRAMP2. Deleting the C-tail from CRLR disrupted AM-evoked cAMP production or receptor internalization, but did not affect [125I]AM binding. We found that CRLR residues 428-439 are required for AM-evoked cAMP production, though deleting this region had little effect on receptor internalization. Moreover, pretreatment with pertussis toxin (100 ng/mL) led to significant increases in AM-induced cAMP production via wild-type CRLR/RAMP2 complexes. This effect was canceled by deleting CRLR residues 454-457, suggesting Gi couples to this region. Flow cytometric analysis revealed that CRLR truncation mutants lacking residues in the Ser/Thr-rich region extending from Ser449 to Ser467 were unable to undergo AM-induced receptor internalization and, in contrast to the effect on wild-type CRLR, overexpression of GPCR kinases-2, -3 and -4 failed to promote internalization of CRLR mutants lacking residues 449-467. Thus, the hCRLR C-tail is crucial for AM-evoked cAMP production and internalization of the CRLR/RAMP2, while the receptor internalization is dependent on the aforementioned GPCR kinases, but not Gs coupling.

  11. Function of the cytoplasmic tail of human calcitonin receptor-like receptor in complex with receptor activity-modifying protein 2

    Energy Technology Data Exchange (ETDEWEB)

    Kuwasako, Kenji, E-mail: kuwasako@fc.miyazaki-u.ac.jp [Frontier Science Research Center, University of Miyazaki, 5200 Kihara, Kiyotake, Miyazaki 889-1692 (Japan); Kitamura, Kazuo; Nagata, Sayaka; Hikosaka, Tomomi [Division of Circulation and Body Fluid Regulation, Faculty of Medicine, University of Miyazaki, 5200 Kihara, Kiyotake, Miyazaki 889-1692 (Japan); Kato, Johji [Frontier Science Research Center, University of Miyazaki, 5200 Kihara, Kiyotake, Miyazaki 889-1692 (Japan)

    2010-02-12

    Receptor activity-modifying protein 2 (RAMP2) enables calcitonin receptor-like receptor (CRLR) to form an adrenomedullin (AM)-specific receptor. Here we investigated the function of the cytoplasmic C-terminal tail (C-tail) of human (h)CRLR by co-transfecting its C-terminal mutants into HEK-293 cells stably expressing hRAMP2. Deleting the C-tail from CRLR disrupted AM-evoked cAMP production or receptor internalization, but did not affect [{sup 125}I]AM binding. We found that CRLR residues 428-439 are required for AM-evoked cAMP production, though deleting this region had little effect on receptor internalization. Moreover, pretreatment with pertussis toxin (100 ng/mL) led to significant increases in AM-induced cAMP production via wild-type CRLR/RAMP2 complexes. This effect was canceled by deleting CRLR residues 454-457, suggesting Gi couples to this region. Flow cytometric analysis revealed that CRLR truncation mutants lacking residues in the Ser/Thr-rich region extending from Ser{sup 449} to Ser{sup 467} were unable to undergo AM-induced receptor internalization and, in contrast to the effect on wild-type CRLR, overexpression of GPCR kinases-2, -3 and -4 failed to promote internalization of CRLR mutants lacking residues 449-467. Thus, the hCRLR C-tail is crucial for AM-evoked cAMP production and internalization of the CRLR/RAMP2, while the receptor internalization is dependent on the aforementioned GPCR kinases, but not Gs coupling.

  12. Quantitative structure-activity relationships of selective antagonists of glucagon receptor using QuaSAR descriptors.

    Science.gov (United States)

    Manoj Kumar, Palanivelu; Karthikeyan, Chandrabose; Hari Narayana Moorthy, Narayana Subbiah; Trivedi, Piyush

    2006-11-01

    In the present paper, quantitative structure activity relationship (QSAR) approach was applied to understand the affinity and selectivity of a novel series of triaryl imidazole derivatives towards glucagon receptor. Statistically significant and highly predictive QSARs were derived for glucagon receptor inhibition by triaryl imidazoles using QuaSAR descriptors of molecular operating environment (MOE) employing computer-assisted multiple regression procedure. The generated QSAR models revealed that factors related to hydrophobicity, molecular shape and geometry predominantly influences glucagon receptor binding affinity of the triaryl imidazoles indicating the relevance of shape specific steric interactions between the molecule and the receptor. Further, QSAR models formulated for selective inhibition of glucagon receptor over p38 mitogen activated protein (MAP) kinase of the compounds in the series highlights that the same structural features, which influence the glucagon receptor affinity, also contribute to their selective inhibition. PMID:17077558

  13. Activation of NTS A2a adenosine receptors differentially resets baroreflex control of renal vs. adrenal sympathetic nerve activity.

    Science.gov (United States)

    Ichinose, Tomoko K; O'Leary, Donal S; Scislo, Tadeusz J

    2009-04-01

    The role of nucleus of solitary tract (NTS) A(2a) adenosine receptors in baroreflex mechanisms is controversial. Stimulation of these receptors releases glutamate within the NTS and elicits baroreflex-like decreases in mean arterial pressure (MAP), heart rate (HR), and renal sympathetic nerve activity (RSNA), whereas inhibition of these receptors attenuates HR baroreflex responses. In contrast, stimulation of NTS A(2a) adenosine receptors increases preganglionic adrenal sympathetic nerve activity (pre-ASNA), and the depressor and sympathoinhibitory responses are not markedly affected by sinoaortic denervation and blockade of NTS glutamatergic transmission. To elucidate the role of NTS A(2a) adenosine receptors in baroreflex function, we compared full baroreflex stimulus-response curves for HR, RSNA, and pre-ASNA (intravenous nitroprusside/phenylephrine) before and after bilateral NTS microinjections of selective adenosine A(2a) receptor agonist (CGS-21680; 2.0, 20 pmol/50 nl), selective A(2a) receptor antagonist (ZM-241385; 40 pmol/100 nl), and nonselective A(1) + A(2a) receptor antagonist (8-SPT; 1 nmol/100 nl) in urethane/alpha-chloralose anesthetized rats. Activation of A(2a) receptors decreased the range, upper plateau, and gain of baroreflex-response curves for RSNA, whereas these parameters all increased for pre-ASNA, consistent with direct effects of the agonist on regional sympathetic activity. However, no resetting of baroreflex-response curves along the MAP axis occurred despite the marked decreases in baseline MAP. The antagonists had no marked effects on baseline variables or baroreflex-response functions. We conclude that the activation of NTS A(2a) adenosine receptors differentially alters baroreflex control of HR, RSNA, and pre-ASNA mostly via non-baroreflex mechanism(s), and these receptors have virtually no tonic action on baroreflex control of these sympathetic outputs.

  14. Quantitative impedimetric NPY-receptor activation monitoring and signal pathway profiling in living cells.

    Science.gov (United States)

    te Kamp, Verena; Lindner, Ricco; Jahnke, Heinz-Georg; Krinke, Dana; Kostelnik, Katja B; Beck-Sickinger, Annette G; Robitzki, Andrea A

    2015-05-15

    Label-free and non-invasive monitoring of receptor activation and identification of the involved signal pathways in living cells is an ongoing analytic challenge and a great opportunity for biosensoric systems. In this context, we developed an impedance spectroscopy-based system for the activation monitoring of NPY-receptors in living cells. Using an optimized interdigital electrode array for sensitive detection of cellular alterations, we were able for the first time to quantitatively detect the NPY-receptor activation directly without a secondary or enhancer reaction like cAMP-stimulation by forskolin. More strikingly, we could show that the impedimetric based NPY-receptor activation monitoring is not restricted to the Y1-receptor but also possible for the Y2- and Y5-receptor. Furthermore, we could monitor the NPY-receptor activation in different cell lines that natively express NPY-receptors and proof the specificity of the observed impedimetric effect by agonist/antagonist studies in recombinant NPY-receptor expressing cell lines. To clarify the nature of the observed impedimetric effect we performed an equivalent circuit analysis as well as analyzed the role of cell morphology and receptor internalization. Finally, an antagonist based extensive molecular signal pathway analysis revealed small alterations of the actin cytoskeleton as well as the inhibition of at least L-type calcium channels as major reasons for the observed NPY-induced impedance increase. Taken together, our novel impedance spectroscopy based NPY-receptor activation monitoring system offers the opportunity to identify signal pathways as well as for novel versatile agonist/antagonist screening systems for identification of novel therapeutics in the field of obesity and cancer.

  15. Activation of the A2A adenosine G-protein-coupled receptor by conformational selection.

    Science.gov (United States)

    Ye, Libin; Van Eps, Ned; Zimmer, Marco; Ernst, Oliver P; Prosser, R Scott

    2016-05-12

    Conformational selection and induced fit are two prevailing mechanisms to explain the molecular basis for ligand-based activation of receptors. G-protein-coupled receptors are the largest class of cell surface receptors and are important drug targets. A molecular understanding of their activation mechanism is critical for drug discovery and design. However, direct evidence that addresses how agonist binding leads to the formation of an active receptor state is scarce. Here we use (19)F nuclear magnetic resonance to quantify the conformational landscape occupied by the adenosine A2A receptor (A2AR), a prototypical class A G-protein-coupled receptor. We find an ensemble of four states in equilibrium: (1) two inactive states in millisecond exchange, consistent with a formed (state S1) and a broken (state S2) salt bridge (known as 'ionic lock') between transmembrane helices 3 and 6; and (2) two active states, S3 and S3', as identified by binding of a G-protein-derived peptide. In contrast to a recent study of the β2-adrenergic receptor, the present approach allowed identification of a second active state for A2AR. Addition of inverse agonist (ZM241385) increases the population of the inactive states, while full agonists (UK432097 or NECA) stabilize the active state, S3', in a manner consistent with conformational selection. In contrast, partial agonist (LUF5834) and an allosteric modulator (HMA) exclusively increase the population of the S3 state. Thus, partial agonism is achieved here by conformational selection of a distinct active state which we predict will have compromised coupling to the G protein. Direct observation of the conformational equilibria of ligand-dependent G-protein-coupled receptor and deduction of the underlying mechanisms of receptor activation will have wide-reaching implications for our understanding of the function of G-protein-coupled receptor in health and disease. PMID:27144352

  16. Gemfibrozil, a lipid lowering drug, inhibits the activation of primary human microglia via peroxisome proliferator-activated receptor β

    OpenAIRE

    Jana, Malabendu; Pahan, Kalipada

    2012-01-01

    Microglial activation participates in the pathogenesis of various neuroinflammatory and neurodegenerative diseases. However, mechanisms by which microglial activation could be controlled are poorly understood. Peroxisome proliferator-activated receptors (PPAR) are transcription factors belonging to the nuclear receptor super family with diverse effect. This study underlines the importance of PPARβ/δ in mediating the anti-inflammatory effect of gemfibrozil, an FDA-approved lipid-lowering drug,...

  17. Oral administration of protease inhibits enterotoxigenic Escherichia coli receptor activity in piglet small intestine.

    OpenAIRE

    Mynott, T L; Luke, R K; Chandler, D S

    1996-01-01

    The virulence of enterotoxigenic Escherichia coli (ETEC) is attributed to their ability to adhere via fimbrial adhesins to specific receptors located on the intestinal mucosa. A novel approach to preventing ETEC induced diarrhoea would be to prevent attachment of ETEC to intestine by proteolytically modifying the receptor attachment sites. This study aimed to examine the effect of bromelain, a proteolytic extract obtained from pineapple stems, on ETEC receptor activity in porcine small intest...

  18. Cytokines increase endothelin ETB receptor contractile activity in rat cerebral artery

    DEFF Research Database (Denmark)

    Leseth, K H; Adner, M; Berg, H K;

    1999-01-01

    The plasticity of endothelin ETB receptor activity and the influence of pro-inflammatory cytokines was examined in cerebral artery. In fresh rat basilar artery, the selective ETB receptor agonist, sarafotoxin S6c, induced negligible contractions. However, after 1 day of organ culture, fully defined...... receptor expression in cerebral arteries and sensitivity for pro-inflammatory cytokines, suggest a role in inflammatory cerebral diseases such as stroke....

  19. Pharmacology and toxicology of fibrates as hypolipidemic drugs mediated by nuclear receptor peroxisome proliferator—activated receptor

    Institute of Scientific and Technical Information of China (English)

    SugaT

    2002-01-01

    PPAR(peroxisome proliferator-activated receptor) is a family of nuclear receptor.In recent years,it has been focused for the discovery and development of new drugs which are mediated by PPARs.Fibrate hypolipidemic drugs are the specific and potent ligands to PPAR alpha and have been widely used for the treatment of hyperlipidemia.But these drugs induce hepatocarcinogenesis in rodent animals after the long-term administration.However,there are species differences on these phenomena which are not seen in mammals ioncluding human.To clarify the mechanism of carcinogenesis by these drugs in important for the evaluation of safety of these drugs in human.

  20. The urokinase receptor (uPAR) and the uPAR-associated protein (uPARAP/Endo180)

    DEFF Research Database (Denmark)

    Behrendt, Niels

    2004-01-01

    The breakdown of the barriers formed by extracellular matrix proteins is a pre-requisite for all processes of tissue remodeling. Matrix degradation reactions take part in specific physiological events in the healthy organism but also represent a crucial step in cancer invasion. These degradation...

  1. The formyl peptide receptor like-1 and scavenger receptor MARCO are involved in glial cell activation in bacterial meningitis

    Directory of Open Access Journals (Sweden)

    Jansen Sandra

    2011-02-01

    Full Text Available Abstract Background Recent studies have suggested that the scavenger receptor MARCO (macrophage receptor with collagenous structure mediates activation of the immune response in bacterial infection of the central nervous system (CNS. The chemotactic G-protein-coupled receptor (GPCR formyl-peptide-receptor like-1 (FPRL1 plays an essential role in the inflammatory responses of host defence mechanisms and neurodegenerative disorders such as Alzheimer's disease (AD. Expression of the antimicrobial peptide cathelicidin CRAMP/LL-37 is up-regulated in bacterial meningitis, but the mechanisms underlying CRAMP expression are far from clear. Methods Using a rat meningitis model, we investigated the influence of MARCO and FPRL1 on rCRAMP (rat cathelin-related antimicrobial peptide expression after infection with bacterial supernatants of Streptococcus pneumoniae (SP and Neisseria meningitides (NM. Expression of FPRL1 and MARCO was analyzed by immunofluorescence and real-time RT-PCR in a rat meningitis model. Furthermore, we examined the receptor involvement by real-time RT-PCR, extracellular-signal regulated kinases 1/2 (ERK1/2 phosphorylation and cAMP level measurement in glial cells (astrocytes and microglia and transfected HEK293 cells using receptor deactivation by antagonists. Receptors were inhibited by small interference RNA and the consequences in NM- and SP-induced Camp (rCRAMP gene expression and signal transduction were determined. Results We show an NM-induced increase of MARCO expression by immunofluorescence and real-time RT-PCR in glial and meningeal cells. Receptor deactivation by antagonists and small interfering RNA (siRNA verified the importance of FPRL1 and MARCO for NM- and SP-induced Camp and interleukin-1β expression in glial cells. Furthermore, we demonstrated a functional interaction between FPRL1 and MARCO in NM-induced signalling by real-time RT-PCR, ERK1/2 phosphorylation and cAMP level measurement and show differences between

  2. Receptor interconversion model of hormone action. 3. Estrogen receptor mediated repression of reporter gene activity in A431 cells.

    Science.gov (United States)

    Nag, A; Park, I; Krust, A; Smith, R G

    1990-03-20

    The chicken estrogen receptor exists in three interconvertible forms, two of which bind estradiol with high affinity and one which lacks the capacity to bind estradiol. Interconversion is regulated by reactions involving ATP/Mg2+. By cotransfecting into A431 cells estrogen receptor cDNA in an expression vector together with the pA2 (-821/-87) tk-CAT vitellogenin construct, we demonstrate that constitutive expression of chloramphenicol acetyltransferase (CAT) activity can be regulated either by selection of ligand or by modifying phosphorylation reactions in the recipient cells. In the presence of estrogen receptors, constitutive expression of CAT activity is inhibited in three situations: (i) in the absence of an estrogenic ligand; (ii) in the presence of an anti-estrogen; and (iii) in the presence of an estrogenic ligand together with 12-O-tetradecanoylphorbol 13-acetate (TPA). Estrogen receptor mediated repression of constitutive CAT activity is not observed with the pA2 (-331/-87) tk-CAT construct, indicating that DNA sequences required for repression are located between -821 and -331 base pairs upstream of the transcription initiation site. PMID:2346742

  3. β-Arrestin biosensors reveal a rapid, receptor-dependent activation/deactivation cycle.

    Science.gov (United States)

    Nuber, Susanne; Zabel, Ulrike; Lorenz, Kristina; Nuber, Andreas; Milligan, Graeme; Tobin, Andrew B; Lohse, Martin J; Hoffmann, Carsten

    2016-03-31

    (β-)Arrestins are important regulators of G-protein-coupled receptors (GPCRs). They bind to active, phosphorylated GPCRs and thereby shut off 'classical' signalling to G proteins, trigger internalization of GPCRs via interaction with the clathrin machinery and mediate signalling via 'non-classical' pathways. In addition to two visual arrestins that bind to rod and cone photoreceptors (termed arrestin1 and arrestin4), there are only two (non-visual) β-arrestin proteins (β-arrestin1 and β-arrestin2, also termed arrestin2 and arrestin3), which regulate hundreds of different (non-visual) GPCRs. Binding of these proteins to GPCRs usually requires the active form of the receptors plus their phosphorylation by G-protein-coupled receptor kinases (GRKs). The binding of receptors or their carboxy terminus as well as certain truncations induce active conformations of (β-)arrestins that have recently been solved by X-ray crystallography. Here we investigate both the interaction of β-arrestin with GPCRs, and the β-arrestin conformational changes in real time and in living human cells, using a series of fluorescence resonance energy transfer (FRET)-based β-arrestin2 biosensors. We observe receptor-specific patterns of conformational changes in β-arrestin2 that occur rapidly after the receptor-β-arrestin2 interaction. After agonist removal, these changes persist for longer than the direct receptor interaction. Our data indicate a rapid, receptor-type-specific, two-step binding and activation process between GPCRs and β-arrestins. They further indicate that β-arrestins remain active after dissociation from receptors, allowing them to remain at the cell surface and presumably signal independently. Thus, GPCRs trigger a rapid, receptor-specific activation/deactivation cycle of β-arrestins, which permits their active signalling. PMID:27007855

  4. Activity of protease-activated receptors in primary cultured human myenteric neurons

    Directory of Open Access Journals (Sweden)

    Eva Maria Kugler

    2012-09-01

    Full Text Available Activity of the four known protease-activated receptors (PARs has been well studied in rodent enteric nervous system and results in animal models established an important role for neuronal PAR2. We recently demonstrated that, unlike in rodents, PAR1 is the dominant neuronal protease receptor in the human submucous plexus. With this study we investigated whether this also applies to the human myenteric plexus. We used voltage sensitive dye recordings to detect action potential discharge in primary cultures of human myenteric neurons in response to PAR activating peptides (AP. Application of the PAR1-AP (TFLLR or PAR4-AP (GYPGQV evoked spike discharge in 79% or 23% of myenteric neurons, respectively. The PAR1-AP response was mimicked by the endogenous PAR1 activator thrombin and blocked by the PAR1 antagonists SCH79797. Human myenteric neurons did not respond to PAR2-AP. This was not due to culture conditions because all three PAR-APs evoked action potentials in cultured guinea pig myenteric neurons. Consecutive application of PAR-APs revealed coexpression (relative to the population responding to PAR-APs of PAR1/PAR2 in 51%, PAR1/PAR4 in 43% and of PAR2/PAR4 in 29% of guinea pig myenteric neurons. Our study provided further evidence for the prominent role of neuronal PAR1 in the human enteric nervous system.

  5. Xenobiotic-induced hepatocyte proliferation associated with constitutive active/androstane receptor (CAR or peroxisome proliferator-activated receptor α (PPARα is enhanced by pregnane X receptor (PXR activation in mice.

    Directory of Open Access Journals (Sweden)

    Ryota Shizu

    Full Text Available Xenobiotic-responsive nuclear receptors pregnane X receptor (PXR, constitutive active/androstane receptor (CAR and peroxisome proliferator-activated receptor α (PPARα play pivotal roles in the metabolic functions of the liver such as xenobiotics detoxification and energy metabolism. While CAR or PPARα activation induces hepatocyte proliferation and hepatocarcinogenesis in rodent models, it remains unclear whether PXR activation also shows such effects. In the present study, we have investigated the role of PXR in the xenobiotic-induced hepatocyte proliferation with or without CAR activation by 1,4-bis[2-(3,5-dichloropyridyloxy]benzene (TCPOBOP and phenobarbital, or PPARα activation by Wy-14643 in mice. Treatment with TCPOBOP or phenobarbital increased the percentage of Ki-67-positive nuclei as well as mRNA levels of cell proliferation-related genes in livers as expected. On the other hand, treatment with the PXR activator pregnenolone 16α-carbonitrile (PCN alone showed no such effects. Surprisingly, PCN co-treatment significantly augmented the hepatocyte proliferation induced by CAR activation with TCPOBOP or phenobarbital in wild-type mice but not in PXR-deficient mice. Intriguingly, PXR activation also augmented the hepatocyte proliferation induced by Wy-14643 treatment. Moreover, PCN treatment increased the RNA content of hepatocytes, suggesting the induction of G0/G1 transition, and reduced mRNA levels of Cdkn1b and Rbl2, encoding suppressors of cell cycle initiation. Our present findings indicate that xenobiotic-induced hepatocyte proliferation mediated by CAR or PPARα is enhanced by PXR co-activation despite that PXR activation alone does not cause the cell proliferation in mouse livers. Thus PXR may play a novel and unique role in the hepatocyte/liver hyperplasia upon exposure to xenobiotics.

  6. Molecular photoacoustic tomography of breast cancer using receptor targeted magnetic iron oxide nanoparticles as contrast agents.

    Science.gov (United States)

    Xi, Lei; Grobmyer, Stephen R; Zhou, Guangyin; Qian, Weiping; Yang, Lily; Jiang, Huabei

    2014-06-01

    In this report, we present a breast imaging technique combining high-resolution near-infrared (NIR) light induced photoacoustic tomography (PAT) with NIR dye-labeled amino-terminal fragments of urokinase plasminogen activator receptor (uPAR) targeted magnetic iron oxide nanoparticles (NIR830-ATF-IONP) for breast cancer imaging using an orthotopic mouse mammary tumor model. We show that accumulation of the targeted nanoparticles in the tumor led to photoacoustic contrast enhancement due to the high absorption of iron oxide nanoparticles (IONP). NIR fluorescence images were used to validate specific delivery of NIR830-ATF-IONP to mouse mammary tumors. We found that systemic delivery of the targeted IONP produced 4- and 10-fold enhancement in photoacoustic signals in the tumor, compared to the tumor of the mice that received non-targeted IONP or control mice. The use of targeted nanoparticles allowed imaging of tumors located as deep as 3.1 cm beneath the normal tissues. Our study indicates the potential of the combination of photoacoustic tomography and receptor-targeted NIR830-ATF-IONP as a clinical tool that can provide improved specificity and sensitivity for breast cancer detection.

  7. Analysis of the epidermal growth factor receptor specific transcriptome: effect of receptor expression level and an activating mutation

    DEFF Research Database (Denmark)

    Pedersen, Mikkel W; Pedersen, Nina; Damstrup, Lars;

    2005-01-01

    moderately expressed or overexpressed at an in-itself transforming level. These changes were compared to those induced by the naturally occurring constitutively active variant EGFRvIII. This study provides novel insight on the activities and mechanisms of EGFRvIII and EGFR mediated transformation, as genes...... by interferons. Expression of this module was absent in the EGFRvIII-expressing cell line and the parental cell line. Treatment with the specific EGFR inhibitor AG1478 indicated that the regulations were primary, receptor-mediated events. Furthermore, activation of this module correlated with activation of STAT1...

  8. A2A adenosine receptor antagonism enhances synaptic and motor effects of cocaine via CB1 cannabinoid receptor activation.

    Directory of Open Access Journals (Sweden)

    Alessandro Tozzi

    Full Text Available BACKGROUND: Cocaine increases the level of endogenous dopamine (DA in the striatum by blocking the DA transporter. Endogenous DA modulates glutamatergic inputs to striatal neurons and this modulation influences motor activity. Since D2 DA and A2A-adenosine receptors (A2A-Rs have antagonistic effects on striatal neurons, drugs targeting adenosine receptors such as caffeine-like compounds, could enhance psychomotor stimulant effects of cocaine. In this study, we analyzed the electrophysiological effects of cocaine and A2A-Rs antagonists in striatal slices and the motor effects produced by this pharmacological modulation in rodents. PRINCIPAL FINDINGS: Concomitant administration of cocaine and A2A-Rs antagonists reduced glutamatergic synaptic transmission in striatal spiny neurons while these drugs failed to produce this effect when given in isolation. This inhibitory effect was dependent on the activation of D2-like receptors and the release of endocannabinoids since it was prevented by L-sulpiride and reduced by a CB1 receptor antagonist. Combined application of cocaine and A2A-R antagonists also reduced the firing frequency of striatal cholinergic interneurons suggesting that changes in cholinergic tone might contribute to this synaptic modulation. Finally, A2A-Rs antagonists, in the presence of a sub-threshold dose of cocaine, enhanced locomotion and, in line with the electrophysiological experiments, this enhanced activity required activation of D2-like and CB1 receptors. CONCLUSIONS: The present study provides a possible synaptic mechanism explaining how caffeine-like compounds could enhance psychomotor stimulant effects of cocaine.

  9. Enzymatic Regulation of Steroidogenesis and Nuclear Receptor Activation : Special Focus on Vitamin D and Sex Hormones

    OpenAIRE

    LUNDQVIST, JOHAN

    2011-01-01

    Enzyme-catalyzed reactions are important to regulate steroidogenesis and nuclear receptor activation. The present investigation examines the role of steroid metabolism catalyzed by CYP7B1 for regulation of hormone receptor activation and the effects of vitamin D on enzymatic regulation of steroidogenesis. The study reports data indicating that CYP7B1 can regulate estrogenic signaling by converting estrogens into inactive or less active metabolites. Similar results were obtained for CYP7B1-med...

  10. Interactions between Human Liver Fatty Acid Binding Protein and Peroxisome Proliferator Activated Receptor Selective Drugs

    OpenAIRE

    Tony Velkov

    2013-01-01

    Fatty acid binding proteins (FABPs) act as intracellular shuttles for fatty acids as well as lipophilic xenobiotics to the nucleus, where these ligands are released to a group of nuclear receptors called the peroxisome proliferator activated receptors (PPARs). PPAR mediated gene activation is ultimately involved in maintenance of cellular homeostasis through the transcriptional regulation of metabolic enzymes and transporters that target the activating ligand. Here we show that liver- (L-) FA...

  11. Modulation of visceral pain and inflammation by protease-activated receptors

    OpenAIRE

    Vergnolle, Nathalie

    2004-01-01

    The gastrointestinal (GI) tract is exposed to a large array of proteases, under both physiological and pathophysiological conditions. The discovery of G protein-coupled receptors activated by proteases, the protease-activated receptors (PARs), has highlighted new signaling functions for proteases in the GI tract, particularly in the domains of inflammation and pain mechanisms. Activation of PARs by selective peptidic agonists in the intestine or the pancreas leads to inflammatory events and c...

  12. Effect of highly bioaccumulated polychlorinated biphenyl congeners on estrogen and androgen receptor activity

    DEFF Research Database (Denmark)

    Bonefeld-Jørgensen, E.C.; Andersen, H. R.; Rasmussen, T.H.;

    2001-01-01

    Polychlorinated biphenyls (PCBs) are ubiquitous environmental persistent contaminants giving rise to potential health hazard. Some PCBs exert dioxin-like activities mediated through the aryl hydrocarbon receptor. Although reports on interaction with other nuclear receptors are sparce, some...... pleiotropic effects on the estrogen- and androgen-receptor. In MCF-7 cells a slightly increased cell proliferation was observed at low concentrations (1-10 nM) in cells co-treated with 0.01 nM 17 beta -Estradiol. whereas the compounds inhibited cell growth significantly at 1 and 10 muM. In reporter gene (ERE......-tk-CAT) analysis the three congeners exhibited a significantly estrogen receptor-ligand mediated decrease of the chloramphenicol transferase activity in both control and 10 nM 17 beta -estradiol induced MCF-7 cells. In addition, PCB # 138 elicited a dose-dependent antagonistic effect on androgen receptor activity...

  13. GABA-A receptor-mediated signaling alters the structure of spontaneous activity in the developing retina

    OpenAIRE

    Wang, Chih-Tien; Blankenship, Aaron G.; Anishchenko, Anastasia; Elstrott, Justin; Fikhman, Michael; Nakanishi, Shigetada; Feller, Marla B

    2007-01-01

    Ambient GABA modulates firing patterns in adult neural circuits by tonically activating extrasynaptic GABA-A receptors. Here, we demonstrate that during a developmental period when activation of GABA-A receptors causes membrane depolarization, tonic activation of GABA-A receptors blocks all spontaneous activity recorded in retinal ganglion cells (RGCs) and starburst amacrine cells (SACs). Bath application of the GABA-A receptor agonist muscimol blocked spontaneous correlated increases in intr...

  14. Expression of protease-activated receptor 1 and 2 and anti-tubulogenic activity of protease-activated receptor 1 in human endothelial colony-forming cells.

    Directory of Open Access Journals (Sweden)

    Tiago M Fortunato

    Full Text Available Endothelial colony-forming cells (ECFCs are obtained from the culture of human peripheral blood mononuclear cell (hPBMNC fractions and are characterised by high proliferative and pro-vasculogenic potential, which makes them of great interest for cell therapy. Here, we describe the detection of protease-activated receptor (PAR 1 and 2 amongst the surface proteins expressed in ECFCs. Both receptors are functionally coupled to extracellular signal-regulated kinase (ERK 1 and 2, which become activated and phosphorylated in response to selective PAR1- or PAR2-activating peptides. Specific stimulation of PAR1, but not PAR2, significantly inhibits capillary-like tube formation by ECFCs in vitro, suggesting that tubulogenesis is negatively regulated by proteases able to stimulate PAR1 (e.g. thrombin. The activation of ERKs is not involved in the regulation of tubulogenesis in vitro, as suggested by use of the MEK inhibitor PD98059 and by the fact that PAR2 stimulation activates ERKs without affecting capillary tube formation. Both qPCR and immunoblotting showed a significant downregulation of vascular endothelial growth factor 2 (VEGFR2 in response to PAR1 stimulation. Moreover, the addition of VEGF (50-100 ng/ml but not basic Fibroblast Growth Factor (FGF (25-100 ng/ml rescued tube formation by ECFCs treated with PAR1-activating peptide. Therefore, we propose that reduction of VEGF responsiveness resulting from down-regulation of VEGFR2 is underlying the anti-tubulogenic effect of PAR1 activation. Although the role of PAR2 remains elusive, this study sheds new light on the regulation of the vasculogenic activity of ECFCs and suggests a potential link between adult vasculogenesis and the coagulation cascade.

  15. An angiotensin II type 1 receptor activation switch patch revealed through evolutionary trace analysis

    DEFF Research Database (Denmark)

    Bonde, Marie Mi; Yao, Rong; Ma, Jian-Nong;

    2010-01-01

    Seven transmembrane (7TM) or G protein-coupled receptors constitute a large superfamily of cell surface receptors sharing a structural motif of seven transmembrane spanning alpha helices. Their activation mechanism most likely involves concerted movements of the transmembrane helices, but remains...

  16. An Angiotensin II type 1 receptor activation switch patch revealed through Evolutionary Trace analysis

    DEFF Research Database (Denmark)

    Bonde, Marie Mi; Yao, Rong; Ma, Jian-Nong;

    2010-01-01

    Seven transmembrane (7TM) or G protein-coupled receptors constitute a large superfamily of cell surface receptors sharing a structural motif of seven transmembrane spanning alpha helices. Their activation mechanism most likely involves concerted movements of the transmembrane helices, but remains...

  17. Halogen bonding-enhanced electrochemical halide anion sensing by redox-active ferrocene receptors.

    Science.gov (United States)

    Lim, Jason Y C; Cunningham, Matthew J; Davis, Jason J; Beer, Paul D

    2015-10-01

    The first examples of halogen bonding redox-active ferrocene receptors and their anion electrochemical sensing properties are reported. Halogen bonding was found to significantly amplify the magnitude of the receptor's metallocene redox-couple's voltammetric responses for halide sensing compared to their hydrogen bonding analogues in both acetonitrile and aqueous-acetonitrile solvent media. PMID:26289779

  18. The role of transmembrane segment II in 7TM receptor activation

    DEFF Research Database (Denmark)

    Benned-Jensen, Tau; Rosenkilde, M M

    2009-01-01

    , accumulating evidence emphasize that this is not the case. In this review, we focus on TM-II with an emphasis on position II:20/2.60, and present data from structure-activity studies on a range of Family A 7TM receptors including chemokine, ghrelin and melanocortin receptors in addition to the orphan EBI2...

  19. The role of GH receptor tyrosine phosphorylation in Stat5 activation

    DEFF Research Database (Denmark)

    Hansen, J A; Hansen, L H; Wang, X;

    1997-01-01

    . Mutated GH receptors lacking all but one of these three tyrosines are able to mediate a transcriptional response when transiently transfected into CHO cells together with a Spi 2.1 promoter/luciferase construct. Similarly, these GH receptors were found to be able to mediate activation of Stat5 DNA...

  20. Tumorigenesis induced by the HHV8-encoded chemokine receptor requires ligand modulation of high constitutive activity

    DEFF Research Database (Denmark)

    Holst, P J; Rosenkilde, M M; Manfra, D;

    2001-01-01

    ORF74 (or KSHV-vGPCR) is a highly constitutively active G protein-coupled receptor encoded by HHV8 that is regulated both positively and negatively by endogenous chemokines. When expressed in transgenic mice, this chemokine receptor induces an angioproliferative disease closely resembling Kaposi...

  1. Tumor-Suppressive Activity of Lunatic Fringe in Prostate through Differential Modulation of Notch Receptor Activation

    Directory of Open Access Journals (Sweden)

    Shubing Zhang

    2014-02-01

    Full Text Available Elevated Notch ligand and receptor expression has been associated with aggressive forms of prostate cancer, suggesting a role for Notch signaling in regulation of prostate tumor initiation and progression. Here, we report a critical role for Lunatic Fringe (Lfng, which encodes an O-fucosylpeptide 3-ß-N-acetylglucosaminyltransferase known to modify epidermal growth factor repeats of Notch receptor proteins, in regulation of prostate epithelial differentiation and proliferation, as well as in prostate tumor suppression. Deletion of Lfng in mice caused altered Notch activation in the prostate, associated with elevated accumulation of Notch1, Notch2, and Notch4 intracellular domains, decreased levels of the putative Notch3 intracellular fragment, as well as increased expression of Hes1, Hes5, and Hey2. Loss of Lfng resulted in expansion of the basal layer, increased proliferation of both luminal and basal cells, and ultimately, prostatic intraepithelial neoplasia. The Lfng-null prostate showed down-regulation of prostatic tumor suppressor gene NKX3.1 and increased androgen receptor expression. Interestingly, expression of LFNG and NKX3.1 were positively correlated in publically available human prostate cancer data sets. Knockdown of LFNG in DU-145 prostate cancer cells led to expansion of CD44+CD24− and CD49f+CD24− stem/progenitor-like cell population associated with enhanced prostatosphere-forming capacity. Taken together, these data revealed a tumor-suppressive role for Lfng in the prostate through differential regulation of Notch signaling.

  2. P2Y2 receptor activation decreases blood pressure and increases renal Na+ excretion

    OpenAIRE

    Rieg, Timo; Gerasimova, Maria; Boyer, José L.; Insel, Paul A.; Vallon, Volker

    2011-01-01

    ATP and UTP are endogenous agonists of P2Y2/4 receptors. To define the in vivo effects of P2Y2 receptor activation on blood pressure and urinary excretion, we compared the response to INS45973, a P2Y2/4 receptor agonist and UTP analog, in wild-type (WT) and P2Y2 receptor knockout (P2Y2−/−) mice. INS45973 was administered intravenously as a bolus injection or continuous infusion to determine effects on blood pressure and renal function, respectively. Within seconds, bolus application of INS459...

  3. Activation of peroxisome proliferator-activated receptor-α enhances fatty acid oxidation in human adipocytes

    International Nuclear Information System (INIS)

    Highlights: → PPARα activation increased mRNA expression levels of adipocyte differentiation marker genes and GPDH activity in human adipocytes. → PPARα activation also increased insulin-dependent glucose uptake in human adipocytes. → PPARα activation did not affect lipid accumulation in human adipocytes. → PPARα activation increased fatty acid oxidation through induction of fatty acid oxidation-related genes in human adipocytes. -- Abstract: Peroxisome proliferator-activated receptor-α (PPARα) is a key regulator for maintaining whole-body energy balance. However, the physiological functions of PPARα in adipocytes have been unclarified. We examined the functions of PPARα using human multipotent adipose tissue-derived stem cells as a human adipocyte model. Activation of PPARα by GW7647, a potent PPARα agonist, increased the mRNA expression levels of adipocyte differentiation marker genes such as PPARγ, adipocyte-specific fatty acid-binding protein, and lipoprotein lipase and increased both GPDH activity and insulin-dependent glucose uptake level. The findings indicate that PPARα activation stimulates adipocyte differentiation. However, lipid accumulation was not changed, which is usually observed when PPARγ is activated. On the other hand, PPARα activation by GW7647 treatment induced the mRNA expression of fatty acid oxidation-related genes such as CPT-1B and AOX in a PPARα-dependent manner. Moreover, PPARα activation increased the production of CO2 and acid soluble metabolites, which are products of fatty acid oxidation, and increased oxygen consumption rate in human adipocytes. The data indicate that activation of PPARα stimulates both adipocyte differentiation and fatty acid oxidation in human adipocytes, suggesting that PPARα agonists could improve insulin resistance without lipid accumulation in adipocytes. The expected effects of PPARα activation are very valuable for managing diabetic conditions accompanied by obesity, because PPAR

  4. Activation of multiple mitogen-activated protein kinases by recombinant calcitonin gene-related peptide receptor.

    Science.gov (United States)

    Parameswaran, N; Disa, J; Spielman, W S; Brooks, D P; Nambi, P; Aiyar, N

    2000-02-18

    Calcitonin gene-related peptide is a 37-amino-acid neuropeptide and a potent vasodilator. Although calcitonin gene-related peptide has been shown to have a number of effects in a variety of systems, the mechanisms of action and the intracellular signaling pathways, especially the regulation of mitogen-activated protien kinase (MAPK) pathway, is not known. In the present study we investigated the role of calcitonin gene-related peptide in the regulation of MAPKs in human embryonic kidney (HEK) 293 cells stably transfected with a recombinant porcine calcitonin gene-related peptide-1 receptor. Calcitonin gene-related peptide caused a significant dose-dependent increase in cAMP response and the effect was inhibited by calcitonin gene-related peptide(8-37), the calcitonin gene-related peptide-receptor antagonist. Calcitonin gene-related peptide also caused a time- and concentration-dependent increase in extracellular signal-regulated kinase (ERK) and P38 mitogen-activated protein kinase (P38 MAPK) activities, with apparently no significant change in cjun-N-terminal kinase (JNK) activity. Forskolin, a direct activator of adenylyl cyclase also stimulated ERK and P38 activities in these cells suggesting the invovement of cAMP in this process. Calcitonin gene-related peptide-stimulated ERK and P38 MAPK activities were inhibited significantly by calcitonin gene-related peptide receptor antagonist, calcitonin gene-related peptide-(8-37) suggesting the involvement of calcitonin gene-related peptide-1 receptor. Preincubation of the cells with the cAMP-dependent protein kinase inhibitor, H89 [¿N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, hydrochloride¿] inhibited calcitonin gene-related peptide-mediated activation of ERK and p38 kinases. On the other hand, preincubation of the cells with wortmannin ¿[1S-(1alpha,6balpha,9abeta,11alpha, 11bbeta)]-11-(acetyloxy)-1,6b,7,8,9a,10,11, 11b-octahydro-1-(methoxymethyl)-9a,11b-dimethyl-3H-furo[4,3, 2-de]indeno[4,5-h]-2

  5. PROKR2 missense mutations associated with Kallmann syndrome impair receptor signalling activity.

    Science.gov (United States)

    Monnier, Carine; Dodé, Catherine; Fabre, Ludovic; Teixeira, Luis; Labesse, Gilles; Pin, Jean-Philippe; Hardelin, Jean-Pierre; Rondard, Philippe

    2009-01-01

    Kallmann syndrome (KS) combines hypogonadism due to gonadotropin-releasing hormone deficiency, and anosmia or hyposmia, related to defective olfactory bulb morphogenesis. In a large series of KS patients, ten different missense mutations (p.R85C, p.R85H, p.R164Q, p.L173R, p.W178S, p.Q210R, p.R268C, p.P290S, p.M323I, p.V331M) have been identified in the gene encoding the G protein-coupled receptor prokineticin receptor-2 (PROKR2), most often in the heterozygous state. Many of these mutations were, however, also found in clinically unaffected individuals, thus raising the question of their actual implication in the KS phenotype. We reproduced each of the ten mutations in a recombinant murine Prokr2, and tested their effects on the signalling activity in transfected HEK-293 cells, by measuring intracellular calcium release upon ligand-activation of the receptor. We found that all mutated receptors except one (M323I) had decreased signalling activities. These could be explained by different defective mechanisms. Three mutations (L173R, W178S, P290S) impaired cell surface-targeting of the receptor. One mutation (Q210R) abolished ligand-binding. Finally, five mutations (R85C, R85H, R164Q, R268C, V331M) presumably impaired G protein-coupling of the receptor. In addition, when wild-type and mutant receptors were coexpressed in HEK-293 cells, none of the mutant receptors that were retained within the cells did affect cell surface-targeting of the wild-type receptor, and none of the mutant receptors properly addressed at the plasma membrane did affect wild-type receptor signalling activity. This argues against a dominant negative effect of the mutations in vivo. PMID:18826963

  6. DMPD: Proximal effects of Toll-like receptor activation in dendritic cells. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 17142025 Proximal effects of Toll-like receptor activation in dendritic cells. Watt...) (.svg) (.html) (.csml) Show Proximal effects of Toll-like receptor activation in dendritic cells. PubmedID... 17142025 Title Proximal effects of Toll-like receptor activation in dendritic ce

  7. Allosteric activation of membrane-bound glutamate receptors using coordination chemistry within living cells

    Science.gov (United States)

    Kiyonaka, Shigeki; Kubota, Ryou; Michibata, Yukiko; Sakakura, Masayoshi; Takahashi, Hideo; Numata, Tomohiro; Inoue, Ryuji; Yuzaki, Michisuke; Hamachi, Itaru

    2016-10-01

    The controlled activation of proteins in living cells is an important goal in protein-design research, but to introduce an artificial activation switch into membrane proteins through rational design is a significant challenge because of the structural and functional complexity of such proteins. Here we report the allosteric activation of two types of membrane-bound neurotransmitter receptors, the ion-channel type and the G-protein-coupled glutamate receptors, using coordination chemistry in living cells. The high programmability of coordination chemistry enabled two His mutations, which act as an artificial allosteric site, to be semirationally incorporated in the vicinity of the ligand-binding pockets. Binding of Pd(2,2‧-bipyridine) at the allosteric site enabled the active conformations of the glutamate receptors to be stabilized. Using this approach, we were able to activate selectively a mutant glutamate receptor in live neurons, which initiated a subsequent signal-transduction pathway.

  8. Activating Effect of Benzbromarone, a Uricosuric Drug, on Peroxisome Proliferator-Activated Receptors

    Directory of Open Access Journals (Sweden)

    Chiyoko Kunishima

    2007-01-01

    Full Text Available Benzbromarone, a uricosuric drug, reportedly causes hepatic hypertrophy accompanied by proliferation of peroxisomes in rats. To elucidate the mechanisms underlying induction of peroxisome proliferation by benzbromarone, we examined binding affinity for peroxisome proliferator-activated receptor α (PPARα and γ (PPARγ, and effects on the binding activity of PPARs with peroxisome proliferation-responsive element (PPRE and expression of the PPARs target protein. Binding affinity of benzbromarone for PPARα and PPARγ was examined by reporter gene assay. Binding activity of PPARs with PPRE was determined by electric mobility shift assay, and expression of lipoprotein lipase (LPL and acyl-CoA synthetase (ACS by Western blot method. Benzbromarone displayed affinity for PPARα and PPARγ, and promoted binding of PPARs to PPRE. Furthermore, cultured cells with benzbromarone added showed upregulated expression of LPL and ACS. These results suggest that benzbromarone induces peroxisome proliferation in hepatocytes by binding to PPARs, and controls expression of proteins related to lipid metabolism.

  9. Transcriptional activation by the thyroid hormone receptor through ligand-dependent receptor recruitment and chromatin remodelling

    DEFF Research Database (Denmark)

    Grøntved, Lars; Waterfall, Joshua J; Kim, Dong Wook;

    2015-01-01

    A bimodal switch model is widely used to describe transcriptional regulation by the thyroid hormone receptor (TR). In this model, the unliganded TR forms stable, chromatin-bound complexes with transcriptional co-repressors to repress transcription. Binding of hormone dissociates co-repressors and...

  10. Research Resource: Androgen Receptor Activity Is Regulated Through the Mobilization of Cell Surface Receptor Networks.

    Science.gov (United States)

    Hsiao, Jordy J; Ng, Brandon H; Smits, Melinda M; Martinez, Harryl D; Jasavala, Rohini J; Hinkson, Izumi V; Fermin, Damian; Eng, Jimmy K; Nesvizhskii, Alexey I; Wright, Michael E

    2015-08-01

    The aberrant expression of androgen receptor (AR)-dependent transcriptional programs is a defining pathology of the development and progression of prostate cancers. Transcriptional cofactors that bind AR are critical determinants of prostate tumorigenesis. To gain a deeper understanding of the proteins linked to AR-dependent gene transcription, we performed a DNA-affinity chromatography-based proteomic screen designed to identify proteins involved in AR-mediated gene transcription in prostate tumor cells. Functional experiments validated the coregulator roles of known AR-binding proteins in AR-mediated transcription in prostate tumor cells. More importantly, novel coregulatory functions were detected in components of well-established cell surface receptor-dependent signal transduction pathways. Further experimentation demonstrated that components of the TNF, TGF-β, IL receptor, and epidermal growth factor signaling pathways modulated AR-dependent gene transcription and androgen-dependent proliferation in prostate tumor cells. Collectively, our proteomic dataset demonstrates that the cell surface receptor- and AR-dependent pathways are highly integrated, and provides a molecular framework for understanding how disparate signal-transduction pathways can influence AR-dependent transcriptional programs linked to the development and progression of human prostate cancers.

  11. GABA-B receptor activation and conflict behavior

    Energy Technology Data Exchange (ETDEWEB)

    Ketelaars, C.E.J.; Bollen, E.L.; Rigter, H.; Bruinvels, J.

    1988-01-01

    Baclofen and oxazepam enhance extinction of conflict behavior in the Geller-Seifter test while baclofen and diazepam release punished behavior in Vogel's conflict test. In order to investigate the possibility that the effect of the selective GABA-B receptor agonist baclofen is mediated indirectly via the GABA-A/benzodiazepine receptor complex, the effect of pretreatment of rats with baclofen on (/sup 3/H)-diazepam binding to washed and unwashed cortical and cerebellar membranes of rats has been studied. Baclofen pretreatment increase Bmax in washed cerebellar membranes when bicuculline was present in the incubation mixture. No effect was seen in cortical membranes. The present results render it unlikely that the effect of baclofen on extinction of conflict behavior and punished drinking is mediated via the GABA-A/benzodiazepine receptor complex. 50 references, 1 figure, 4 tables.

  12. GABA-B receptor activation and conflict behavior

    International Nuclear Information System (INIS)

    Baclofen and oxazepam enhance extinction of conflict behavior in the Geller-Seifter test while baclofen and diazepam release punished behavior in Vogel's conflict test. In order to investigate the possibility that the effect of the selective GABA-B receptor agonist baclofen is mediated indirectly via the GABA-A/benzodiazepine receptor complex, the effect of pretreatment of rats with baclofen on [3H]-diazepam binding to washed and unwashed cortical and cerebellar membranes of rats has been studied. Baclofen pretreatment increase Bmax in washed cerebellar membranes when bicuculline was present in the incubation mixture. No effect was seen in cortical membranes. The present results render it unlikely that the effect of baclofen on extinction of conflict behavior and punished drinking is mediated via the GABA-A/benzodiazepine receptor complex. 50 references, 1 figure, 4 tables

  13. G protein activation by G protein coupled receptors: ternary complex formation or catalyzed reaction?

    Science.gov (United States)

    Roberts, David J; Waelbroeck, Magali

    2004-09-01

    G protein coupled receptors catalyze the GDP/GTP exchange on G proteins, thereby activating them. The ternary complex model, designed to describe agonist binding in the absence of GTP, is often extended to G protein activation. This is logically unsatisfactory as the ternary complex does not accumulate when G proteins are activated by GTP. Extended models taking into account nucleotide binding exist, but fail to explain catalytic G protein activation. This review puts forward an enzymatic model of G protein activation and compares its predictions with the ternary complex model and with observed receptor phenomenon. This alternative model does not merely provide a new set of formulae but leads to a new philosophical outlook and more readily accommodates experimental observations. The ternary complex model implies that, HRG being responsible for efficient G protein activation, it should be as stable as possible. In contrast, the enzyme model suggests that although a limited stabilization of HRG facilitates GDP release, HRG should not be "too stable" as this might trap the G protein in an inactive state and actually hinder G protein activation. The two models also differ completely in the definition of the receptor "active state": the ternary complex model implies that the active state corresponds to a single active receptor conformation (HRG); in contrast, the catalytic model predicts that the active receptor state is mobile, switching smoothly through various conformations with high and low affinities for agonists (HR, HRG, HRGGDP, HRGGTP, etc.).

  14. Molecular basis for activation of G protein-coupled receptor kinases

    Energy Technology Data Exchange (ETDEWEB)

    Boguth, Cassandra A.; Singh, Puja; Huang, Chih-chin; Tesmer, John J.G. (Michigan)

    2012-03-16

    G protein-coupled receptor (GPCR) kinases (GRKs) selectively recognize and are allosterically regulated by activated GPCRs, but the molecular basis for this interaction is not understood. Herein, we report crystal structures of GRK6 in which regions known to be critical for receptor phosphorylation have coalesced to stabilize the kinase domain in a closed state and to form a likely receptor docking site. The crux of this docking site is an extended N-terminal helix that bridges the large and small lobes of the kinase domain and lies adjacent to a basic surface of the protein proposed to bind anionic phospholipids. Mutation of exposed, hydrophobic residues in the N-terminal helix selectively inhibits receptor, but not peptide phosphorylation, suggesting that these residues interact directly with GPCRs. Our structural and biochemical results thus provide an explanation for how receptor recognition, phospholipid binding, and kinase activation are intimately coupled in GRKs.

  15. Roles of activin receptor-like kinase 7 signaling and its target, peroxisome proliferator-activated receptor γ, in lean and obese adipocytes

    OpenAIRE

    Yogosawa, Satomi; Izumi, Tetsuro

    2013-01-01

    We recently discovered a novel signaling pathway involving activin receptor-like kinase 7 (ALK7), one of the type I transforming growth factor-β receptors. ALK7 and activated Smads 2, 3, and 4 inhibit the master regulators of adipogenesis, CCAAT/enhancer-binding protein α (C/EBPα) and peroxisome proliferator-activated receptor γ (PPARγ) specifically in differentiated adipocytes, but surprisingly increase both the adipocyte size and lipid content by suppressing lipolysis. Here, we show that, a...

  16. Chronic regulation of colonic epithelial secretory function by activation of G protein-coupled receptors.

    LENUS (Irish Health Repository)

    Toumi, F

    2011-02-01

    Enteric neurotransmitters that act at G protein-coupled receptors (GPCRs) are well known to acutely promote epithelial Cl(-) and fluid secretion. Here we examined if acute GPCR activation might have more long-term consequences for epithelial secretory function.

  17. Effects related to gene-gene interactions of peroxisome proliferator-activated receptor on essential hypertension

    Institute of Scientific and Technical Information of China (English)

    俞浩

    2013-01-01

    Objective To explore the impact of the gene-gene interaction among the single nucleotide polymorphisms(SNPs) of peroxisome proliferator-activated receptorα/δ/γ on essential hypertension(EH).Methods

  18. Direct activation of GABAA receptors by substances in the organic acid fraction of Japanese sake.

    Science.gov (United States)

    Izu, Hanae; Shigemori, Kensuke; Eguchi, Masaya; Kawane, Shuhei; Fujii, Shouko; Kitamura, Yuji; Aoshima, Hitoshi; Yamada, Yasue

    2017-01-01

    We investigated the effect of substances present in Japanese sake on the response of ionotropic γ-aminobutyric acid (GABA)A receptors expressed in Xenopus oocytes. Sake was fractionated by ion-exchange chromatography. The fraction containing organic acids (OA fraction) showed agonist activities on the GABAA receptor. OA fractions from sake were analyzed by capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS). Of the 64 compounds identified, 13 compounds showed GABAA receptor agonist activities. Especially, l-lactic acid showed high agonist activity and its EC50 value was 37μM. Intraperitoneal injections of l-lactic acid, gluconic acid, and pyruvic acid (10, 10, and 5mg/kg BW, respectively), which showed agonistic activity on the GABAA receptor, led to significant anxiolytic effects during an elevated plus-maze test in mice. PMID:27507485

  19. Differential Requirement of the Extracellular Domain in Activation of Class B G Protein-coupled Receptors.

    Science.gov (United States)

    Zhao, Li-Hua; Yin, Yanting; Yang, Dehua; Liu, Bo; Hou, Li; Wang, Xiaoxi; Pal, Kuntal; Jiang, Yi; Feng, Yang; Cai, Xiaoqing; Dai, Antao; Liu, Mingyao; Wang, Ming-Wei; Melcher, Karsten; Xu, H Eric

    2016-07-15

    G protein-coupled receptors (GPCRs) from the secretin-like (class B) family are key players in hormonal homeostasis and are important drug targets for the treatment of metabolic disorders and neuronal diseases. They consist of a large N-terminal extracellular domain (ECD) and a transmembrane domain (TMD) with the GPCR signature of seven transmembrane helices. Class B GPCRs are activated by peptide hormones with their C termini bound to the receptor ECD and their N termini bound to the TMD. It is thought that the ECD functions as an affinity trap to bind and localize the hormone to the receptor. This in turn would allow the hormone N terminus to insert into the TMD and induce conformational changes of the TMD to activate downstream signaling. In contrast to this prevailing model, we demonstrate that human class B GPCRs vary widely in their requirement of the ECD for activation. In one group, represented by corticotrophin-releasing factor receptor 1 (CRF1R), parathyroid hormone receptor (PTH1R), and pituitary adenylate cyclase activating polypeptide type 1 receptor (PAC1R), the ECD requirement for high affinity hormone binding can be bypassed by induced proximity and mass action effects, whereas in the other group, represented by glucagon receptor (GCGR) and glucagon-like peptide-1 receptor (GLP-1R), the ECD is required for signaling even when the hormone is covalently linked to the TMD. Furthermore, the activation of GLP-1R by small molecules that interact with the intracellular side of the receptor is dependent on the presence of its ECD, suggesting a direct role of the ECD in GLP-1R activation. PMID:27226600

  20. Artificial sweeteners and salts producing a metallic taste sensation activate TRPV1 receptors.

    Science.gov (United States)

    Riera, Céline E; Vogel, Horst; Simon, Sidney A; le Coutre, Johannes

    2007-08-01

    Throughout the world many people use artificial sweeteners (AS) for the purpose of reducing caloric intake. The most prominently used of these molecules include saccharin, aspartame (Nutrasweet), acesulfame-K, and cyclamate. Despite the caloric advantage they provide, one key concern in their use is their aversive aftertaste that has been characterized on a sensory level as bitter and/or metallic. Recently, it has been shown that the activation of particular T2R bitter taste receptors is partially involved with the bitter aftertaste sensation of saccharin and acesulfame-K. To more fully understand the biology behind these phenomena we have addressed the question of whether AS could stimulate transient receptor potential vanilloid-1 (TRPV1) receptors, as these receptors are activated by a large range of structurally different chemicals. Moreover, TRPV1 receptors and/or their variants are found in taste receptor cells and in nerve terminals throughout the oral cavity. Hence, TRPV1 activation could be involved in the AS aftertaste or even contribute to the poorly understood metallic taste sensation. Using Ca(2+) imaging on TRPV1 receptors heterologously expressed in the human embryonic kidney (HEK) 293 cells and on dissociated primary sensory neurons, we find that in both systems, AS activate TRPV1 receptors, and, moreover, they sensitize these channels to acid and heat. We also found that TRPV1 receptors are activated by CuSO(4), ZnSO(4), and FeSO(4), three salts known to produce a metallic taste sensation. In summary, our results identify a novel group of compounds that activate TRPV1 and, consequently, provide a molecular mechanism that may account for off tastes of sweeteners and metallic tasting salts. PMID:17567713

  1. Activation of mitogen activated protein kinases via complement receptor type 2

    Institute of Scientific and Technical Information of China (English)

    LUO Min-hua 罗敏华; CHEN Ming-liang 陈明亮; Heribert Stoiber; Manfred P Dierich

    2004-01-01

    Background Complement receptor type 2 (CR2) is the receptor for C3d and C3dg and for Epstein-Barr virus. The aim of our study was to explore whether CR2 can independently mediate the activation of mitogen-activated protein kinases (MAPKs, including ERK, JNK, and p38MAPK), and to highlight the molecular mechanism of CD4+ cell deletion in AIDS.Results FACS results showed that the positive rates of HOS-CR2 and HOS-CD4CR2 cells were greater than 96%, and Western blot showed that the CR2 expression levels on HOS-CR2 and HOS-CD4CR2 cells were high. Activation and blocking tests of MAPKs (ERK, JNK, and p38MAPK) were carried out in HOS-CR2, HOS-CD4, and HOS-CD4CR2 cells. The activation of MAPKs in HOS-CR2 cells stimulated with PMA (100 ng/ml) and NHS (10%) was identical. The activation of MAPKs increased at 5 minutes, reached a peak at 10 minutes, and decreased to baseline within 30 minutes, all in a time-dependent manner; the activation of MAPKs was blocked by anti-CR2 McAb, PD98059 (inhibitor of ERK), and Wortmanin (inhibitor of PI-3K), respectively. In HOS-CD4 cells, MAPKs were activated by HIV-gp160. In HOS-CD4CR2 cells, MAPK activation was induced by HIV-gp160, 10% NHS, and HIV-gp160+10%NHS; phosphorylation of p38MAPK was dramatically induced by HIV-gp160+NHS, and lasted for 1 hour. The cell proliferation results showed that HIV-gp160 inhibited the proliferation of HOS-CD4 and HOS-CD4CR2 cells (P<0.01) and that NHS enhanced the effect of HIV-gp160 (P<0.01).Conclusions The activation of MAPKs is independently mediated by CR2 and that anti-CR2 McAb, PD98059, and Wortmanin block the activation of MAPKs, respectively. The results of the signal transduction and cell proliferation assays of HOS-CD4CR2 cells show that CR2 plays a role in the pathogenesis of HIV infection, especially in the inhibition of CD4+ cell proliferation.

  2. EPO-independent functional EPO receptor in breast cancer enhances estrogen receptor activity and promotes cell proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Reinbothe, Susann; Larsson, Anna-Maria; Vaapil, Marica; Wigerup, Caroline [Department of Laboratory Medicine, Translational Cancer Research, Medicon Village, Lund University, SE-223 81 Lund (Sweden); CREATE Health, Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv (Israel); Sun, Jianmin [Department of Laboratory Medicine, Translational Cancer Research, Medicon Village, Lund University, SE-223 81 Lund (Sweden); Jögi, Annika [Department of Laboratory Medicine, Translational Cancer Research, Medicon Village, Lund University, SE-223 81 Lund (Sweden); CREATE Health, Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv (Israel); Neumann, Drorit [Department of Cell and Developmental Biology, Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv (Israel); Rönnstrand, Lars [Department of Laboratory Medicine, Translational Cancer Research, Medicon Village, Lund University, SE-223 81 Lund (Sweden); Påhlman, Sven, E-mail: sven.pahlman@med.lu.se [Department of Laboratory Medicine, Translational Cancer Research, Medicon Village, Lund University, SE-223 81 Lund (Sweden); CREATE Health, Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv (Israel)

    2014-02-28

    Highlights: • New anti-human EPOR antibody confirms full-length EPOR expression in breast cancer cells. • Proliferation of breast cancer cells is not affected by rhEPO treatment in vitro. • EPOR knockdown impairs proliferation of ERa positive breast cancer cells. • EPOR knockdown reduces AKT phosphorylation and ERa activity. - Abstract: The main function of Erythropoietin (EPO) and its receptor (EPOR) is the stimulation of erythropoiesis. Recombinant human EPO (rhEPO) is therefore used to treat anemia in cancer patients. However, clinical trials have indicated that rhEPO treatment might promote tumor progression and has a negative effect on patient survival. In addition, EPOR expression has been detected in several cancer forms. Using a newly produced anti-EPOR antibody that reliably detects the full-length isoform of the EPOR we show that breast cancer tissue and cells express the EPOR protein. rhEPO stimulation of cultured EPOR expressing breast cancer cells did not result in increased proliferation, overt activation of EPOR (receptor phosphorylation) or a consistent activation of canonical EPOR signaling pathway mediators such as JAK2, STAT3, STAT5, or AKT. However, EPOR knockdown experiments suggested functional EPO receptors in estrogen receptor positive (ERα{sup +}) breast cancer cells, as reduced EPOR expression resulted in decreased proliferation. This effect on proliferation was not seen in ERα negative cells. EPOR knockdown decreased ERα activity further supports a mechanism by which EPOR affects proliferation via ERα-mediated mechanisms. We show that EPOR protein is expressed in breast cancer cells, where it appears to promote proliferation by an EPO-independent mechanism in ERα expressing breast cancer cells.

  3. EPO-independent functional EPO receptor in breast cancer enhances estrogen receptor activity and promotes cell proliferation

    International Nuclear Information System (INIS)

    Highlights: • New anti-human EPOR antibody confirms full-length EPOR expression in breast cancer cells. • Proliferation of breast cancer cells is not affected by rhEPO treatment in vitro. • EPOR knockdown impairs proliferation of ERa positive breast cancer cells. • EPOR knockdown reduces AKT phosphorylation and ERa activity. - Abstract: The main function of Erythropoietin (EPO) and its receptor (EPOR) is the stimulation of erythropoiesis. Recombinant human EPO (rhEPO) is therefore used to treat anemia in cancer patients. However, clinical trials have indicated that rhEPO treatment might promote tumor progression and has a negative effect on patient survival. In addition, EPOR expression has been detected in several cancer forms. Using a newly produced anti-EPOR antibody that reliably detects the full-length isoform of the EPOR we show that breast cancer tissue and cells express the EPOR protein. rhEPO stimulation of cultured EPOR expressing breast cancer cells did not result in increased proliferation, overt activation of EPOR (receptor phosphorylation) or a consistent activation of canonical EPOR signaling pathway mediators such as JAK2, STAT3, STAT5, or AKT. However, EPOR knockdown experiments suggested functional EPO receptors in estrogen receptor positive (ERα+) breast cancer cells, as reduced EPOR expression resulted in decreased proliferation. This effect on proliferation was not seen in ERα negative cells. EPOR knockdown decreased ERα activity further supports a mechanism by which EPOR affects proliferation via ERα-mediated mechanisms. We show that EPOR protein is expressed in breast cancer cells, where it appears to promote proliferation by an EPO-independent mechanism in ERα expressing breast cancer cells

  4. Therapy for acute pancreatitis with platelet-activating factor receptor antagonists

    OpenAIRE

    Chen, Chong; Xia, Shi-hai; Chen, Hong; Li, Xiao-Hong

    2008-01-01

    Acute pancreatitis (AP) causes release of platelet-activating factor (PAF), which induces systemic effects that contribute to circulatory disturbances and multiple organ failure. PAF is a cell surface secretion of bioactive lipid, which could produce physiological and pathological effects by binding to its cell surface receptor called platelet-activating factor receptor (PAF-R). Studies showed that PAF participates in the occurrence and development of AP and administration of platelet-activat...

  5. Farnesoid X Receptor Inhibits the Transcriptional Activity of Carbohydrate Response Element Binding Protein in Human Hepatocytes

    OpenAIRE

    Caron, Sandrine; Huaman Samanez, Carolina; Dehondt, Hélène; Ploton, Maheul; Briand, Olivier; Lien, Fleur; Dorchies, Emilie; Dumont, Julie; Postic, Catherine; Cariou, Bertrand; Lefebvre, Philippe; Staels, Bart

    2013-01-01

    The glucose-activated transcription factor carbohydrate response element binding protein (ChREBP) induces the expression of hepatic glycolytic and lipogenic genes. The farnesoid X receptor (FXR) is a nuclear bile acid receptor controlling bile acid, lipid, and glucose homeostasis. FXR negatively regulates hepatic glycolysis and lipogenesis in mouse liver. The aim of this study was to determine whether FXR regulates the transcriptional activity of ChREBP in human hepatocytes and to unravel the...

  6. Constitutive Activity among Orphan Class-A G Protein Coupled Receptors

    OpenAIRE

    Martin, Adam L.; Michael A Steurer; Aronstam, Robert S.

    2015-01-01

    The purpose of this study was to evaluate the extent of constitutive activity among orphan class-A G protein coupled receptors within the cAMP signaling pathway. Constitutive signaling was revealed by changes in gene expression under control of the cAMP response element. Gene expression was measured in Chinese hamster ovary cells transiently co-transfected with plasmids containing a luciferase reporter and orphan receptor. Criteria adopted for defining constitutive activation were: 1) 200% el...

  7. Evaluation on Potential Contributions of Protease Activated Receptors Related Mediators in Allergic Inflammation

    OpenAIRE

    Huiyun Zhang; Xiaoning Zeng; Shaoheng He

    2014-01-01

    Protease activated receptors (PARs) have been recognized as a distinctive four-member family of seven transmembrane G protein-coupled receptors (GPCRs) that can be cleaved by certain serine proteases. In recent years, there has been considerable interest in the role of PARs in allergic inflammation, the fundamental pathologic changes of allergy, but the potential roles of PARs in allergy remain obscure. Since many of these proteases are produced and actively involved in the pathologic process...

  8. Receptor activator of nuclear factor kappa B ligand and osteoprotegerin levels in gingival crevicular fluid

    OpenAIRE

    Fatemeh Sarlati; Mandana Sattari; Shilan Razzaghi; Malihe Nasiri

    2012-01-01

    Background: Osteoclastogenesis is coordinated by the interaction of three members of the tumor necrosis factor (TNF) superfamily: Osteoprotegerin (OPG)/receptor activator of nuclear factor kappa B ligand (RANKL)/receptor activator of nuclear factor kappa B (RANK). The aim of this study was to investigate RANKL and OPG levels, and their relative ratio in gingival crevicular fluid (GCF) of patients with chronic and aggressive periodontitis, as well as healthy controls. Materials and Methods:...

  9. Identification of Functionally Relevant Lysine Residues That Modulate Human Farnesoid X Receptor Activation

    OpenAIRE

    Sun, An-Qiang; Luo, Yuhuan; Backos, Donald S.; Xu, Shuhua; Balasubramaniyan, Natarajan; Reigan, Philip; Suchy, Frederick J.

    2013-01-01

    Base amino acid lysine residues play an important role in regulation of nuclear receptors [e.g., farnesyl X receptor (FXR)], leading to enhanced or suppressed biologic activity. To understand the molecular mechanisms and the subsequent effects in modulating FXR functions in diverse biologic processes, we individually replaced eight highly conserved lysine residues of human FXR (hFXR) with arginine. The effects of each mutated FXR on target gene activation, subcellular localization, protein-pr...

  10. Steroid receptor RNA activator (SRA1): unusual bifaceted gene products with suspected relevance to breast cancer

    OpenAIRE

    Leygue, Etienne

    2007-01-01

    The steroid receptor RNA activator (SRA) is a unique modulator of steroid receptor transcriptional activity, as it is able to mediate its coregulatory effects as a RNA molecule. Recent findings, however, have painted a more complex picture of the SRA gene (SRA1) products. Indeed, even though SRA was initially thought to be noncoding, several RNA isoforms have now been found to encode an endogenous protein (SRAP), which is well conserved among Chordata. Although the function of SRAP remains la...

  11. Structure-activity relationships for the antifungal activity of selective estrogen receptor antagonists related to tamoxifen.

    Directory of Open Access Journals (Sweden)

    Arielle Butts

    Full Text Available Cryptococcosis is one of the most important invasive fungal infections and is a significant contributor to the mortality associated with HIV/AIDS. As part of our program to repurpose molecules related to the selective estrogen receptor modulator (SERM tamoxifen as anti-cryptococcal agents, we have explored the structure-activity relationships of a set of structurally diverse SERMs and tamoxifen derivatives. Our data provide the first insights into the structural requirements for the antifungal activity of this scaffold. Three key molecular characteristics affecting anti-cryptococcal activity emerged from our studies: 1 the presence of an alkylamino group tethered to one of the aromatic rings of the triphenylethylene core; 2 an appropriately sized aliphatic substituent at the 2 position of the ethylene moiety; and 3 electronegative substituents on the aromatic rings modestly improved activity. Using a cell-based assay of calmodulin antagonism, we found that the anti-cryptococcal activity of the scaffold correlates with calmodulin inhibition. Finally, we developed a homology model of C. neoformans calmodulin and used it to rationalize the structural basis for the activity of these molecules. Taken together, these data and models provide a basis for the further optimization of this promising anti-cryptococcal scaffold.

  12. Rapid effect of stress concentration corticosterone on glutamate receptor and its subtype NMDA receptor activity in cultured hippocampal neurons

    Institute of Scientific and Technical Information of China (English)

    刘玲; 孙继虎; 王春安

    2003-01-01

    Objective:To study the rapid effect of glucocorticoids(GCs)on NMDA receptor activity in hippocampal neurons in stress and to elucidate its underlying probable membrane mechanisms.Methods:Whole-cell patch-clamp recording was used to assess the effect of stress concentration corticosterone(B)on the responses of cultured hippocampal neurons to glutamate and NMDA(N-methy-D-asparatic acid).To make clear the target of B,intracellular dialysis of B(10 μ mol/L)through patch pipette and extracellular application of bovine serum albumin-conjugated corticosterone(B-BSA,10 μmol/L)were carried out to observe their influence on peak amplitude of NMDA-evoked current.Results:B had a rapid,reversible and inhibitory effect on peak amplitude of GLU- or NMDA-evoked current in cultured hippocampal neurons.Furthermore,B-BSA had the inhibitory effect on INMDA as that of B,but intracellularly dialyzed B had no significant effect on INMDA.Conclusion:These results suggest that under the condition of stress,GCs may rapidly,negatively regulate excitatory synaptic receptors-glutamate receptors(GluRs),especially NMDA receptor(NMDAR)in central nervous system,which is mediated by rapid membrane mechanisms,but not by classical,genomic mechanisms.

  13. The DEAD-Box Protein DP103 (Ddx20 or Gemin-3) Represses Orphan Nuclear Receptor Activity via SUMO Modification

    OpenAIRE

    Lee, Martin B.; Lebedeva, Lioudmila A.; Suzawa, Miyuki; Wadekar, Subhagya A.; Desclozeaux, Marion; Ingraham, Holly A.

    2005-01-01

    Structural analysis of nuclear receptor subfamily V orphan nuclear receptors suggests that ligand-independent mechanisms must regulate this subclass of receptors. Here, we report that steroidogenic factor 1 (SF-1) and liver receptor homolog 1 are repressed via posttranslational SUMO modification at conserved lysines within the hinge domain. Indeed, mutating these lysines or adding the SUMO isopeptidase SENP1 dramatically increased both native and Gal4-chimera receptor activities. The mechanis...

  14. Activation of Nuclear Receptors RAR, RXR, and LXR Does Not Reduce Cuprizone-Induced Demyelination in Mice

    OpenAIRE

    Davina Kruczek; Tim Clarner; Cordian Beyer; Markus Kipp; Jörg Mey

    2015-01-01

    Experiments with animal models of multiple sclerosis have shown that the expression of retinoid X receptors (RXR) increases during demyelination and that RXR is involved in the regulation of remyelination. After ligand binding RXRs form heterodimeric transcription factors with other nuclear receptor (NR) families including the retinoic acid receptors (RAR) and liver X receptors (LXR). We tested whether activation of these nuclear receptor complexes reduces pathological demyelination using the...

  15. Increased amphetamine-induced locomotor activity, sensitization and accumbal dopamine release in M5 muscarinic receptor knockout mice

    OpenAIRE

    Schmidt, Lene S.; Miller, Anthony D.; Lester, Deranda B.; Bay-Richter, Cecilie; Schülein, Christina; Schmidt, Henriette F.; Wess, Jürgen; Blaha, Charles D.; Woldbye, David P.D.; Fink-Jensen, Anders; Wortwein, Gitta

    2009-01-01

    Muscarinic M5 receptors are the only muscarinic receptor subtype expressed by dopamine-containing neurons of the ventral tegmental area. These cells play an important role for the reinforcing properties of psychostimulants and M5 receptors modulate their activity. Previous studies showed that M5 receptor knockout (M5−/−) mice are less sensitive to the reinforcing properties of addictive drugs. Here we investigate the role of M5 receptors in the effects of amphetamine and cocaine on locomotor ...

  16. GIRK channel activation via adenosine or muscarinic receptors has similar effects on rat atrial electrophysiology.

    Science.gov (United States)

    Wang, Xiaodong; Liang, Bo; Skibsbye, Lasse; Olesen, Søren-Peter; Grunnet, Morten; Jespersen, Thomas

    2013-08-01

    G protein-coupled inwardly rectifying K⁺ channels (GIRK) are important in the regulation of heart rate and atrial electrophysiology. GIRK channels are activated by G protein-coupled receptors, including muscarinic M₂ receptors and adenosine A₁ receptors. The aim of this study was to characterize and compare the electrophysiological effects of acetylcholine (ACh) and adenosine on GIRK channels in rat atria. Action potential duration at 90% repolarization (APD₉₀), effective refractory period (ERP), and resting membrane potential (RMP) were investigated in isolated rat atria by intracellular recordings. Both the adenosine analog N6-cyclopentyladenosine (CPA) and ACh profoundly shortened APD₉₀ and ERP and hyperpolarized the RMP. No additive or synergistic effect of CPA and ACh coapplication was observed. To antagonize GIRK channel activation, the specific inhibitor rTertiapin Q (TTQ) was applied. The coapplication of TTQ reversed the CPA and ACh-induced effects. When TTQ was applied without exogenous receptor activator, both APD₉₀ and ERP were prolonged and RMP was depolarized, confirming a basal activity of the GIRK current. The results reveal that activation of A₁ and M₂ receptors has a profound and equal effect on the electrophysiology in rat atrium. This effect is to a major extent mediated through GIRK channels. Furthermore, these results support the notion that atrial GIRK currents from healthy hearts have a basal component and additional activation can be mediated via at least 2 different receptor mechanisms. PMID:23609329

  17. Activation of the Retinoid X Receptor Modulates Angiotensin II-Induced Smooth Muscle Gene Expression and Inflammation in Vascular Smooth Muscle Cells

    OpenAIRE

    Lehman, Allison M.B.; Montford, John R.; Horita, Henrick; Ostriker, Allison C.; Weiser-Evans, Mary C. M.; Nemenoff, Raphael A.; Furgeson, Seth B.

    2014-01-01

    The retinoid X receptor (RXR) partners with numerous nuclear receptors, such as the peroxisome proliferator activated receptor (PPAR) family, liver X receptors (LXRs), and farnesoid X receptor (FXR). Although each heterodimer can be activated by specific ligands, a subset of these receptors, defined as permissive nuclear receptors, can also be activated by RXR agonists known as rexinoids. Many individual RXR heterodimers have beneficial effects in vascular smooth muscle cells (SMCs). Because ...

  18. Selective androgen receptor modulator activity of a steroidal antiandrogen TSAA-291 and its cofactor recruitment profile.

    Science.gov (United States)

    Hikichi, Yukiko; Yamaoka, Masuo; Kusaka, Masami; Hara, Takahito

    2015-10-15

    Selective androgen receptor modulators (SARMs) specifically bind to the androgen receptor and exert agonistic or antagonistic effects on target organs. In this study, we investigated the SARM activity of TSAA-291, previously known as a steroidal antiandrogen, in mice because TSAA-291 was found to possess partial androgen receptor agonist activity in reporter assays. In addition, to clarify the mechanism underlying its tissue selectivity, we performed comprehensive cofactor recruitment analysis of androgen receptor using TSAA-291 and dihydrotestosterone (DHT), an endogenous androgen. The androgen receptor agonistic activity of TSAA-291 was more obvious in reporter assays using skeletal muscle cells than in those using prostate cells. In castrated mice, TSAA-291 increased the weight of the levator ani muscle without increasing the weight of the prostate and seminal vesicle. Comprehensive cofactor recruitment analysis via mammalian two-hybrid methods revealed that among a total of 112 cofactors, 12 cofactors including the protein inhibitor of activated STAT 1 (PIAS1) were differently recruited to androgen receptor in the presence of TSAA-291 and DHT. Prostate displayed higher PIAS1 expression than skeletal muscle. Forced expression of the PIAS1 augmented the transcriptional activity of the androgen receptor, and silencing of PIAS1 by siRNAs suppressed the secretion of prostate-specific antigen, an androgen responsive marker. Our results demonstrate that TSAA-291 has SARM activity and suggest that TSAA-291 may induce different conformational changes of the androgen receptor and recruitment profiles of cofactors such as PIAS1, compared with DHT, to exert tissue-specific activity.

  19. The Relaxin Receptor (RXFP1) Utilizes Hydrophobic Moieties on a Signaling Surface of Its N-terminal Low Density Lipoprotein Class A Module to Mediate Receptor Activation*

    Science.gov (United States)

    Kong, Roy C. K.; Petrie, Emma J.; Mohanty, Biswaranjan; Ling, Jason; Lee, Jeremy C. Y.; Gooley, Paul R.; Bathgate, Ross A. D.

    2013-01-01

    The peptide hormone relaxin is showing potential as a treatment for acute heart failure. Although it is known that relaxin mediates its actions through the G protein-coupled receptor relaxin family peptide receptor 1 (RXFP1), little is known about the molecular mechanisms by which relaxin binding results in receptor activation. Previous studies have highlighted that the unique N-terminal low density lipoprotein class A (LDLa) module of RXFP1 is essential for receptor activation, and it has been hypothesized that this module is the true “ligand” of the receptor that directs the conformational changes necessary for G protein coupling. In this study, we confirmed that an RXFP1 receptor lacking the LDLa module binds ligand normally but cannot signal through any characterized G protein-coupled receptor signaling pathway. Furthermore, we comprehensively examined the contributions of amino acids in the LDLa module to RXFP1 activity using both gain-of-function and loss-of-function mutational analysis together with NMR structural analysis of recombinant LDLa modules. Gain-of-function studies with an inactive RXFP1 chimera containing the LDLa module of the human LDL receptor (LB2) demonstrated two key N-terminal regions of the module that were able to rescue receptor signaling. Loss-of-function mutations of residues in these regions demonstrated that Leu-7, Tyr-9, and Lys-17 all contributed to the ability of the LDLa module to drive receptor activation, and judicious amino acid substitutions suggested this involves hydrophobic interactions. Our results demonstrate that these key residues contribute to interactions driving the active receptor conformation, providing further evidence of a unique mode of G protein-coupled receptor activation. PMID:23926099

  20. The relaxin receptor (RXFP1) utilizes hydrophobic moieties on a signaling surface of its N-terminal low density lipoprotein class A module to mediate receptor activation.

    Science.gov (United States)

    Kong, Roy C K; Petrie, Emma J; Mohanty, Biswaranjan; Ling, Jason; Lee, Jeremy C Y; Gooley, Paul R; Bathgate, Ross A D

    2013-09-27

    The peptide hormone relaxin is showing potential as a treatment for acute heart failure. Although it is known that relaxin mediates its actions through the G protein-coupled receptor relaxin family peptide receptor 1 (RXFP1), little is known about the molecular mechanisms by which relaxin binding results in receptor activation. Previous studies have highlighted that the unique N-terminal low density lipoprotein class A (LDLa) module of RXFP1 is essential for receptor activation, and it has been hypothesized that this module is the true "ligand" of the receptor that directs the conformational changes necessary for G protein coupling. In this study, we confirmed that an RXFP1 receptor lacking the LDLa module binds ligand normally but cannot signal through any characterized G protein-coupled receptor signaling pathway. Furthermore, we comprehensively examined the contributions of amino acids in the LDLa module to RXFP1 activity using both gain-of-function and loss-of-function mutational analysis together with NMR structural analysis of recombinant LDLa modules. Gain-of-function studies with an inactive RXFP1 chimera containing the LDLa module of the human LDL receptor (LB2) demonstrated two key N-terminal regions of the module that were able to rescue receptor signaling. Loss-of-function mutations of residues in these regions demonstrated that Leu-7, Tyr-9, and Lys-17 all contributed to the ability of the LDLa module to drive receptor activation, and judicious amino acid substitutions suggested this involves hydrophobic interactions. Our results demonstrate that these key residues contribute to interactions driving the active receptor conformation, providing further evidence of a unique mode of G protein-coupled receptor activation. PMID:23926099

  1. Estrogen activation of the nuclear orphan receptor CAR (constitutive active receptor) in induction of the mouse Cyp2b10 gene.

    Science.gov (United States)

    Kawamoto, T; Kakizaki, S; Yoshinari, K; Negishi, M

    2000-11-01

    The nuclear orphan receptor CAR (constitutively active receptor or constitutive androstane receptor) can be activated in response to xenochemical exposure, such as activation by phenobarbital of a response element called NR1 found in the CYP2B gene. Here various steroids were screened for potential endogenous chemicals that may activate CAR, using the NR1 enhancer and Cyp2b10 induction in transfected HepG2 cell and/or in mouse primary hepatocytes as the experimental criteria. 17beta-Estradiol and estrone activated NR1, whereas estriol, estetrol, estradiol sulfate, and the synthetic estrogen diethylstilbestrol did not. On the other hand, progesterone and androgens repressed NR1 activity in HepG2 cells, and the repressed NR1 activity was fully restored by estradiol. Moreover, estrogen treatment elicited nuclear accumulation of CAR in the mouse livers, as well as primary hepatocytes, and induced the endogenous Cyp2b10 gene. Ovariectomy did not affect either the basal or induced level of CAR in the nucleus of the female livers, while castration slightly increased the basal and greatly increased the induced levels in the liver nucleus of male mice. Thus, endogenous estrogen appears not to regulate CAR in female mice, whereas endogenous androgen may be the repressive factor in male mice. Estrogen at pharmacological levels is an effective activator of CAR in both female and male mice, suggesting a biological and/or toxicological role of this receptor in estrogen metabolism. In addition to mouse CAR, estrogens activated rat CAR, whereas human CAR did not respond well to the estrogens under the experimental conditions. PMID:11075820

  2. Cellular, Molecular Consequences of Peroxisome Proliferator- Activated ReceptorActivation in Ovarian Cancer Cells

    Directory of Open Access Journals (Sweden)

    Sara Vignati

    2006-10-01

    Full Text Available Peroxisome proliferator-activated receptor-δ (PPAR-δ is a ligand-activated transcription factor. In addition to its canonical role in lipid, glucose metabolism, PPAR-δ controls cell proliferation, death, differentiation in several tissues. Here we have examined the expression of PPAR-δ in ovarian tumors, the cellular, molecular consequences of its activation in ovarian cancer cells. PPAR-δ was expressed in a large number of epithelial ovarian tumors, cell lines. The PPAR-δ lig, ciglitazone inhibited the growth, clonogenic survival of ovarian cancer cells, inducing cell cycle arrest, cell death. Growth inhibition by ciglitazone was reversed by the PPAR-δ antagonist GW9662, indicating the involvement of PPAR-δ- dependent mechanisms. Microarray-based gene profiling revealed complex changes in the transcriptional program of ovarian cancer cells on treatment with ciglitazone, identified multiple pathways that may contribute to PPAR-δ ligands' antitumor activity. Genes upregulated by ciglitazone were predominantly associated with metabolic, differentiation, tumorsuppressor pathways, whereas downregulated genes were involved in cell proliferation, cell cycle, cell organization, steroid biosynthesis. Collectively, our data indicate that PPAR-δ activation by selective agonists is a valid strategy for ovarian cancer therapy, prevention, should be tested alone, in combination with other anticancer drugs.

  3. Pharmacological activation of CB1 receptor modulates long term potentiation by interfering with protein synthesis.

    Science.gov (United States)

    Navakkode, Sheeja; Korte, Martin

    2014-04-01

    Cognitive impairment is one of the most important side effects associated with cannabis drug abuse, as well as the serious issue concerning the therapeutic use of cannabinoids. Cognitive impairments and neuropsychiatric symptoms are caused by early synaptic dysfunctions, such as loss of synaptic connections in different brain structures including the hippocampus, a region that is believed to play an important role in certain forms of learning and memory. We report here that metaplastic priming of synapses with a cannabinoid type 1 receptor (CB1 receptor) agonist, WIN55,212-2 (WIN55), significantly impaired long-term potentiation in the apical dendrites of CA1 pyramidal neurons. Interestingly, the CB1 receptor exerts its effect by altering the balance of protein synthesis machinery towards higher protein production. Therefore the activation of CB1 receptor, prior to strong tetanization, increased the propensity to produce new proteins. In addition, WIN55 priming resulted in the expression of late-LTP in a synaptic input that would have normally expressed early-LTP, thus confirming that WIN55 priming of LTP induces new synthesis of plasticity-related proteins. Furthermore, in addition to the effects on protein translation, WIN55 also induced synaptic deficits due to the ability of CB1 receptors to inhibit the release of acetylcholine, mediated by both muscarinic and nicotinic acetylcholine receptors. Taken together this supports the notion that the modulation of cholinergic activity by CB1 receptor activation is one mechanism that regulates the synthesis of plasticity-related proteins.

  4. Effect of Cannabinoid Receptor Activation on Spreading Depression

    OpenAIRE

    Kazemi, Hadi; Rahgozar, Mehdi; Speckmann, Erwin-Josef; Gorji, Ali

    2012-01-01

    Objective(s):The objective of this study was to evaluate the effect of cannabinoid on cortical spreading depression (CSD) in rat brain. Cannabis has been used for centuries for both symptomatic and prophylactic treatment of different types of headaches including migraine. CSD is believed to be a putative neuronal mechanism underlying migraine aura and subsequent pain. Materials and Methods:The effects of Delta9-tetrahydrocannabinol (THC), as well as, cannabinoid CB1 and CB2 receptor agonists ...

  5. Structural basis for AMPA receptor activation and ligand selectivity

    DEFF Research Database (Denmark)

    Hogner, A; Kastrup, Jette Sandholm Jensen; Jin, R;

    2002-01-01

    and binding experiments, has been used to increase our knowledge concerning the ionotropic glutamate receptor GluR2 at the molecular level. Five high-resolution X-ray structures of the ligand-binding domain of GluR2 (S1S2J) complexed with the three agonists (S)-2-amino-3-[3-hydroxy-5-(2-methyl-2H-tetrazol-5...

  6. Structural basis for activation of G-protein-coupled receptors

    DEFF Research Database (Denmark)

    Gether, Ulrik; Asmar, Fazila; Meinild, Anne Kristine;

    2002-01-01

    Our understanding of how G-protein-coupled receptors (GPCRs) operate at the molecular level has been considerably improved over the last few years. The application of advanced biophysical techniques as well as the availability of high-resolution structural information has allowed insight both int......-expression with the cAMP sensitive Cl- channel CFTR (cystic fibrosis transmembrane conductance regulator) and electrophysiological measurements....

  7. Stress Exacerbates Neuropathic Pain via Glucocorticoid and NMDA Receptor Activation

    OpenAIRE

    Alexander, Jessica K.; DeVries, A Courtney; KIGERL, KRISTINA A.; Dahlman, Jason M.; G.Popovich, Phillip

    2009-01-01

    There is growing recognition that psychological stress influences pain. Hormones that comprise the physiological response to stress (e.g. corticosterone; CORT) may interact with effectors of neuropathic pain. To test this hypothesis, mice received a spared nerve injury (SNI) after exposure to 60 min restraint stress. In stressed mice, allodynia was consistently increased. The mechanism(s) underlying the exacerbated pain response involves CORT acting via glucocorticoid receptors (GRs); RU486, ...

  8. Modulation by group I mGLU receptor activation and group III mGLU receptor blockade of locomotor responses induced by D1-like and D2-like receptor agonists in the nucleus accumbens.

    Science.gov (United States)

    Rouillon, Christophe; Degoulet, Mickael; Chevallier, Karine; Abraini, Jacques H; David, Hélène N

    2008-03-10

    Evidence for functional motor interactions between group I and group III metabotropic glutamatergic (mGlu) receptors and dopamine neurotransmission is now clearly established [David, H.N., Abraini, J.H., 2001a. The group I metabotropic glutamate receptor antagonist S-4-CPG modulates the locomotor response produced by the activation of D1-like, but not D2-like, dopamine receptors in the rat nucleus accumbens. Eur. J. Neurosci. 15, 2157-2164, David, H.N., Abraini, J.H., 2002. Group III metabotropic glutamate receptors and D1-like and D2-like dopamine receptors interact in the rat nucleus accumbens to influence locomotor activity. Eur. J. Neurosci. 15, 869-875]. Nevertheless, whether or not and how, activation of group I and blockade of group III mGlu receptors modulate the motor responses induced by the activation of dopaminergic receptors in the NAcc still remains unknown. Answering this question needs to be assessed since functional interactions between neurotransmitters in the NAcc are well known to depend upon the level of activation of glutamatergic and/or dopaminergic receptors and because the effects of glutamatergic receptor agonists and antagonists on dopaminergic receptor-mediated locomotor responses are not always reciprocal as shown in previous studies. Our results show that activation of group I mGlu receptors by DHPG in the NAcc potentiated the locomotor response induced by intra-NAcc activation of D1-like receptors and blocked those induced by D2-like presynaptic or postsynaptic receptors. Alternatively, blockade of group III mGlu receptors by MPPG in the NAcc potentiated the locomotor responses mediated by D1-like receptors and by D2-like postsynaptic receptors and inhibited that induced by D2-like presynaptic receptors. These results compiled with previous data demonstrate that group I mGlu receptors and group III mGlu receptors can modulate the locomotor responses produced by D1-like and/or D2-like receptor agonists in a complex phasic and tonic

  9. Pharmacology of the hypothermic response to 5-HT1A receptor activation in humans.

    Science.gov (United States)

    Lesch, K P; Poten, B; Söhnle, K; Schulte, H M

    1990-01-01

    The selective 5-HT1A receptor ligand ipsapirone (IPS) caused dose-related hypothermia in humans. The response was attenuated by the nonselective 5-HT1/2 receptor antagonist metergoline and was completely antagonized by the nonselective beta-adrenoceptor antagonist pindolol, which interacts stereoselectively with the 5-HT1A receptor. The selective beta 1-adrenergic antagonist betaxolol had no effect. The findings indicate that IPS-induced hypothermia specifically involves activation of (presynaptic) 5-HT1A receptors. Therefore, the hypothermic response to IPS may provide a convenient in vivo paradigma to assess the function of the presynaptic 5-HT receptor in affective disorders and its involvement in the effects of psychotropic drugs. PMID:1980461

  10. Pharmacology of the hypothermic response to 5-HT1A receptor activation in humans.

    Science.gov (United States)

    Lesch, K P; Poten, B; Söhnle, K; Schulte, H M

    1990-01-01

    The selective 5-HT1A receptor ligand ipsapirone (IPS) caused dose-related hypothermia in humans. The response was attenuated by the nonselective 5-HT1/2 receptor antagonist metergoline and was completely antagonized by the nonselective beta-adrenoceptor antagonist pindolol, which interacts stereoselectively with the 5-HT1A receptor. The selective beta 1-adrenergic antagonist betaxolol had no effect. The findings indicate that IPS-induced hypothermia specifically involves activation of (presynaptic) 5-HT1A receptors. Therefore, the hypothermic response to IPS may provide a convenient in vivo paradigma to assess the function of the presynaptic 5-HT receptor in affective disorders and its involvement in the effects of psychotropic drugs.

  11. Activation of peroxisome proliferator-activated receptor-{alpha} enhances fatty acid oxidation in human adipocytes

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Joo-Young; Hashizaki, Hikari; Goto, Tsuyoshi; Sakamoto, Tomoya; Takahashi, Nobuyuki [Laboratory of Molecular Function of Food, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011 (Japan); Kawada, Teruo, E-mail: fat@kais.kyoto-u.ac.jp [Laboratory of Molecular Function of Food, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011 (Japan)

    2011-04-22

    Highlights: {yields} PPAR{alpha} activation increased mRNA expression levels of adipocyte differentiation marker genes and GPDH activity in human adipocytes. {yields} PPAR{alpha} activation also increased insulin-dependent glucose uptake in human adipocytes. {yields} PPAR{alpha} activation did not affect lipid accumulation in human adipocytes. {yields} PPAR{alpha} activation increased fatty acid oxidation through induction of fatty acid oxidation-related genes in human adipocytes. -- Abstract: Peroxisome proliferator-activated receptor-{alpha} (PPAR{alpha}) is a key regulator for maintaining whole-body energy balance. However, the physiological functions of PPAR{alpha} in adipocytes have been unclarified. We examined the functions of PPAR{alpha} using human multipotent adipose tissue-derived stem cells as a human adipocyte model. Activation of PPAR{alpha} by GW7647, a potent PPAR{alpha} agonist, increased the mRNA expression levels of adipocyte differentiation marker genes such as PPAR{gamma}, adipocyte-specific fatty acid-binding protein, and lipoprotein lipase and increased both GPDH activity and insulin-dependent glucose uptake level. The findings indicate that PPAR{alpha} activation stimulates adipocyte differentiation. However, lipid accumulation was not changed, which is usually observed when PPAR{gamma} is activated. On the other hand, PPAR{alpha} activation by GW7647 treatment induced the mRNA expression of fatty acid oxidation-related genes such as CPT-1B and AOX in a PPAR{alpha}-dependent manner. Moreover, PPAR{alpha} activation increased the production of CO{sub 2} and acid soluble metabolites, which are products of fatty acid oxidation, and increased oxygen consumption rate in human adipocytes. The data indicate that activation of PPAR{alpha} stimulates both adipocyte differentiation and fatty acid oxidation in human adipocytes, suggesting that PPAR{alpha} agonists could improve insulin resistance without lipid accumulation in adipocytes. The expected

  12. Evidence for Activation of Toll-Like Receptor and Receptor for Advanced Glycation End Products in Preterm Birth

    Directory of Open Access Journals (Sweden)

    Taketoshi Noguchi

    2010-01-01

    Full Text Available Objective. Individuals with inflammation have a myriad of pregnancy aberrations including increasing their preterm birth risk. Toll-like receptors (TLRs and receptor for advanced glycation end products (RAGE and their ligands were all found to play a key role in inflammation. In the present study, we reviewed TLR and RAGE expression, their ligands, and signaling in preterm birth. Research Design and Methods. A systematic search was performed in the electronic databases PubMed and ScienceDirect up to July 2010, combining the keywords “preterm birth,” “TLR”, “RAGE”, “danger signal”, “alarmin”, “genomewide,” “microarray,” and “proteomics” with specific expression profiles of genes and proteins. Results. This paper provides data on TLR and RAGE levels and critical downstream signaling events including NF-kappaB-dependent proinflammatory cytokine expression in preterm birth. About half of the genes and proteins specifically present in preterm birth have the properties of endogenous ligands “alarmin” for receptor activation. The interactions between the TLR-mediated acute inflammation and RAGE-mediated chronic inflammation have clear implications for preterm birth via the TLR and RAGE system, which may be acting collectively. Conclusions. TLR and RAGE expression and their ligands, signaling, and functional activation are increased in preterm birth and may contribute to the proinflammatory state.

  13. A high-content EMT screen identifies multiple receptor tyrosine kinase inhibitors with activity on TGFβ receptor

    Science.gov (United States)

    Ackerknecht, Sabine; Lehembre, François; Fink, Tobias; Stritt, Manuel; Wirth, Matthias; Pavan, Simona; Bill, Ruben; Regenass, Urs; Christofori, Gerhard; Meyer-Schaller, Nathalie

    2016-01-01

    An epithelial to mesenchymal transition (EMT) enables epithelial tumor cells to break out of the primary tumor mass and to metastasize. Understanding the molecular mechanisms driving EMT in more detail will provide important tools to interfere with the metastatic process. To identify pharmacological modulators and druggable targets of EMT, we have established a novel multi-parameter, high-content, microscopy-based assay and screened chemical compounds with activities against known targets. Out of 3423 compounds, we have identified 19 drugs that block transforming growth factor beta (TGFβ)-induced EMT in normal murine mammary gland epithelial cells (NMuMG). The active compounds include inhibitors against TGFβ receptors (TGFBR), Rho-associated protein kinases (ROCK), myosin II, SRC kinase and uridine analogues. Among the EMT-repressing compounds, we identified a group of inhibitors targeting multiple receptor tyrosine kinases, and biochemical profiling of these multi-kinase inhibitors reveals TGFBR as a thus far unknown target of their inhibitory spectrum. These findings demonstrate the feasibility of a multi-parameter, high-content microscopy screen to identify modulators and druggable targets of EMT. Moreover, the newly discovered “off-target” effects of several receptor tyrosine kinase inhibitors have important consequences for in vitro and in vivo studies and might beneficially contribute to the therapeutic effects observed in vivo. PMID:27036020

  14. Class A scavenger receptor promotes osteoclast differentiation via the enhanced expression of receptor activator of NF-{kappa}B (RANK)

    Energy Technology Data Exchange (ETDEWEB)

    Takemura, Kenichi [Department of Cell Pathology, Graduate School of Medical Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto 860-8556 (Japan); Department of Orthopaedic and Neuro-Musculoskeletal Surgery, Graduate School of Medical Sciences, Kumamoto University, Kumamoto (Japan); Sakashita, Naomi; Fujiwara, Yukio; Komohara, Yoshihiro; Lei, XiaoFeng; Ohnishi, Koji [Department of Cell Pathology, Graduate School of Medical Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto 860-8556 (Japan); Suzuki, Hiroshi [National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Hokkaido (Japan); Kodama, Tatsuhiko [Department of Molecular Biology and Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo (Japan); Mizuta, Hiroshi [Department of Orthopaedic and Neuro-Musculoskeletal Surgery, Graduate School of Medical Sciences, Kumamoto University, Kumamoto (Japan); Takeya, Motohiro, E-mail: takeya@kumamoto-u.ac.jp [Department of Cell Pathology, Graduate School of Medical Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto 860-8556 (Japan)

    2010-01-22

    Osteoclasts originate from bone marrow monocyte/macrophage lineage cells, and their differentiation depends on macrophage colony-stimulating factor (M-CSF) and receptor activator nuclear factor kappa B (RANK) ligand. Class A scavenger receptor (SR-A) is one of the principal functional molecules of macrophages, and its level of expression declines during osteoclast differentiation. To investigate the role of SR-A in osteoclastogenesis, we examined pathological changes in femoral bone and the expression levels of osteoclastogenesis-related molecules in SR-A{sup -/-} mice. The femoral osseous density of SR-A{sup -/-} mice was higher than that of SR-A{sup +/+} mice, and the number of multinucleated osteoclasts was significantly decreased. An in vitro differentiation assay revealed that the differentiation of multinucleated osteoclasts from bone marrow-derived progenitor cells is impaired in SR-A{sup -/-} mice. Elimination of SR-A did not alter the expression level of the M-CSF receptor, c-fms; however, the expression levels of RANK and RANK-related osteoclast-differentiation molecules such as nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1 (NFATc1) and microphthalmia-associated transcription factor (MITF) significantly decreased. Furthermore, acetylated low-density lipoprotein (AcLDL), an SR-A ligand, significantly increased the expression level of RANK and MITF during osteoclast differentiation. These data indicate that SR-A promotes osteoclastogenesis via augmentation of the expression level of RANK and its related molecules.

  15. Peroxisome Proliferator-Activated Receptor γ Activity is Required for Appropriate Cardiomyocyte Differentiation

    Directory of Open Access Journals (Sweden)

    Maryam Peymani

    2016-07-01

    Full Text Available Objective Peroxisome proliferator-activated receptor γ (PPARγ is a member of the PPAR nuclear receptor superfamily. Although PPARγ acts as a master transcription factor in adipocyte differentiation, it is also associated with a variety of cell functions including carbohydrate and lipid metabolism, glucose homeostasis, cell proliferation and cell differentiation. This study aimed to assess the expression level of PPARγ in order to address its role in cardiac cell differentiation of mouse embryonic stem cells (mESCs. Materials and Methods In this an intervening study, mESCs were subjected to cardiac differentiation. Total RNA was extracted from the cells and quantitative real time polymerase chain reaction (qPCR was carried out to estimate level of gene expression. Furthermore, the requirement of PPARγ in cardiac differentiation of mESCs, during cardiac progenitor cells (CPCs formation, was examined by applying the respective agonist and antagonist. Results The obtained data revealed an elevation in the expression level of PPARγ during spontaneous formation of CPCs and cardiomyocytes. Our results indicated that during CPC formation, PPARγ inactivation via treatment with GW9662 (GW reduced expression of CPC and cardiac markers. Conclusion We conclude that PPARγ modulation has an effective role on cardiac differentiation of mESCs at the early stage of cardiomyogenesis.

  16. Transition of arrestin into the active receptor-binding state requires an extended interdomain hinge.

    Science.gov (United States)

    Vishnivetskiy, Sergey A; Hirsch, Joel A; Velez, Maria-Gabriela; Gurevich, Yulia V; Gurevich, Vsevolod V

    2002-11-15

    Arrestins selectively bind to the phosphorylated activated form of G protein-coupled receptors, thereby blocking further G protein activation. Structurally, arrestins consist of two domains topologically connected by a 12-residue long loop, which we term the "hinge" region. Both domains contain receptor-binding elements. The relative size and shape of arrestin and rhodopsin suggest that dramatic changes in arrestin conformation are required to bring all of its receptor-binding elements in contact with the cytoplasmic surface of the receptor. Here we use the visual arrestin/rhodopsin system to test the hypothesis that the transition of arrestin into its active receptor-binding state involves a movement of the two domains relative to each other that might be limited by the length of the hinge. We have introduced three insertions and 24 deletions in the hinge region and measured the binding of all of these mutants to light-activated phosphorylated (P-Rh*), dark phosphorylated (P-Rh), dark unphosphorylated (Rh), and light-activated unphosphorylated rhodopsin (Rh*). The addition of 1-3 extra residues to the hinge has no effect on arrestin function. In contrast, sequential elimination of 1-8 residues results in a progressive decrease in P-Rh* binding without changing arrestin selectivity for P-Rh*. These results suggest that there is a minimum length of the hinge region necessary for high affinity binding, consistent with the idea that the two domains move relative to each other in the process of arrestin transition into its active receptor-binding state. The same length of the hinge is also necessary for the binding of "constitutively active" arrestin mutants to P-Rh*, dark P-Rh, and Rh*, suggesting that the active (receptor-bound) arrestin conformation is essentially the same in both wild type and mutant forms.

  17. Soluble Urokinase Receptors in Focal Segmental Glomerulosclerosis: A Review on the Scientific Point of View

    Science.gov (United States)

    Saleem, Moin A.; Meijers, Björn

    2016-01-01

    Focal segmental glomerulosclerosis (FSGS) is one of the primary glomerular disorders in both children and adults which can progress to end-stage renal failure. Although there are genetic and secondary causes, circulating factors have also been regarded as an important factor in the pathogenesis of FSGS, because about 40% of the patients with FSGS have recurrence after renal transplantation. Soluble urokinase-type plasminogen activator receptor (suPAR) is a soluble form of uPAR, which is a membrane-bound protein linked to GPI in various immunologically active cells, including podocytes. It has recently been suggested as a potential circulating factor in FSGS by in vitro podocyte experiments, in vivo mice models, and human studies. However, there have also been controversies on this issue, because subsequent studies showed conflicting results. suPAR levels were also increased in patients with other glomerular diseases and were inversely correlated with estimated glomerular filtration rate. Nevertheless, there has been no balanced review on this issue. In this review, we compare the conflicting data on the involvement of suPAR in the pathogenesis of FSGS and shed light on interpretation by taking into account many points and the potential variables and confounders influencing serum suPAR levels.

  18. Soluble Urokinase Receptors in Focal Segmental Glomerulosclerosis: A Review on the Scientific Point of View

    Directory of Open Access Journals (Sweden)

    Andreas Kronbichler

    2016-01-01

    Full Text Available Focal segmental glomerulosclerosis (FSGS is one of the primary glomerular disorders in both children and adults which can progress to end-stage renal failure. Although there are genetic and secondary causes, circulating factors have also been regarded as an important factor in the pathogenesis of FSGS, because about 40% of the patients with FSGS have recurrence after renal transplantation. Soluble urokinase-type plasminogen activator receptor (suPAR is a soluble form of uPAR, which is a membrane-bound protein linked to GPI in various immunologically active cells, including podocytes. It has recently been suggested as a potential circulating factor in FSGS by in vitro podocyte experiments, in vivo mice models, and human studies. However, there have also been controversies on this issue, because subsequent studies showed conflicting results. suPAR levels were also increased in patients with other glomerular diseases and were inversely correlated with estimated glomerular filtration rate. Nevertheless, there has been no balanced review on this issue. In this review, we compare the conflicting data on the involvement of suPAR in the pathogenesis of FSGS and shed light on interpretation by taking into account many points and the potential variables and confounders influencing serum suPAR levels.

  19. Angiotensin type 2-receptor (AT2R) activation induces hypotension in apolipoprotein E-deficient mice by activating peroxisome proliferator-activated receptor

    Science.gov (United States)

    Li, Ming; Tejada, Thor; Lambert, Jonathan P; Nicholson, Chad K; Yahiro, Eiji; Ambai, Vats T; Ali, Syeda F; Bradley, Eddie W; Graham, Robert M; Dell’Italia, Louis J; Calvert, John W; Naqvi, Nawazish

    2016-01-01

    Angiotensin II (Ang II) modulates blood pressure and atherosclerosis development through its vascular type-1 (AT1R) and type-2 (AT2R) receptors, which have opposing effects. AT2R activation produces hypotension, and is anti-atherogenic. Targeted overexpression of AT2Rs in vascular smooth muscle cells (VSMCs) indicates that these effects are due to increased nitric oxide (NO) generation. However, the role of endogenous VSMC AT2Rs in these events is unknown. Effect of 7-day low-dose Ang II-infusion (12 µg/kg/hr) on blood pressure was tested in 9-week-old apoE(-/-) mice fed a low or high cholesterol diet (LCD or HCD, respectively). Cardiac output was measured by echocardiography. Immunohistochemistry was performed to localize and quantify AT2Rs and p-Ser1177-endothelial nitric oxide synthase (eNOS) levels in the aortic arch. PD123319 and GW-9662 were used to selectively block the AT2R and peroxisome proliferator-activated receptor-γ (PPAR-γ), respectively. Ang II infusion decreased blood pressure by 12 mmHg (P LCD/apoE(-/-) mice without altering cardiac output; a response blocked by PD123319. Although, AT2R stimulation neither activated eNOS (p-Ser1177-eNOS) nor changed plasma NO metabolites, it caused an ~6-fold increase in VSMC PPAR-γ levels (P < 0.001) and the AT2R-mediated hypotension was abolished by GW-9662. AT2R-mediated hypotension was also inhibited by HCD, which selectively decreased VSMC AT2R expression by ~6-fold (P < 0.01). These findings suggest a novel pathway for the Ang II/AT2R-mediated hypotensive response that involves PPAR-γ, and is down regulated by a HCD. PMID:27679746

  20. Angiotensin type 2-receptor (AT2R) activation induces hypotension in apolipoprotein E-deficient mice by activating peroxisome proliferator-activated receptor-γ.

    Science.gov (United States)

    Li, Ming; Tejada, Thor; Lambert, Jonathan P; Nicholson, Chad K; Yahiro, Eiji; Ambai, Vats T; Ali, Syeda F; Bradley, Eddie W; Graham, Robert M; Dell'Italia, Louis J; Calvert, John W; Naqvi, Nawazish

    2016-01-01

    Angiotensin II (Ang II) modulates blood pressure and atherosclerosis development through its vascular type-1 (AT1R) and type-2 (AT2R) receptors, which have opposing effects. AT2R activation produces hypotension, and is anti-atherogenic. Targeted overexpression of AT2Rs in vascular smooth muscle cells (VSMCs) indicates that these effects are due to increased nitric oxide (NO) generation. However, the role of endogenous VSMC AT2Rs in these events is unknown. Effect of 7-day low-dose Ang II-infusion (12 µg/kg/hr) on blood pressure was tested in 9-week-old apoE((-/-)) mice fed a low or high cholesterol diet (LCD or HCD, respectively). Cardiac output was measured by echocardiography. Immunohistochemistry was performed to localize and quantify AT2Rs and p-Ser(1177)-endothelial nitric oxide synthase (eNOS) levels in the aortic arch. PD123319 and GW-9662 were used to selectively block the AT2R and peroxisome proliferator-activated receptor-γ (PPAR-γ), respectively. Ang II infusion decreased blood pressure by 12 mmHg (P < 0.001) in LCD/apoE((-/-)) mice without altering cardiac output; a response blocked by PD123319. Although, AT2R stimulation neither activated eNOS (p-Ser(1177)-eNOS) nor changed plasma NO metabolites, it caused an ~6-fold increase in VSMC PPAR-γ levels (P < 0.001) and the AT2R-mediated hypotension was abolished by GW-9662. AT2R-mediated hypotension was also inhibited by HCD, which selectively decreased VSMC AT2R expression by ~6-fold (P < 0.01). These findings suggest a novel pathway for the Ang II/AT2R-mediated hypotensive response that involves PPAR-γ, and is down regulated by a HCD. PMID:27679746

  1. Involvement of Ca2+-activated K+ Channels in Receptor-Regulated Sperm Motility in Rats

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Previous voltage-clamp studies have demonstrated the modulation of sperm Ca2+-activated K+ (KCa) channels expressed in Xenopus oocytes by angiotensin Ⅱ (Ang Ⅱ)and extracellular ATP via AT1 receptor and P2U receptor, respectively. In the presentstudy, we investigated the involvement of KCa channels in receptor-regulated spermmotility of the rat using a computer-aided sperm analysis system, HTM-IVOS, in con-junction with Ca2+-mobilizing agents, receptor agonists/antagonists and KCa channelsblockers.The percentage of motile sperm was increased by ionomycin (0. 5 μmol/L), whichcould be inhibited by K+ channel blockers, tetraethylammonium (TEA 1 μmol/L ) orcharybdotoxin (ChTX, 300 nmol/L) indicating the presence of KCa channels. AngⅡ, at low concentration, 10 nmol/L, was found to increase motility, however, athigher concentration, 1 μmol/L, percentage of motility was found to be suppressed.Both stimulatory and inhibitory effects of Ang Ⅱ could be reversed by losartan, aspecific antagonist of AT 1 receptors, but not AT 2 antagonist PD123177, indicating theinvolvement of AT1 but not AT2 receptor in mediating both effects. ChTX also abol-ished both stimulatory and inhibitory effects of Ang H, suggesting the involvement ofKCa channels. The percentage of motility was also enhanced by extracellular ATP, afactor known to be involved in sperm activation. The ATP-enhanced sperm motilitywas mimicked by UTP , and inhibited by ChTX and reactive blue, an antagonist of P2receptor, indicating the involvement of both P2U and KCa channels. RT-PCR studywas also conducted to confirm the expression of KCa channels, AT1 receptors and P2Ureceptor, but not AT2 receptor, in rat caudal epididymal sperm. The present findingssuggest an important role of KCa channels in the regulation of sperm motility by AT1and P 2U receptors.

  2. Acanthamoeba protease activity promotes allergic airway inflammation via protease-activated receptor 2.

    Directory of Open Access Journals (Sweden)

    Mi Kyung Park

    Full Text Available Acanthamoeba is a free-living amoeba commonly present in the environment and often found in human airway cavities. Acanthamoeba possesses strong proteases that can elicit allergic airway inflammation. To our knowledge, the aeroallergenicity of Acanthamoeba has not been reported. We repeatedly inoculated mice with Acanthamoeba trophozoites or excretory-secretory (ES proteins intra-nasally and evaluated symptoms and airway immune responses. Acanthamoeba trophozoites or ES proteins elicited immune responses in mice that resembled allergic airway inflammation. ES proteins had strong protease activity and activated the expression of several chemokine genes (CCL11, CCL17, CCL22, TSLP, and IL-25 in mouse lung epithelial cells. The serine protease inhibitor phenyl-methane-sulfonyl fluoride (PMSF inhibited ES protein activity. ES proteins also stimulated dendritic cells and enhanced the differentiation of naive T cells into IL-4-secreting T cells. After repeated inoculation of the protease-activated receptor 2 knockout mouse with ES proteins, airway inflammation and Th2 immune responses were markedly reduced, but not to basal levels. Furthermore, asthma patients had higher Acanthamoeba-specific IgE titers than healthy controls and we found Acanthamoeba specific antigen from house dust in typical living room. Our findings suggest that Acanthamoeba elicits allergic airway symptoms in mice via a protease allergen. In addition, it is possible that Acanthamoeba may be one of the triggers human airway allergic disease.

  3. Structural plasticity of GABAergic axons is regulated by network activity and GABAA receptor activation

    Directory of Open Access Journals (Sweden)

    Anne eSchuemann

    2013-06-01

    Full Text Available Coordinated changes at excitatory and inhibitory synapses are essential for normal brain development and function. It is well established that excitatory neurons undergo structural changes, but our knowledge about inhibitory structural plasticity is rather scarce. Here we present a quantitative analysis of the dynamics of GABAergic boutons in the dendritic region of the hippocampal CA1 area using time-lapse two-photon imaging in organotypic hippocampal cultures from GAD65-GFP mice. We show that ~20% of inhibitory boutons are not stable. They are appearing, disappearing and reappearing at specific locations along the inhibitory axon and reflect immature or incomplete synapses. Furthermore, we observed that persistent boutons show large volume fluctuations over several hours, suggesting that presynaptic content of inhibitory synapses is not constant. Our data show that inhibitory boutons are highly dynamic structures and suggest that inhibitory axons are continuously probing potential locations for inhibitory synapse formation by redistributing presynaptic material along the axon.In addition, we found that neuronal activity affects the exploratory dynamics of inhibitory axons. Blocking network activity rapidly reduces the number of transient boutons, whereas enhancing activity reduces the number of persistent inhibitory boutons, possibly reflecting enhanced competition between boutons along the axon. The latter effect requires signaling through GABAA receptors. We propose that activity-dependent regulation of bouton dynamics contributes to inhibitory synaptic plasticity.

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

    Science.gov (United States)

    Sjöqvist, Marika; Antfolk, Daniel; Ferraris, Saima; Rraklli, Vilma; Haga, Cecilia; Antila, Christian; Mutvei, Anders; Imanishi, Susumu Y; Holmberg, Johan; Jin, Shaobo; Eriksson, John E; Lendahl, Urban; Sahlgren, Cecilia

    2014-04-01

    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 central nervous system and in cultured myoblasts, Notch-stimulated cells were allowed to undergo differentiation. PKCζ phosphorylates membrane-tethered forms of Notch and regulates two distinct routing steps, depending on the Notch activation state. When Notch is activated, PKCζ promotes re-localization of Notch from late endosomes to the nucleus and enhances production of the Notch intracellular domain, which leads to increased Notch activity. In the non-activated state, PKCζ instead facilitates Notch receptor internalization, accompanied with increased ubiquitylation and interaction with the endosomal sorting protein Hrs. Collectively, these data identify PKCζ as a key regulator of Notch trafficking and demonstrate that distinct steps in intracellular routing are differentially modulated depending on Notch signaling status.

  5. Activation of muscarinic receptors in porcine airway smooth muscle elicits a transient increase in phospholipase D activity.

    Science.gov (United States)

    Mamoon, A M; Smith, J; Baker, R C; Farley, J M

    1999-01-01

    Phospholipase D (PLD) is a phosphodiesterase that catalyses hydrolysis of phosphatidylcholine to produce phosphatidic acid and choline. In the presence of ethanol, PLD also catalyses the formation of phosphatidylethanol, which is a unique characteristic of this enzyme. Muscarinic receptor-induced changes in the activity of PLD were investigated in porcine tracheal smooth muscle by measuring the formation of [3H]phosphatidic acid ([3H]PA) and [3H]phosphatidylethanol ([3H]PEth) after labeling the muscle strips with [3H]palmitic acid. The cholinergic receptor agonist acetylcholine (Ach) significantly but transiently increased formation of both [3H]PA and [3H]PEth in a concentration-dependent manner (>105-400% vs. controls in the presence of 10(-6) to 10(-4) M Ach) when pretreated with 100 mM ethanol. The Ach receptor-mediated increase in PLD activity was inhibited by atropine (10(-6) M), indicating that activation of PLD occurred via muscarinic receptors. Activation of protein kinase C (PKC) by phorbol-12-myristate-13-acetate (PMA) increased PLD activity that was effectively blocked by the PKC inhibitors calphostin C (10(-8) to 10(-6) M) and GFX (10(-8) to 10(-6) M). Ach-induced increases in PLD activity were also significantly, but incompletely, inhibited by both GFX and calphostin C. From the present data, we conclude that in tracheal smooth muscle, muscarinic acetylcholine receptor-induced PLD activation is transient in nature and coupled to these receptors via PKC. However, PKC activation is not solely responsible for Ach-induced activation of PLD in porcine tracheal smooth muscle.

  6. P2X receptors mediate ATP-induced primary nociceptive neurone activation.

    Science.gov (United States)

    Bland-Ward, P A; Humphrey, P P

    2000-07-01

    ATP-gated P2X ion-channel receptors are localised throughout the mammalian nervous system and have been identified on neurones which participate in conduction of nociceptive information from the periphery to, and within, the CNS. This article briefly reviews recently published research describing the role that ATP and P2X receptors may play in pain perception, highlighting the importance of the P2X(3) receptor in this process. The P2X(3) receptor subunit is almost exclusively expressed on a subset of small and medium diameter sensory neurones innervating cutaneous and visceral tissue. Activation of P2X receptors present on the peripheral terminals of primary afferents results in neuronal depolarisation and, in conscious animals, leads to the manifestation of acute nociceptive behaviour. Recent animal studies have also shown that P2X(3) receptor expression is increased in sensory ganglia following acute neuronal injury, hinting that similar plasticity in the expression of this receptor subtype could underlie the mechanisms involved in a range of conditions characterised by sensory hypersensitivity in man. It is apparent from the evidence available that functional antagonists at specific P2X receptor subtypes could represent an important class of novel analgesic agents.

  7. Receptor density is key to the alpha2/beta interferon differential activities.

    Science.gov (United States)

    Moraga, Ignacio; Harari, Daniel; Schreiber, Gideon; Uzé, Gilles; Pellegrini, Sandra

    2009-09-01

    Multiple type I interferons (IFN-alpha/beta) elicit Jak/Stat activation, rapid gene induction, and pleiotropic effects, such as differentiation, antiviral protection, and blocks in proliferation, which are dependent on the IFN subtype and the cellular context. To date, ligand- and receptor-specific molecular determinants underlying IFN-alpha/beta differential activities or potencies have been well characterized. To analyze cellular determinants that impact subtype-specific potency, human fibrosarcoma U5A-derived clones, exhibiting a gradient of IFN sensitivity by virtue of increasing receptor levels, were monitored for Jak/Stat signaling, gene induction, cell cycle lengthening, and apoptosis. In cells with scarce receptors, IFN-beta was more potent than IFN-alpha2 in antiproliferative activity, while the two subtypes were equipotent in all other readouts. Conversely, in cells with abundant receptors, IFN-alpha2 matched or even surpassed IFN-beta in all readouts tested. Our results suggest that the differential activities of the IFN subtypes are dictated not only by the intrinsic ligand/receptor binding kinetics but also by the density of cell surface receptor components.

  8. Ligand Perception, Activation, and Early Signaling of Plant Steroid Receptor Brassinosteroid Insensitive 1

    Institute of Scientific and Technical Information of China (English)

    Jianjun Jiang; Chi Zhang; Xuelu Wang

    2013-01-01

    Leucine-rich repeat receptor-like kinases (LRR-RLKs) belong to a large group of cell surface proteins involved in many aspects of plant development and environmental responses in both monocots and dicots. Brassinosteroid insensitive 1 (BRI1), a member of the LRR X subfamily, was first identified through several forward genetic screenings for mutants insensitive to brassinosteroids (BRs), which are a class of plant-specific steroid hormones. Since its identification, BRI1 and its homologs had been proved as receptors perceiving BRs and initiating BR signaling. The co-receptor BRI1-associated kinase 1 and its homologs, and other BRI1 interacting proteins such as its inhibitor BRI1 kinase inhibitor 1 (BKI1) were identified by genetic and biochemical approaches. The detailed mechanisms of BR perception by BRI1 and the activation of BRI1 receptor complex have also been elucidated. Moreover, several mechanisms for termination of the activated BRI1 signaling were also discovered. In this review, we will focus on the recent advances on the mechanism of BRI1 phosphorylation and activation, the regulation of its receptor complex, the structure basis of BRI1 ectodomain and BR recognition, its direct substrates, and the termination of the activated BRI1 receptor complex.

  9. Activation of the kinin B1 receptor attenuates melanoma tumor growth and metastasis.

    Directory of Open Access Journals (Sweden)

    Patricia Dillenburg-Pilla

    Full Text Available Melanoma is a very aggressive tumor that does not respond well to standard therapeutic approaches, such as radio- and chemotherapies. Furthermore, acquiring the ability to metastasize in melanoma and many other tumor types is directly related to incurable disease. The B1 kinin receptor participates in a variety of cancer-related pathophysiological events, such as inflammation and angiogenesis. Therefore, we investigated whether this G protein-coupled receptor plays a role in tumor progression. We used a murine melanoma cell line that expresses the kinin B1 receptor and does not express the kinin B2 receptor to investigate the precise contribution of activation of the B1 receptor in tumor progression and correlated events using various in vitro and in vivo approaches. Activation of the kinin B1 receptor in the absence of B2 receptor inhibits cell migration in vitro and decreases tumor formation in vivo. Moreover, tumors formed from cells stimulated with B1-specific agonist showed several features of decreased aggressiveness, such as smaller size and infiltration of inflammatory cells within the tumor area, higher levels of pro-inflammatory cytokines implicated in the host anti-tumor immune response, lower number of cells undergoing mitosis, a poorer vascular network, no signs of invasion of surrounding tissues or metastasis and increased animal survival. Our findings reveal that activation of the kinin B1 receptor has a host protective role during murine melanoma tumor progression, suggesting that the B1 receptor could be a new anti-tumor GPCR and provide new opportunities for therapeutic targeting.

  10. Analgesic tone conferred by constitutively active mu opioid receptors in mice lacking β-arrestin 2

    Directory of Open Access Journals (Sweden)

    Hales Tim G

    2011-04-01

    Full Text Available Abstract Hedonic reward, dependence and addiction are unwanted effects of opioid analgesics, linked to the phasic cycle of μ opioid receptor activation, tolerance and withdrawal. In vitro studies of recombinant G protein coupled receptors (GPCRs over expressed in cell lines reveal an alternative tonic signaling mechanism that is independent of agonist. Such studies demonstrate that constitutive GPCR signaling can be inhibited by inverse agonists but not by neutral antagonists. However, ligand-independent activity has been difficult to examine in vivo, at the systems level, due to relatively low levels of constitutive activity of most GPCRs including μ receptors, often necessitating mutagenesis or pharmacological manipulation to enhance basal signaling. We previously demonstrated that the absence of β-arrestin 2 (β-arr2 augments the constitutive coupling of μ receptors to voltage-activated Ca2+ channels in primary afferent dorsal root ganglion neurons from β-arr2-/- mice. We used this in vitro approach to characterize neutral competitive antagonists and inverse agonists of the constitutively active wild type μ receptors in neurons. We administered these agents to β-arr2-/- mice to explore the role of constitutive μ receptor activity in nociception and hedonic tone. This study demonstrates that the induction of constitutive μ receptor activity in vivo in β-arr2-/- mice prolongs tail withdrawal from noxious heat, a phenomenon that was reversed by inverse agonists, but not by antagonists that lack negative efficacy. By contrast, the aversive effects of inverse agonists were similar in β-arr2-/- and β-arr2+/+ mice, suggesting that hedonic tone was unaffected.

  11. Activation of GLP-1 Receptor Promotes Bone Marrow Stromal Cell Osteogenic Differentiation through β-Catenin

    Directory of Open Access Journals (Sweden)

    Jingru Meng

    2016-04-01

    Full Text Available Glucagon-like peptide 1 (GLP-1 plays an important role in regulating bone remodeling, and GLP-1 receptor agonist shows a positive relationship with osteoblast activity. However, GLP-1 receptor is not found in osteoblast, and the mechanism of GLP-1 receptor agonist on regulating bone remodeling is unclear. Here, we show that the GLP-1 receptor agonist exendin-4 (Ex-4 promoted bone formation and increased bone mass and quality in a rat unloading-induced bone loss model. These functions were accompanied by an increase in osteoblast number and serum bone formation markers, while the adipocyte number was decreased. Furthermore, GLP-1 receptor was detected in bone marrow stromal cells (BMSCs, but not in osteoblast. Activation of GLP-1 receptor by Ex-4 promoted the osteogenic differentiation and inhibited BMSC adipogenic differentiation through regulating PKA/β-catenin and PKA/PI3K/AKT/GSK3β signaling. These findings reveal that GLP-1 receptor regulates BMSC osteogenic differentiation and provide a molecular basis for therapeutic potential of GLP-1 against osteoporosis.

  12. Dimerization by a cytokine receptor is necessary for constitutive activation of JAK2V617F.

    Science.gov (United States)

    Lu, Xiaohui; Huang, Lily Jun-Shen; Lodish, Harvey F

    2008-02-29

    The majority of the BCR-ABL-negative myeloproliferative disorders express the mutant JAK2, JAK2V617F. Previously we showed that constitutive activation of this oncogenic JAK2 mutant in Ba/F3 or 32D cells requires coexpression of a cognate homodimeric cytokine receptor, such as the EpoR. However, overexpression of JAK2V617F in Ba/F3 cells renders them cytokine-independent for growth in the absence of an exogenous cytokine receptor. Here, we demonstrated that JAK2V617F domains required for receptor association are essential for cytokine-independent growth by overexpressed JAK2V617F, suggesting JAK2V617F is binding to an unknown endogenous cytokine receptor(s) for its activation. We further showed that disruption of EpoR dimerization by coexpressing a truncated EpoR disrupted JAK2V617F-mediated transformation, indicating that EpoR dimerization plays an essential role in the activation of JAK2V617F. Interestingly, coexpression of JAK2V617F with EpoR mutants that retain JAK2 binding but are defective in mediating Epo-dependent JAK2 activation due to mutations in a conserved juxtamembrane motif does lead to cytokine-independent activation of JAK2V617F. Overall, these findings confirm that JAK2V617F requires binding to a dimerized cytokine receptor for its activation, and that the key EpoR juxtamembrane regulatory motif essential for Epo-dependent JAK2 activation is not essential for the activation of JAK2V617F. The structure of the activated JAK2V617F is thus likely to be different from that of the activated wild-type JAK2, raising the possibility of developing a specifically targeted therapy for myeloproliferative disorders.

  13. Salt Bridges Overlapping the Gonadotropin-Releasing Hormone Receptor Agonist Binding Site Reveal a Coincidence Detector for G Protein-Coupled Receptor Activation

    OpenAIRE

    Janovick, Jo Ann; Pogozheva, Irina D.; Mosberg, Henry I.; Conn, P. Michael

    2011-01-01

    G protein-coupled receptors (GPCRs) play central roles in most physiological functions, and mutations in them cause heritable diseases. Whereas crystal structures provide details about the structure of GPCRs, there is little information that identifies structural features that permit receptors to pass the cellular quality control system or are involved in transition from the ground state to the ligand-activated state. The gonadotropin-releasing hormone receptor (GnRHR), because of its small s...

  14. Spontaneous Synaptic Activation of Muscarinic Receptors by Striatal Cholinergic Neuron Firing.

    Science.gov (United States)

    Mamaligas, Aphroditi A; Ford, Christopher P

    2016-08-01

    Cholinergic interneurons (CHIs) play a major role in motor and learning functions of the striatum. As acetylcholine does not directly evoke postsynaptic events at most striatal synapses, it remains unclear how postsynaptic cholinergic receptors encode the firing patterns of CHIs in the striatum. To examine the dynamics of acetylcholine release, we used optogenetics and paired recordings from CHIs and medium spiny neurons (MSNs) virally overexpressing G-protein-activated inwardly rectifying potassium (GIRK) channels. Due to the efficient coupling between endogenous muscarinic receptors and GIRK channels, we found that firing of individual CHIs resulted in monosynaptic spontaneous inhibitory post-synaptic currents (IPSCs) in MSNs. Paired CHI-MSN recordings revealed that the high probability of acetylcholine release at these synapses allowed muscarinic receptors to faithfully encode physiological activity patterns from individual CHIs without failure. These results indicate that muscarinic receptors in striatal output neurons reliably decode CHI firing. PMID:27373830

  15. Ligand binding and activation mechanism og the glucagon-like peptide-1 receptor

    DEFF Research Database (Denmark)

    Underwood, Christina Rye

    In recent years, G-protein coupled receptors (GPCRs) have become important drug targets, which makes elucidation of their molecular structure and functional domains increasingly important for designing new and better therapeutic agents. The Glucagon-Like Peptide-1 receptor (GLP-1R) is a GPCR. Its...... molecule-mediated activation of GLP-1R (Study II). A fully functional, cysteine-deprived and Cterminally truncated GLP-1R is developed and characterised in Study III. In Study IV, a cAMP biosensor is used to investigate the cAMP kinetics of GLP-1R upon stimulation with different receptor agonists...

  16. Molecular simulation of receptors of physiologically active compounds for purposes of medical chemistry

    Science.gov (United States)

    Baskin, Igor I.; Palyulin, Vladimir A.; Zefirov, Nikolai S.

    2009-06-01

    The general strategy of the molecular simulation of biological receptors and their interaction with ligands is considered. The procedures for construction of 3D protein models, molecular docking, evaluation of model quality, determination of the free energy of protein binding with ligands are discussed. The methods of molecular design of new medicaments based on molecular models of biological targets: virtual screening and de novo design, are presented. Examples of the above-listed approaches for the simulation of a number of pharmacologically significant receptors, analysis of receptor-ligand interactions and design of new biologically active organic compounds are given.

  17. Evidence that leptin-induced weight loss requires activation of both forebrain and hindbrain receptors

    OpenAIRE

    Ruth B.S. Harris

    2013-01-01

    Previous studies with chronic decerebrate rats and rats infused with leptin into the 4th ventricle suggest that hindbrain leptin receptors attenuate the catabolic effect of forebrain leptin receptor activation. To test this further rats were fitted with both 3rd and 4th ventricle cannulae. They were infused for 12 days with different combinations of saline, low dose leptin or leptin receptor antagonist (leptin mutein protein). Infusion of 0.1 μg leptin/day into the 3rd ventricle or 0.6 μg lep...

  18. Soluble urokinase plasminogen activator receptor is a marker of dysmetabolism in HIV-infected patients receiving highly active antiretroviral therapy

    DEFF Research Database (Denmark)

    Andersen, Ove; Eugen-Olsen, Jesper; Kofoed, Kristian;

    2008-01-01

    Circulating soluble urokinase plasminogen activator receptor (suPAR) reflects the immune and pro-inflammatory status of the HIV-infected patient. Highly active antiretroviral therapy (HAART) suppresses suPAR. Independent of the immune response to HAART, suPAR remains elevated in some HIV-infected...

  19. Activation of the Farnesoid X Receptor Provides Protection against Acetaminophen-Induced Hepatic Toxicity

    OpenAIRE

    Lee, Florence Ying; de Aguiar Vallim, Thomas Quad; Chong, Hansook Kim; Zhang, Yanqiao; Liu, Yaping; Jones, Stacey A.; Osborne, Timothy F.; Edwards, Peter A.

    2010-01-01

    The nuclear receptor, farnesoid X receptor (FXR, NR1H4), is known to regulate cholesterol, bile acid, lipoprotein, and glucose metabolism. In the current study, we provide evidence to support a role for FXR in hepatoprotection from acetaminophen (APAP)-induced toxicity. Pharmacological activation of FXR induces the expression of several genes involved in phase II and phase III xenobiotic metabolism in wild-type, but not Fxr−/− mice. We used chromatin immunoprecipitation-based genome-wide resp...

  20. Morbilliviruses Use Signaling Lymphocyte Activation Molecules (CD150) as Cellular Receptors

    OpenAIRE

    Tatsuo, Hironobu; Ono, Nobuyuki; Yanagi, Yusuke

    2001-01-01

    Morbilliviruses comprise measles virus, canine distemper virus, rinderpest virus, and several other viruses that cause devastating human and animal diseases accompanied by severe immunosuppression and lymphopenia. Recently, we have shown that human signaling lymphocyte activation molecule (SLAM) is a cellular receptor for measles virus. In this study, we examined whether canine distemper and rinderpest viruses also use canine and bovine SLAMs, respectively, as cellular receptors. The Onderste...

  1. Peroxisome Proliferator-Activated Receptors (PPARs) as Potential Inducers of Antineoplastic Effects in CNS Tumors

    OpenAIRE

    Lars Tatenhorst; Eric Hahnen; Heneka, Michael T

    2008-01-01

    The peroxisome proliferator-activated receptors (PPARs) are ligand-inducible transcription factors which belong to the superfamily of nuclear hormone receptors. In recent years it turned out that natural as well as synthetic PPAR agonists exhibit profound antineoplastic as well as redifferentiation effects in tumors of the central nervous system (CNS). The molecular understanding of the underlying mechanisms is still emerging, with partially controverse findings reported by a number of studie...

  2. BDNF Selectively Regulates GABAA Receptor Transcription by Activation of the JAK/STAT Pathway

    OpenAIRE

    Lund, Ingrid V.; Hu, Yinghui; Raol, YogendraSinh H.; Benham, Rebecca S.; Faris, Ramona; Russek, Shelley J.; Brooks-Kayal, Amy R.

    2008-01-01

    The γ-aminobutyric acid (GABA) type A receptor (GABAAR) is the major inhibitory neurotransmitter receptor in the brain. Its multiple subunits show regional, developmental, and disease-related plasticity of expression; however, the regulatory networks controlling GABAAR subunit expression remain poorly understood. We report that the seizure-induced decrease in GABAAR α1 subunit expression associated with epilepsy is mediated by the Janus kinase (JAK)/signal transducer and activator of transcri...

  3. PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR (PPAR) AGONISTS AS PROMISING NEW MEDICATIONS FOR DRUG ADDICTION: PRECLINICAL EVIDENCE

    OpenAIRE

    Foll, Bernard Le; Ciano, Patricia Di; Panlilio, Leigh V; Goldberg, Steven R.; Ciccocioppo, Roberto

    2013-01-01

    This review examines the growing literature on the role of peroxisome proliferator-activated receptors (PPARs) in addiction. There are two subtypes of PPAR receptors that have been studied in addiction: PPAR-α and PPAR-γ. The role of each PPAR subtype in common models of addictive behavior, mainly pre-clinical models, is summarized. In particular, studies are reviewed that investigated the effects of PPAR-α agonists on relapse, sensitization, conditioned place preference, withdrawal and drug ...

  4. Activation of NTS A(1) adenosine receptors inhibits regional sympathetic responses evoked by activation of cardiopulmonary chemoreflex.

    Science.gov (United States)

    Ichinose, Tomoko K; Minic, Zeljka; Li, Cailian; O'Leary, Donal S; Scislo, Tadeusz J

    2012-09-01

    Previously we have shown that adenosine operating via the A(1) receptor subtype may inhibit glutamatergic transmission in the baroreflex arc within the nucleus of the solitary tract (NTS) and differentially increase renal (RSNA), preganglionic adrenal (pre-ASNA), and lumbar (LSNA) sympathetic nerve activity (ASNA>RSNA≥LSNA). Since the cardiopulmonary chemoreflex and the arterial baroreflex are mediated via similar medullary pathways, and glutamate is a primary transmitter in both pathways, it is likely that adenosine operating via A(1) receptors in the NTS may differentially inhibit regional sympathetic responses evoked by activation of cardiopulmonary chemoreceptors. Therefore, in urethane-chloralose-anesthetized rats (n = 37) we compared regional sympathoinhibition evoked by the cardiopulmonary chemoreflex (activated with right atrial injections of serotonin 5HT(3) receptor agonist phenylbiguanide, PBG, 1-8 μg/kg) before and after selective stimulation of NTS A(1) adenosine receptors [microinjections of N(6)-cyclopentyl adenosine (CPA), 0.033-330 pmol/50 nl]. Activation of cardiopulmonary chemoreceptors evoked differential, dose-dependent sympathoinhibition (RSNA>ASNA>LSNA), and decreases in arterial pressure and heart rate. These differential sympathetic responses were uniformly attenuated in dose-dependent manner by microinjections of CPA into the NTS. Volume control (n = 11) and blockade of adenosine receptor subtypes in the NTS via 8-(p-sulfophenyl)theophylline (8-SPT, 1 nmol in 100 nl) (n = 9) did not affect the reflex responses. We conclude that activation of NTS A(1) adenosine receptors uniformly inhibits neural and cardiovascular cardiopulmonary chemoreflex responses. A(1) adenosine receptors have no tonic modulatory effect on this reflex under normal conditions. However, when adenosine is released into the NTS (i.e., during stress or severe hypotension/ischemia), it may serve as negative feedback regulator for depressor and sympathoinhibitory reflexes

  5. A common model for cytokine receptor activation: combined scissor-like rotation and self-rotation of receptor dimer induced by class I cytokine.

    Directory of Open Access Journals (Sweden)

    Xiaodong Pang

    Full Text Available The precise mechanism by which the binding of a class I cytokine to the extracellular domain of its corresponding receptor transmits a signal through the cell membrane remains unclear. Receptor activation involves a cytokine-receptor complex with a 1∶2 stoichiometry. Previously we used our transient-complex theory to calculate the rate constant of the initial cytokine-receptor binding to form a 1∶1 complex. Here we computed the binding pathway leading to the 1∶2 activation complex. Three cytokine systems (growth hormone, erythropoietin, and prolactin were studied, and the focus was on the binding of the extracellular domain of the second receptor molecule after forming the 1∶1 complex. According to the transient-complex theory, translational and rotation diffusion of the binding entities bring them together to form a transient complex, which has near-native relative separation and orientation but not the short-range specific native interactions. Subsequently conformational rearrangement leads to the formation of the native complex. We found that the changes in relative orientations between the two receptor molecules from the transient complex to the 1∶2 native complex are similar for the three cytokine-receptor systems. We thus propose a common model for receptor activation by class I cytokines, involving combined scissor-like rotation and self-rotation of the two receptor molecules. Both types of rotations seem essential: the scissor-like rotation separates the intracellular domains of the two receptor molecules to make room for the associated Janus kinase molecules, while the self-rotation allows them to orient properly for transphosphorylation. This activation model explains a host of experimental observations. The transient-complex based approach presented here may provide a strategy for designing antagonists and prove useful for elucidating activation mechanisms of other receptors.

  6. Two Redundant Receptor-Like Cytoplasmic Kinases Function Downstream of Pattern Recognition Receptors to Regulate Activation of SA Biosynthesis.

    Science.gov (United States)

    Kong, Qing; Sun, Tongjun; Qu, Na; Ma, Junling; Li, Meng; Cheng, Yu-Ti; Zhang, Qian; Wu, Di; Zhang, Zhibin; Zhang, Yuelin

    2016-06-01

    Salicylic acid (SA) serves as a critical signaling molecule in plant defense. Two transcription factors, SARD1 and CBP60g, control SA biosynthesis through regulating pathogen-induced expression of Isochorismate Synthase1, which encodes a key enzyme for SA biosynthesis. Here, we report that Pattern-Triggered Immunity Compromised Receptor-like Cytoplasmic Kinase1 (PCRK1) and PCRK2 function as key regulators of SA biosynthesis. In the pcrk1 pcrk2 double mutant, pathogen-induced expression of SARD1, CBP60g, and ICS1 is greatly reduced. The pcrk1 pcrk2 double mutant, but neither of the single mutants, exhibits reduced accumulation of SA and enhanced disease susceptibility to bacterial pathogens. Both PCRK1 and PCRK2 interact with the pattern recognition receptor FLS2, and treatment with pathogen-associated molecular patterns leads to rapid phosphorylation of PCRK2. Our data suggest that PCRK1 and PCRK2 function downstream of pattern recognition receptor in a signal relay leading to the activation of SA biosynthesis. PMID:27208222

  7. Activation of Dopamine Receptors in the Nucleus Accumbens Promotes Sucrose-Reinforced Cued Approach Behavior

    Science.gov (United States)

    du Hoffmann, Johann; Nicola, Saleem M.

    2016-01-01

    Dopamine receptor activation in the nucleus accumbens (NAc) promotes vigorous environmentally-cued food-seeking in hungry rats. Rats fed ad libitum, however, respond to fewer food-predictive cues, particularly when the value of food reward is low. Here, we investigated whether this difference could be due to differences in the degree of dopamine receptor activation in the NAc. First, we observed that although rats given ad libitum access to chow in their home cages approached a food receptacle in response to reward-predictive cues, the number of such approaches declined as animals accumulated food rewards. Intriguingly, cued approach to food occurred in clusters, with several cued responses followed by successive non-responses. This pattern suggested that behavior was dictated by transitions between two states, responsive and non-responsive. Injection of D1 or D2 dopamine receptor agonists into the NAc dose-dependently increased cue responding by promoting transitions to the responsive state and by preventing transitions to the non-responsive state. In contrast, antagonists of either D1 or D2 receptors promoted long bouts of non-responding by inducing transitions to the non-responsive state and by preventing transitions to the responsive state. Moreover, locomotor behavior during the inter-trial interval was correlated with the responsive state, and was also increased by dopamine receptor agonists. These results suggest that activation of NAc dopamine receptors plays an important role in regulating the probability of approach to food under conditions of normative satiety. PMID:27471453

  8. Natural compounds regulate energy metabolism by the modulating the activity of lipid-sensing nuclear receptors.

    Science.gov (United States)

    Goto, Tsuyoshi; Kim, Young-Il; Takahashi, Nobuyuki; Kawada, Teruo

    2013-01-01

    Obesity causes excess fat accumulation in various tissues, most notoriously in the adipose tissue, along with other insulin-responsive organs such as skeletal muscle and the liver, which predisposes an individual to the development of metabolic abnormalities. The molecular mechanisms underlying obesity-induced metabolic abnormalities have not been completely elucidated; however, in recent years, the search for therapies to prevent the development of obesity and obesity-associated metabolic disorders has increased. It is known that several nuclear receptors, when activated by specific ligands, regulate carbohydrate and lipid metabolism at the transcriptional level. The expression of lipid metabolism-related enzymes is directly regulated by the activity of various nuclear receptors via their interaction with specific response elements in promoters of those genes. Many natural compounds act as ligands of nuclear receptors and regulate carbohydrate and lipid metabolism by regulating the activities of these nuclear receptors. In this review, we describe our current knowledge of obesity, the role of lipid-sensing nuclear receptors in energy metabolism, and several examples of food factors that act as agonists or antagonists of nuclear receptors, which may be useful for the management of obesity and the accompanying energy metabolism abnormalities.

  9. Exercise reduces adipose tissue via cannabinoid receptor type 1 which is regulated by peroxisome proliferator-activated receptor

    International Nuclear Information System (INIS)

    Obesity is one major cardiovascular risk factor. We tested effects of endurance exercise on cannabinoid receptor type 1 (CB1) and peroxisome proliferator-activated receptor-δ (PPAR-δ)-dependent pathways in adipose tissue. Male Wistar rats were randomly assigned to standard laboratory chow or a high-fat diet without and with regular endurance exercise. Exercise in rats on high-fat diet significantly reduced visceral fat mass, blood pressure, and adipocyte size (each p < 0.05). Adipocyte hypertrophy induced by high-fat diet was accompanied by increased CB1 expression in adipose tissue, whereas exercise significantly reduced CB1 expression (each p < 0.05). CB1 receptor expression and adipocyte differentiation were directly regulated by PPAR-δ. Adipocyte hypertrophy induced by high-fat diet was accompanied by reduced PPAR-δ. Furthermore, selective silencing of PPAR-δ by RNA interference in 3T3-L1-preadipocyte cells significantly increased CB1 expression from 1.00 ± 0.06 (n = 3) to 1.91 ± 0.06 (n = 3; p < 0.01) and increased adipocyte differentiation, whereas adenovirus-mediated overexpression of PPAR-δ significantly reduced CB1 expression to 0.39 ± 0.03 (n = 3; p < 0.01) and reduced adipocyte differentiation. In the presence of the CB1 antagonist rimonabant adipocyte differentiation in stimulated 3T3 L1 preadipocyte cells was significantly reduced. The study indicates that high-fat diet-induced hypertrophy of adipocytes is associated with increased CB1 receptor expression which is directly regulated by PPAR-δ. Both CB1 and PPAR-δ are intimately involved in therapeutic interventions against a most important cardiovascular risk factor

  10. Biological activities of binding site specific monoclonal antibodies to prolactin receptors of rabbit mammary gland

    International Nuclear Information System (INIS)

    The biological activity of three monoclonal antibodies (mAbs) against the rabbit mammary prolactin (PRL) receptor (M110, A82, and A917) were investigated using explants of rabbit mammary gland. The three mAbs which were all able to inhibit the binding of 125I-ovine prolactin to its receptor had different biological activities. Two mAbs (M110 and A82) were able to prevent the stimulating effect of PRL on casein synthesis when the molar ratio between the mAb and PRL was 100. One mAb (A917) was able to mimic the action of PRL on both casein and DNA ([3H]thymidine incorporation) synthesis, whereas the other two mAbs were without any stimulatory effect. For this stimulatory effect to be observed, bivalency of the antibody was essential, since monovalent fragments, which were able to inhibit PRL binding, had no agonistic activity. The ability of the mAbs to induce a down-regulation of receptors was also studied. These studies suggest that the binding domain of the receptor might be relatively complex, since only a part of this domain recognized by the antibody with PRL-like activity was able to induce hormonal action. Alternatively, only those antibodies able to microaggregate the receptors may possess PRL-like activity

  11. Structural mechanism of ligand activation in human calcium-sensing receptor

    Science.gov (United States)

    Geng, Yong; Mosyak, Lidia; Kurinov, Igor; Zuo, Hao; Sturchler, Emmanuel; Cheng, Tat Cheung; Subramanyam, Prakash; Brown, Alice P; Brennan, Sarah C; Mun, Hee-chang; Bush, Martin; Chen, Yan; Nguyen, Trang X; Cao, Baohua; Chang, Donald D; Quick, Matthias; Conigrave, Arthur D; Colecraft, Henry M; McDonald, Patricia; Fan, Qing R

    2016-01-01

    Human calcium-sensing receptor (CaSR) is a G-protein-coupled receptor (GPCR) that maintains extracellular Ca2+ homeostasis through the regulation of parathyroid hormone secretion. It functions as a disulfide-tethered homodimer composed of three main domains, the Venus Flytrap module, cysteine-rich domain, and seven-helix transmembrane region. Here, we present the crystal structures of the entire extracellular domain of CaSR in the resting and active conformations. We provide direct evidence that L-amino acids are agonists of the receptor. In the active structure, L-Trp occupies the orthosteric agonist-binding site at the interdomain cleft and is primarily responsible for inducing extracellular domain closure to initiate receptor activation. Our structures reveal multiple binding sites for Ca2+ and PO43- ions. Both ions are crucial for structural integrity of the receptor. While Ca2+ ions stabilize the active state, PO43- ions reinforce the inactive conformation. The activation mechanism of CaSR involves the formation of a novel dimer interface between subunits. DOI: http://dx.doi.org/10.7554/eLife.13662.001 PMID:27434672

  12. Differential activation of nitric oxide synthase through muscarinic acetylcholine receptors in rat salivary glands.

    Science.gov (United States)

    Leirós, C P; Rosignoli, F; Genaro, A M; Sales, M E; Sterin-Borda, L; Santiago BordaE

    2000-03-15

    Muscarinic receptors play an important role in secretory and vasodilator responses in rat salivary glands. Nitric oxide synthase (NOS) appears to be one of the multiple effectors coupled to muscarinic receptors in both submandibular and sublingual glands although some differences have been found depending on the gland studied. First, submandibular glands had a lower basal activity of nitric oxide synthase than sublingual glands and the concentration-response curve for carbachol was bell-shaped in the former but not in sublingual glands. Second, cGMP levels displayed a similar profile to that observed for NOS activity in both glands. Third, protein kinase C also coupled to muscarinic receptor activation in the glands might have a regulatory effect on nitric oxide production since its activity was higher in basal conditions in submandibular than sublingual glands and it also increased in the presence of the agonist at a concentration that inhibited NOS activity in submandibular glands. The effects appear to be partly related to the expression of a minor population of M(1) receptors in submandibular glands absent in sublingual as determined in binding and signaling experiments with the muscarinic receptor antagonist pirenzepine.

  13. Structural mechanism of ligand activation in human calcium-sensing receptor

    Energy Technology Data Exchange (ETDEWEB)

    Geng, Yong; Mosyak, Lidia; Kurinov, Igor; Zuo, Hao; Sturchler, Emmanuel; Cheng, Tat Cheung; Subramanyam, Prakash; Brown, Alice P.; Brennan, Sarah C.; Mun, Hee-chang; Bush, Martin; Chen, Yan; Nguyen, Trang X.; Cao, Baohua; Chang, Donald D.; Quick, Matthias; Conigrave, Arthur D.; Colecraft, Henry M.; McDonald, Patricia; Fan, Qing R.

    2016-07-19

    Human calcium-sensing receptor (CaSR) is a G-protein-coupled receptor (GPCR) that maintains extracellular Ca2+homeostasis through the regulation of parathyroid hormone secretion. It functions as a disulfide-tethered homodimer composed of three main domains, the Venus Flytrap module, cysteine-rich domain, and seven-helix transmembrane region. Here, we present the crystal structures of the entire extracellular domain of CaSR in the resting and active conformations. We provide direct evidence that L-amino acids are agonists of the receptor. In the active structure, L-Trp occupies the orthosteric agonist-binding site at the interdomain cleft and is primarily responsible for inducing extracellular domain closure to initiate receptor activation. Our structures reveal multiple binding sites for Ca2+and PO43-ions. Both ions are crucial for structural integrity of the receptor. While Ca2+ions stabilize the active state, PO43-ions reinforce the inactive conformation. The activation mechanism of CaSR involves the formation of a novel dimer interface between subunits.

  14. PKC-dependent Phosphorylation of the H1 Histamine Receptor Modulates TRPC6 Activity.

    Science.gov (United States)

    Chen, Xingjuan; Egly, Christian; Riley, Ashley M; Li, Wennan; Tewson, Paul; Hughes, Thomas E; Quinn, Anne Marie; Obukhov, Alexander G

    2014-01-01

    Transient receptor potential canonical 6 (TRPC6) is a cation selective, DAG-regulated, Ca2+-permeable channel activated by the agonists of Gq-protein-coupled heptahelical receptors. Dysfunctions of TRPC6 are implicated in the pathogenesis of various cardiovascular and kidney conditions such as vasospasm and glomerulosclerosis. When stimulated by agonists of the histamine H1 receptor (H1R), TRPC6 activity decays to the baseline despite the continuous presence of the agonist. In this study, we examined whether H1R desensitization contributes to regulating the decay rate of TRPC6 activity upon receptor stimulation. We employed the HEK expression system and a biosensor allowing us to simultaneously detect the changes in intracellular diacylglycerol (DAG) and Ca2+ concentrations. We found that the histamine-induced DAG response was biphasic, in which a transient peak was followed by maintained elevated plateau, suggesting that desensitization of H1R takes place in the presence of histamine. The application of PKC inhibitor Gö6983 slowed the decay rate of intracellular DAG concentration. Activation of the mouse H1R mutant lacking a putative PKC phosphorylation site, Ser399, responsible for the receptor desensitization, resulted in a prolonged intracellular DAG increase and greater Mn2+ influx through the TRPC6 channel. Thus, our data support the hypothesis that PKC-dependent H1R phosphorylation leads to a reduced production of intracellular DAG that contributes to TRPC6 activity regulation.

  15. PKC-dependent Phosphorylation of the H1 Histamine Receptor Modulates TRPC6 Activity

    Directory of Open Access Journals (Sweden)

    Xingjuan Chen

    2014-04-01

    Full Text Available Transient receptor potential canonical 6 (TRPC6 is a cation selective, DAG-regulated, Ca2+-permeable channel activated by the agonists of Gq-protein-coupled heptahelical receptors. Dysfunctions of TRPC6 are implicated in the pathogenesis of various cardiovascular and kidney conditions such as vasospasm and glomerulosclerosis. When stimulated by agonists of the histamine H1 receptor (H1R, TRPC6 activity decays to the baseline despite the continuous presence of the agonist. In this study, we examined whether H1R desensitization contributes to regulating the decay rate of TRPC6 activity upon receptor stimulation. We employed the HEK expression system and a biosensor allowing us to simultaneously detect the changes in intracellular diacylglycerol (DAG and Ca2+ concentrations. We found that the histamine-induced DAG response was biphasic, in which a transient peak was followed by maintained elevated plateau, suggesting that desensitization of H1R takes place in the presence of histamine. The application of PKC inhibitor Gö6983 slowed the decay rate of intracellular DAG concentration. Activation of the mouse H1R mutant lacking a putative PKC phosphorylation site, Ser399, responsible for the receptor desensitization, resulted in a prolonged intracellular DAG increase and greater Mn2+ influx through the TRPC6 channel. Thus, our data support the hypothesis that PKC-dependent H1R phosphorylation leads to a reduced production of intracellular DAG that contributes to TRPC6 activity regulation.

  16. Protease-Activated Receptor 4 Variant p.Tyr157Cys Reduces Platelet Functional Responses and Alters Receptor Trafficking

    OpenAIRE

    Norman, Jane E; Cunningham, Margaret R; Jones, Matthew L.; Walker, Mary E; Westbury, Sarah K; Sessions, Richard B.; Mundell, Stuart J.; Mumford, Andrew D.

    2016-01-01

    OBJECTIVE: Protease-activated receptor 4 (PAR4) is a key regulator of platelet reactivity and is encoded by F2RL3, which has abundant rare missense variants. We aimed to provide proof of principle that rare F2LR3 variants potentially affect platelet reactivity and responsiveness to PAR1 antagonist drugs and to explore underlying molecular mechanisms.APPROACH AND RESULTS: We identified 6 rare F2RL3 missense variants in 236 cardiac patients, of which the variant causing a tyrosine 157 to cystei...

  17. Analysis of the heat shock response in mouse liver reveals dependence on the Nuclear Receptor Peroxisome Proliferator-Activated Receptor alpha (PPARalpha)

    NARCIS (Netherlands)

    Vallanat, B.; Anderson, S.P.; Brown-Borg, H.M.; Ren, H.; Kersten, A.H.; Jonnalagadda, S.; Srinivasan, S.; Corton, J.C.

    2010-01-01

    Background - The nuclear receptor peroxisome proliferator-activated receptor alpha (PPARa) regulates responses to chemical or physical stress in part by altering expression of genes involved in proteome maintenance. Many of these genes are also transcriptionally regulated by heat shock (HS) through

  18. Peroxisome proliferator-activated receptor gamma regulates expression of the anti-lipolytic G-protein-coupled receptor 81 (GPR81/Gpr81)

    DEFF Research Database (Denmark)

    Jeninga, Ellen H; Bugge, Anne Skovsø; Nielsen, Ronni;

    2009-01-01

    The ligand-inducible nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma) plays a key role in the differentiation, maintenance, and function of adipocytes and is the molecular target for the insulin-sensitizing thiazoledinediones (TZDs). Although a number of PPARgamma...

  19. Identification of functionally important residues in the silkmoth pheromone biosynthesis-activating neuropeptide receptor, an insect ortholog of the vertebrate Neuromedin U Receptor

    Science.gov (United States)

    The biosynthesis of sex pheromone components in many lepidopteran insects is regulated by interactions between pheromone biosynthesis-activating neuropeptide (PBAN) and the PBAN receptor (PBANR), a class-A G-protein-coupled receptor (GPCR). To identify functionally important amino acid residues in t...

  20. Expression of the Inhibitory CD200 Receptor Is Associated with Alternative Macrophage Activation

    NARCIS (Netherlands)

    N. Koning; M. van Eijk; W. Pouwels; M.S.M. Brouwer; D. Voehringer; I. Huitinga; R.M. Hoek; G. Raes; J. Hamann

    2010-01-01

    Classical macrophage activation is inhibited by the CD200 receptor (CD200R). Here, we show that CD200R expression was specifically induced on human in vitro polarized macrophages of the alternatively activated M2a subtype, generated by incubation with IL-4 or IL-13. In mice, peritoneal M2 macrophage

  1. Activity of L-alpha-amino acids at the promiscuous goldfish odorant receptor 5.24

    DEFF Research Database (Denmark)

    Christiansen, Bolette; Wellendorph, Petrine; Bräuner-Osborne, Hans

    2006-01-01

    report the agonist activities of the 20 proteinogenic L-alpha-amino acids, and L-ornithine and L-citrulline, measured in an intracellular calcium release assay in mammalian tsA cells. The results show that receptor 5.24 is broadly activated by 19 of the tested L-alpha-amino acids and displays...

  2. Prediction of in vitro and in vivo oestrogen receptor activity using hierarchical clustering

    Science.gov (United States)

    In this study, hierarchical clustering classification models were developed to predict in vitro and in vivo oestrogen receptor (ER) activity. Classification models were developed for binding, agonist, and antagonist in vitro ER activity and for mouse in vivo uterotrophic ER bindi...

  3. Activated Cdc42-associated kinase Ack1 promotes prostate cancer progression via androgen receptor tyrosine phosphorylation

    OpenAIRE

    Mahajan, Nupam P.; Liu, Yuanbo; Majumder, Samarpan; Warren, Maria R.; Parker, Carol E.; Mohler, James L.; Earp, H. Shelton; Whang, Young E.

    2007-01-01

    Activation of the androgen receptor (AR) may play a role in androgen-independent progression of prostate cancer. Multiple mechanisms of AR activation, including stimulation by tyrosine kinases, have been postulated. We and others have recently shown involvement of activated Cdc42-associated tyrosine kinase Ack1 in advanced human prostate cancer. Here we provide the molecular basis for interplay between Ack1 and AR in prostate cancer cells. Activated Ack1 promoted androgen-independent growth o...

  4. Dopamine D2 receptor-mediated Akt/PKB signalling: initiation by the D2S receptor and role in quinpirole-induced behavioural activation

    OpenAIRE

    Jin‑Chung Chen; Nan‑Yu Ruan; Tzu‑Yung Lin; Han‑Ting Chen

    2012-01-01

    The short and long isoforms of the dopamine D2 receptor (D2S and D2L respectively) are highly expressed in the striatum. Functional D2 receptors activate an intracellular signalling pathway that includes a cAMP-independent route involving Akt/GSK3 (glycogen synthase kinase 3). To investigate the Akt/GSK3 response to the seldom-studied D2S receptor, we established a rat D2S receptor-expressing cell line [HEK (human embryonic kidney)-293/rD2S]. We found that in HEK-293/rD2S cells, the D2/D3 ago...

  5. A peptide antagonist of the ErbB1 receptor inhibits receptor activation, tumor cell growth and migration in vitro and xenograft tumor growth in vivo

    DEFF Research Database (Denmark)

    Xu, Ruodan; Povlsen, Gro Klitgaard; Soroka, Vladislav;

    2010-01-01

    The epidermal growth factor family of receptor tyrosine kinases (ErbBs) plays essential roles in tumorigenesis and cancer disease progression, and therefore has become an attractive target for structure-based drug design. ErbB receptors are activated by ligand-induced homo- and heterodimerization...... lung cancer cell line A549. The Inherbin3 peptide may be a useful tool for investigating the mechanisms of ErbB receptor homo- and heterodimerization. Moreover, the here described biological effects of Inherbin3 suggest that peptide-based targeting of ErbB receptor dimerization is a promising anti...

  6. Increased dopamine D1 receptor binding in the human mesocortical system following central cholinergic activation

    International Nuclear Information System (INIS)

    Full text: The interaction between the cholinergic and dopaminergic system has been implicated in many pathological processes including, Alzheimer's disease, schizophrenia and drug addiction. Little is known about the control of dopamine (DA) release following central cholinergic activation in humans, but experimental studies suggest that endogenously released Acetylcholine (ACh) achieved by the administration of cholinesterase inhibitors, can increase dopamine efflux in different regions of the brain. This leads to the activation of different types of post-synaptic dopaminergic receptors which belong to the family of G-protein coupled receptors (GPCRs). A common paradigm of the GPCRs desensitization is that agonist-induced receptor signaling is rapidly attenuated by receptor internalisation. Several experiments have shown that the activation of Dl receptors in acute conditions leads, within minutes, to translocation of the receptor from the surface of the neurons to the endosomal compartment in the cytoplasm and increased receptor turnover. To assess changes in Dl receptor density following an intravenous infusion of the selective cholinesterase inhibitor physostigmine salicylate (PHY), we studied eleven normal subjects (10 male and 1 female, mean age 36.1 and 61617; 9.9) using [11C]-SCH23390 and PET The binding potential (BP) for SCH23390 was significantly (p0.05). There was no statistically significant difference between baseline and physostigmine Kl ratio (p>0.05) suggesting that BP changes observed were not secondary to regional blood flow changes or to an order effect of the scans. Copyright (2002) The Australian and New Zealand Society of Nuclear Medicine Inc

  7. receptores

    Directory of Open Access Journals (Sweden)

    Salete Regina Daronco Benetti

    2006-01-01

    Full Text Available Se trata de un estudio etnográfico, que tuvo lo objetivo de interpretar el sistema de conocimiento y del significado atribuidos a la sangre referente a la transfusión sanguínea por los donadores y receptores de un banco de sangre. Para la colecta de las informaciones se observaron los participantes y la entrevista etnográfica se realizó el análisis de dominio, taxonómicos y temáticos. Los dominios culturales fueron: la sangre es vida: fuente de vida y alimento valioso; creencias religiosas: fuentes simbólicas de apoyos; donación sanguínea: un gesto colaborador que exige cuidarse, gratifica y trae felicidad; donación sanguínea: fuente simbólica de inseguridad; estar enfermo es una condición para realizar transfusión sanguínea; transfusión sanguínea: esperanza de vida; Creencias populares: transfusión sanguínea como riesgo para la salud; donadores de sangre: personas benditas; donar y recibir sangre: como significado de felicidad. Temática: “líquido precioso que origina, sostiene, modifica la vida, provoca miedo e inseguridad”.

  8. Glucose activation of islets of Langerhans up-regulates Toll-like receptor 5: possible mechanism of protection

    DEFF Research Database (Denmark)

    Weile, Christian Roar Andersen; Josefsen, Knud Elnegaard; Buschard, Karsten Stig

    2011-01-01

    Toll-like receptors are pattern-recognition receptors of the innate immune system that are activated during viral, bacterial or other infections, as well as during disease progression of type 1 and type 2 diabetes. Toll-like receptor 5 (TLR-5) specifically recognizes bacterial infection through b...

  9. Activation of human tonsil and skin mast cells by agonists of proteinase activated receptor-2

    Institute of Scientific and Technical Information of China (English)

    Shao-heng HE; Hua XIE; Yi-ling FU

    2005-01-01

    Aim: To investigate the effects of the agonists of proteinase activated receptor (PAR)-2,and histamine on degranulation of human mast cells. Methods: Human mast cells were enzymatically dispersed from tonsil and skin tissues. The dis persed cells were then cultured with various stimuli, and tryptase and histamine levels in cell supernatants collected from challenge tubes were measured. Results:PAR-2 agonist peptide SLIGKV provoked a dose-dependent release of histamine from skin mast cells. It also induced tryptase release from tonsil mast cells, tcLIGRLO appeared less potent than SLIGKV in induction of release of histamine and tryptase. Trypsin was able to induce a "bell" shape increase in tryptase release from tonsil mast cells. It was also able to induce a dose-dependent release of histamine from both tonsil and skin mast cells. The actions of trypsin on mast cells were inhibited by soy bean trypsin inhibitor (SBTI) or α1-antitrypsin (α1-AT).Time course study revealed that both stimulated tryptase or histamine release initiated within 10 s and reached their peak release between 4 and 6 min. Pretreatment of cells with metabolic inhibitors or pertussis toxin reduced the ability of mast cells to release tryptase or histamine. Conclusion: It was demonstrated that the in vitro tryptase release properties of human tonsil and skin mast cells suggested a novel type of mast cell heterogeneity. The activation of mast cells by PAR-2 agonists indicated a self-amplification mechanism of mast cell degranulation.

  10. Activation of group III metabotropic glutamate receptors is neuroprotective in cortical cultures.

    Science.gov (United States)

    Bruno, V; Copani, A; Bonanno, L; Knoepfel, T; Kuhn, R; Roberts, P J; Nicoletti, F

    1996-08-22

    (RS)-alpha-Methyl-4-phosphonophenylglycine (MPPG) and (S)-alpha-methyl-3-carboxyphenylalanine (M3CPA), two novel preferential antagonists of group III metabotropic glutamate (mGlu) receptors, antagonized the neuroprotective activity of L-2-amino-4-phosphono-butanoate (L-AP4) or L-serine-O-phosphate in mice cultured cortical cells exposed to a toxic pulse of N-methyl-D-aspartate. In contrast, MPPG did not influence the neuroprotective activity of the selective group II mGlu receptor agonist, (2S,1'R,2'R,3'R)-2-(2,3-dicarboxy-cyclopropyl) glycine (DCG-IV). These results indicate that activation of group III mGu receptors exerts neuroprotective activity against excitotoxic neuronal death. At least one of the two major group III mGlu receptor subtypes, i.e. mGlu4 receptor, is expressed by cultured cortical neurons, as shown by immunocytochemical analysis with specific polyclonal antibodies. PMID:8880068

  11. H(1)-Receptor activation triggers the endogenous nitric oxide signalling system in the rat submandibular gland.

    Science.gov (United States)

    Borda, Enri; Stranieri, Graciela; Sterin-Borda, Leonor

    2002-01-01

    BACKGROUND: Histamine is released from mast cells by immunologic and non-immunologic stimuli during salivary gland inflammation, regulating salivary secretion. The receptor-secretory mechanism has not been studied in detail. AIMS: The studies reported were directed toward elucidating signal transduction/second messenger pathways within the rat submandibular gland associated with 2-thiazolylethylamine (ThEA)-induced H(1)-receptor responses. MATERIALS AND METHODS: To assess the H(1) receptor subtype expression in the rat submandibular gland, a radioligand binding assay was performed. The study also included inositolphosphates and cyclic GMP accumulation, protein kinase C and nitric oxide synthase activities, and amylase release. RESULTS: The histamine H(1) receptor subtype is expressed on the rat submandibular gland with high-affinity binding sites. The ThEA effect was associated with activation of phosphoinositide-specific phospholipase C, translocation of protein kinase C, stimulation of nitric oxide synthase activity and increased production of cyclic GMP. ThEA stimulation of nitric oxide synthase and cyclic GMP was blunted by agents able to interfere with calcium movilization, while a protein kinase C inhibitor was able to stimulate ThEA action. On the other hand, ThEA stimulation evoked amylase release via the H1 receptor but was not followed by the L-arginine/nitric oxide pathway activation. CONCLUSIONS: These results suggest that, apart from the effect of ThEA on amylase release, it also appears to be a vasoactive chemical mediator that triggers vasodilatation, modulating the course of inflammation. PMID:12581497

  12. Improper activation of D1 and D2 receptors leads to excess noise in prefrontal cortex

    Directory of Open Access Journals (Sweden)

    Michael eAvery

    2015-03-01

    Full Text Available The dopaminergic system has been shown to control the amount of noise in the prefrontal cortex (PFC and likely plays an important role in working memory and the pathophysiology of schizophrenia. We developed a model that takes into account the known receptor distributions of D1 and D2 receptors, the changes these receptors have on neuron response properties, as well as identified circuitry involved in working memory. Our model suggests that D1 receptor under-stimulation in supragranular layers gates internal noise into the PFC leading to cognitive symptoms as has been proposed in attention disorders, while D2 over-stimulation gates noise into the PFC by over-activation of cortico-striatal projecting neurons in infragranular layers. We apply this model in the context of a memory-guided saccade paradigm and show deficits similar to those observed in schizophrenic patients. We also show set-shifting impairments similar to those observed in rodents with D1 and D2 receptor manipulations. We discuss how the introduction of noise through changes in D1 and D2 receptor activation may account for many of the symptoms of schizophrenia depending on where this dysfunction occurs in the PFC.

  13. Castor oil induces laxation and uterus contraction via ricinoleic acid activating prostaglandin EP3 receptors.

    Science.gov (United States)

    Tunaru, Sorin; Althoff, Till F; Nüsing, Rolf M; Diener, Martin; Offermanns, Stefan

    2012-06-01

    Castor oil is one of the oldest drugs. When given orally, it has a laxative effect and induces labor in pregnant females. The effects of castor oil are mediated by ricinoleic acid, a hydroxylated fatty acid released from castor oil by intestinal lipases. Despite the wide-spread use of castor oil in conventional and folk medicine, the molecular mechanism by which ricinoleic acid acts remains unknown. Here we show that the EP(3) prostanoid receptor is specifically activated by ricinoleic acid and that it mediates the pharmacological effects of castor oil. In mice lacking EP(3) receptors, the laxative effect and the uterus contraction induced via ricinoleic acid are absent. Although a conditional deletion of the EP(3) receptor gene in intestinal epithelial cells did not affect castor oil-induced diarrhea, mice lacking EP(3) receptors only in smooth-muscle cells were unresponsive to this drug. Thus, the castor oil metabolite ricinoleic acid activates intestinal and uterine smooth-muscle cells via EP(3) prostanoid receptors. These findings identify the cellular and molecular mechanism underlying the pharmacological effects of castor oil and indicate a role of the EP(3) receptor as a target to induce laxative effects.

  14. Phasic and Tonic mGlu7 Receptor Activity Modulates the Thalamocortical Network.

    Science.gov (United States)

    Tassin, Valériane; Girard, Benoît; Chotte, Apolline; Fontanaud, Pierre; Rigault, Delphine; Kalinichev, Mikhail; Perroy, Julie; Acher, Francine; Fagni, Laurent; Bertaso, Federica

    2016-01-01

    Mutation of the metabotropic glutamate receptor type 7 (mGlu7) induces absence-like epileptic seizures, but its precise role in the somatosensory thalamocortical network remains unknown. By combining electrophysiological recordings, optogenetics, and pharmacology, we dissected the contribution of the mGlu7 receptor at mouse thalamic synapses. We found that mGlu7 is functionally expressed at both glutamatergic and GABAergic synapses, where it can inhibit neurotransmission and regulate short-term plasticity. These effects depend on the PDZ-ligand of the receptor, as they are lost in mutant mice. Interestingly, the very low affinity of mGlu7 receptors for glutamate raises the question of how it can be activated, namely at GABAergic synapses and in basal conditions. Inactivation of the receptor activity with the mGlu7 negative allosteric modulator (NAM), ADX71743, enhances thalamic synaptic transmission. In vivo administration of the NAM induces a lethargic state with spindle and/or spike-and-wave discharges accompanied by a behavioral arrest typical of absence epileptic seizures. This provides evidence for mGlu7 receptor-mediated tonic modulation of a physiological function in vivo preventing synchronous and potentially pathological oscillations. PMID:27199672

  15. Phasic and tonic mGlu7 receptor activity modulates the thalamocortical network

    Directory of Open Access Journals (Sweden)

    Valériane eTassin

    2016-04-01

    Full Text Available Mutation of the metabotropic glutamate receptor type 7 (mGlu7 induces absence-like epileptic seizures, but its precise role in the somatosensory thalamocortical network remains unknown. By combining electrophysiological recordings, optogenetics and pharmacology we dissected the contribution of the mGlu7 receptor at mouse thalamic synapses. We found that mGlu7 is functionally expressed at both glutamatergic and GABAergic synapses, where it can inhibit neurotransmission and regulate short-term plasticity. These effects depend on the PDZ-ligand of the receptor, as they are lost in mutant mice. Interestingly, the very low affinity of mGlu7 receptors for glutamate raises the question of how it can be activated, namely at GABAergic synapses and in basal conditions. Inactivation of the receptor activity with the mGlu7 negative allosteric modulator (NAM, ADX71743, enhances thalamic synaptic transmission. In vivo administration of the NAM induces a lethargic state with spindle and/or spike-and-wave discharges accompanied by a behavioral arrest typical of absence epileptic seizures. This provides evidence for mGlu7 receptor-mediated tonic modulation of a physiological function in vivo preventing synchronous and potentially pathological oscillations.

  16. A quantitative method to assess extrasynaptic NMDA receptor function in the protective effect of synaptic activity against neurotoxicity

    Directory of Open Access Journals (Sweden)

    Bading Hilmar

    2008-01-01

    Full Text Available Abstract Background Extrasynaptic NMDA receptors couple to a CREB shut-off pathway and cause cell death, whereas synaptic NMDA receptors and nuclear calcium signaling promote CREB-mediated transcription and neuronal survival. The distribution of NMDA receptors (synaptic versus extrasynaptic may be an important parameter that determines the susceptibility of neurons to toxic insults. Changes in receptor surface expression towards more extrasynaptic NMDA receptors may lead to neurodegeneration, whereas a reduction of extrasynaptic NMDA receptors may render neurons more resistant to death. A quantitative assessment of extrasynaptic NMDA receptors in individual neurons is needed in order to investigate the role of NMDA receptor distribution in neuronal survival and death. Results Here we refined and verified a protocol previously used to isolate the effects of extrasynaptic NMDA receptors using the NMDA receptor open channel blocker, MK-801. Using this method we investigated the possibility that the known neuroprotective shield built up in hippocampal neurons after a period of action potential bursting and stimulation of synaptic NMDA receptors is due to signal-induced trafficking of extrasynaptic NMDA receptors or a reduction in extrasynaptic NMDA receptor function. We found that extrasynaptic NMDA receptor-mediated calcium responses and whole cell currents recorded under voltage clamp were surprisingly invariable and did not change even after prolonged (16 to 24 hours periods of bursting and synaptic NMDA receptor activation. Averaging a large number of calcium imaging traces yielded a small (6% reduction of extrasynaptic NMDA receptor-mediated responses in hippocampal neurons that were pretreated with prolonged bursting. Conclusion The slight reduction in extrasynaptic NMDA receptor function following action potential bursting and synaptic NMDA receptor stimulation could contribute to but is unlikely to fully account for activity

  17. Enhanced Food Anticipatory Activity Associated with Enhanced Activation of Extrahypothalamic Neural Pathways in Serotonin2C Receptor Null Mutant Mice

    OpenAIRE

    Mistlberger, Ralph; Hsu, Jennifer; Yu, Lisa; Bowman, Melody; Tecott, Laurence; Sullivan, Elinor

    2010-01-01

    The ability to entrain circadian rhythms to food availability is important for survival. Food-entrained circadian rhythms are characterized by increased locomotor activity in anticipation of food availability (food anticipatory activity). However, the molecular components and neural circuitry underlying the regulation of food anticipatory activity remain unclear. Here we show that serotonin2C receptor (5-HT2CR) null mutant mice subjected to a daytime restricted feeding schedule exhibit enhanc...

  18. Enhanced Food Anticipatory Activity Associated with Enhanced Activation of Extrahypothalamic Neural Pathways in Serotonin2C Receptor Null Mutant Mice

    OpenAIRE

    Hsu, Jennifer L.; Lisa Yu; Elinor Sullivan; Melodi Bowman; Mistlberger, Ralph E.; Tecott, Laurence H.

    2010-01-01

    The ability to entrain circadian rhythms to food availability is important for survival. Food-entrained circadian rhythms are characterized by increased locomotor activity in anticipation of food availability (food anticipatory activity). However, the molecular components and neural circuitry underlying the regulation of food anticipatory activity remain unclear. Here we show that serotonin(2C) receptor (5-HT2CR) null mutant mice subjected to a daytime restricted feeding schedule exhibit enha...

  19. Functional Selectivity and Antidepressant Activity of Serotonin 1A Receptor Ligands

    Directory of Open Access Journals (Sweden)

    Zdzisław Chilmonczyk

    2015-08-01

    Full Text Available Serotonin (5-HT is a monoamine neurotransmitter that plays an important role in physiological functions. 5-HT has been implicated in sleep, feeding, sexual behavior, temperature regulation, pain, and cognition as well as in pathological states including disorders connected to mood, anxiety, psychosis and pain. 5-HT1A receptors have for a long time been considered as an interesting target for the action of antidepressant drugs. It was postulated that postsynaptic 5-HT1A agonists could form a new class of antidepressant drugs, and mixed 5-HT1A receptor ligands/serotonin transporter (SERT inhibitors seem to possess an interesting pharmacological profile. It should, however, be noted that 5-HT1A receptors can activate several different biochemical pathways and signal through both G protein-dependent and G protein-independent pathways. The variables that affect the multiplicity of 5-HT1A receptor signaling pathways would thus result from the summation of effects specific to the host cell milieu. Moreover, receptor trafficking appears different at pre- and postsynaptic sites. It should also be noted that the 5-HT1A receptor cooperates with other signal transduction systems (like the 5-HT1B or 5-HT2A/2B/2C receptors, the GABAergic and the glutaminergic systems, which also contribute to its antidepressant and/or anxiolytic activity. Thus identifying brain specific molecular targets for 5-HT1A receptor ligands may result in a better targeting, raising a hope for more effective medicines for various pathologies.

  20. The orphan nuclear receptor Rev-Erbalpha is a peroxisome proliferator-activated receptor (PPAR) gamma target gene and promotes PPARgamma-induced adipocyte differentiation

    DEFF Research Database (Denmark)

    Fontaine, Coralie; Dubois, Guillaume; Duguay, Yannick;

    2003-01-01

    Rev-Erbalpha (NR1D1) is an orphan nuclear receptor encoded on the opposite strand of the thyroid receptor alpha gene. Rev-Erbalpha mRNA is induced during adipocyte differentiation of 3T3-L1 cells, and its expression is abundant in rat adipose tissue. Peroxisome proliferator-activated receptor gamma...... (PPARgamma) (NR1C3) is a nuclear receptor controlling adipocyte differentiation and insulin sensitivity. Here we show that Rev-Erbalpha expression is induced by PPARgamma activation with rosiglitazone in rat epididymal and perirenal adipose tissues in vivo as well as in 3T3-L1 adipocytes in vitro...... for this nuclear receptor as a promoter of adipocyte differentiation....

  1. Similar activation of signal transduction pathways by the herpesvirus-encoded chemokine receptors US28 and ORF74

    DEFF Research Database (Denmark)

    McLean, Katherine A; Holst, Peter J; Martini, Lene;

    2004-01-01

    The virally encoded chemokine receptors US28 from human cytomegalovirus and ORF74 from human herpesvirus 8 are both constitutively active. We show that both receptors constitutively activate the transcription factors nuclear factor of activated T cells (NFAT) and cAMP response element binding pro...

  2. DMPD: Toll receptors, CD14, and macrophage activation and deactivation by LPS. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 12106783 Toll receptors, CD14, and macrophage activation and deactivation by LPS. D...ceptors, CD14, and macrophage activation and deactivation by LPS. PubmedID 12106783 Title Toll receptors, CD14, and macrophage activa...tion and deactivation by LPS. Authors Dobrovolskaia MA,

  3. Ultraslow Water-Mediated Transmembrane Interactions Regulate the Activation of A2A Adenosine Receptor.

    Science.gov (United States)

    Lee, Yoonji; Kim, Songmi; Choi, Sun; Hyeon, Changbong

    2016-09-20

    Water molecules inside a G-protein coupled receptor (GPCR) have recently been spotlighted in a series of crystal structures. To decipher the dynamics and functional roles of internal water molecules in GPCR activity, we studied the A2A adenosine receptor using microsecond molecular-dynamics simulations. Our study finds that the amount of water flux across the transmembrane (TM) domain varies depending on the receptor state, and that the water molecules of the TM channel in the active state flow three times more slowly than those in the inactive state. Depending on the location in solvent-protein interface as well as the receptor state, the average residence time of water in each residue varies from ∼O(10(2)) ps to ∼O(10(2)) ns. Especially, water molecules, exhibiting ultraslow relaxation (∼O(10(2)) ns) in the active state, are found around the microswitch residues that are considered activity hotspots for GPCR function. A continuous allosteric network spanning the TM domain, arising from water-mediated contacts, is unique in the active state, underscoring the importance of slow water molecules in the activation of GPCRs. PMID:27653477

  4. Exchange factors directly activated by cAMP mediate melanocortin 4 receptor-induced gene expression

    Science.gov (United States)

    Glas, Evi; Mückter, Harald; Gudermann, Thomas; Breit, Andreas

    2016-01-01

    Gs protein-coupled receptors regulate many vital body functions by activation of cAMP response elements (CRE) via cAMP-dependent kinase A (PKA)-mediated phosphorylation of the CRE binding protein (CREB). Melanocortin 4 receptors (MC4R) are prototypical Gs-coupled receptors that orchestrate the hypothalamic control of food-intake and metabolism. Remarkably, the significance of PKA for MC4R-induced CRE-dependent transcription in hypothalamic cells has not been rigorously interrogated yet. In two hypothalamic cell lines, we observed that blocking PKA activity had only weak or no effects on reporter gene expression. In contrast, inhibitors of exchange factors directly activated by cAMP-1/2 (EPAC-1/2) mitigated MC4R-induced CRE reporter activation and mRNA induction of the CREB-dependent genes c-fos and thyrotropin-releasing hormone. Furthermore, we provide first evidence that extracellular-regulated kinases-1/2 (ERK-1/2) activated by EPACs and not PKA are the elusive CREB kinases responsible for MC4R-induced CREB/CRE activation in hypothalamic cells. Overall, these data emphasize the pivotal role of EPACs rather than PKA in hypothalamic gene expression elicited by a prototypical Gs-coupled receptor. PMID:27612207

  5. Exchange factors directly activated by cAMP mediate melanocortin 4 receptor-induced gene expression.

    Science.gov (United States)

    Glas, Evi; Mückter, Harald; Gudermann, Thomas; Breit, Andreas

    2016-01-01

    Gs protein-coupled receptors regulate many vital body functions by activation of cAMP response elements (CRE) via cAMP-dependent kinase A (PKA)-mediated phosphorylation of the CRE binding protein (CREB). Melanocortin 4 receptors (MC4R) are prototypical Gs-coupled receptors that orchestrate the hypothalamic control of food-intake and metabolism. Remarkably, the significance of PKA for MC4R-induced CRE-dependent transcription in hypothalamic cells has not been rigorously interrogated yet. In two hypothalamic cell lines, we observed that blocking PKA activity had only weak or no effects on reporter gene expression. In contrast, inhibitors of exchange factors directly activated by cAMP-1/2 (EPAC-1/2) mitigated MC4R-induced CRE reporter activation and mRNA induction of the CREB-dependent genes c-fos and thyrotropin-releasing hormone. Furthermore, we provide first evidence that extracellular-regulated kinases-1/2 (ERK-1/2) activated by EPACs and not PKA are the elusive CREB kinases responsible for MC4R-induced CREB/CRE activation in hypothalamic cells. Overall, these data emphasize the pivotal role of EPACs rather than PKA in hypothalamic gene expression elicited by a prototypical Gs-coupled receptor. PMID:27612207

  6. TFIIB-directed transcriptional activation by the orphan nuclear receptor hepatocyte nuclear factor 4.

    OpenAIRE

    Malik, S; Karathanasis, S K

    1996-01-01

    The orphan nuclear receptor hepatocyte nuclear factor 4 (HNF-4) is required for development and maintenance of the liver phenotype. HNF-4 activates several hepatocyte-specific genes, including the gene encoding apolipoprotein AI (apoAI), the major protein component of plasma high-density lipoprotein. The apoAI gene is activated by HNF-4 through a nuclear receptor binding element (site A) located in its liver-specific enhancer. To decipher the mechanism whereby HNF-4 enhances apoAI gene transc...

  7. CLE Peptides in Plants: Proteolytic Processing,Structure-Activity Relationship, and Ligand-Receptor Interaction

    Institute of Scientific and Technical Information of China (English)

    Xiaoming Gao; Yongfeng Guo

    2012-01-01

    Ligand-receptor signaling initiated by the CLAVATA3/ENDOSPERM SURROUNDING REGION (CLE) family peptides is critical in regulating cell division and differentiation in meristematic tissues in plants.Biologically active CLE peptides are released from precursor proteins via proteolytic processing.The mature form of CLE ligands consists of 12-13 amino acids with several post-translational modifications.This review summarizes recent progress toward understanding the proteolytic activities that cleave precursor proteins to release CLE peptides,the molecular structure and function of mature CLE ligands,and interactions between CLE ligands and corresponding leucine-rich repeat (LRR) receptor-like kinases (RLKs).

  8. Activation and block of recombinant GABAA receptors by pentobarbitone: a single-channel study

    OpenAIRE

    Akk, Gustav; Steinbach, Joe Henry

    2000-01-01

    Recombinant GABAA receptors (α1β2γ2L) were transiently expressed in HEK 293 cells. We have investigated activation and block of these receptors by pentobarbitone (PB) using cell-attached single-channel patch clamp.Clusters of single-channel activity elicited by 500 μM PB were analysed to estimate rate constants for agonist binding and channel gating. The minimal model able to describe the kinetic data involved two sequential binding steps, followed by channel opening. The estimated channel op...

  9. Recruitment of epidermal growth factor receptors into coated pits requires their activated tyrosine kinase

    OpenAIRE

    1995-01-01

    EGF-receptor (EGF-R) tyrosine kinase is required for the down- regulation of activated EGF-R. However, controversy exists as to whether ligand-induced activation of the EGF-R tyrosine kinase is required for internalization or for lysosomal targeting. We have addressed this issue using a cell-free assay that selectively measures the recruitment of EGF-R into coated pits. Here we show that EGF bound to wild-type receptors is efficiently sequestered in coated pits. In contrast, sequestration of ...

  10. Synthesis, structure-activity relationships, and characterization of novel nonsteroidal and selective androgen receptor modulators.

    Science.gov (United States)

    Schlienger, Nathalie; Lund, Birgitte W; Pawlas, Jan; Badalassi, Fabrizio; Bertozzi, Fabio; Lewinsky, Rasmus; Fejzic, Alma; Thygesen, Mikkel B; Tabatabaei, Ali; Bradley, Stefania Risso; Gardell, Luis R; Piu, Fabrice; Olsson, Roger

    2009-11-26

    Herein we describe the discovery of ACP-105 (1), a novel and potent nonsteroidal selective androgen receptor modulator (SARM) with partial agonist activity relative to the natural androgen testosterone. Compound 1 was developed from a series of compounds found in a HTS screen using the receptor selection and amplification technology (R-SAT). In vivo, 1 improved anabolic parameters in a 2-week chronic study in castrated male rats. In addition to compound 1, a number of potent antiandrogens were discovered from the same series of compounds whereof one compound, 13, had antagonist activity at the AR T877A mutant involved in prostate cancer.

  11. Expression and activation of proteases in co-cultures.

    Science.gov (United States)

    Paduch, Roman; Kandefer-Szerszeń, Martyna

    2011-01-01

    The present study concerned the expression and activation of metalloproteinase-2 (MMP-2), metalloproteinase-9 (MMP-9) and the urokinase plasminogen activator/urokinase plasminogen activator receptor (uPA/uPAR) system in co-cultures of human colon carcinoma cell spheroids (HT29, LS180, SW948) with human normal colon epithelium (CCD 841 CoTr), myofibroblasts (CCD-18Co) and endothelial cells (HUVEC). Additionally, the influence of monensin on the production and function of the proteases was tested. Tumor cells expressed small amounts of MMP-2, MMP-9 and uPA. Normal cells generally produced proportionally higher concentrations of these proteases (especially MMP-2, compared with significantly smaller yields of MMP-9 and significantly lower amounts of uPAR than tumors. In co-cultures of tumor spheroids with normal cell monolayers, the concentration of the proteases was equal to the sum of the enzymes produced in monocultures of both types of cells. The highest activity of uPA, measured as the reduction of the chromogenic substrate (S-2444), was detected in supernatants and lysates of endothelial cells. Interestingly, in normal cells, the higher expression of proteases, mainly uPA, measured as the level of protein concentration, was closely linked with their lower activity and inversely, in tumor cells, the low level of the expression of the enzymes correlated with their high enzymatic activity. In zymography analysis, mainly pro-MMPs were detected both in culture supernatants and cell lysates. The highest amounts of active forms of the MMPs were detected in tumor spheroids co-cultured with endothelial cells. Monensin inhibited MMPs and uPA secretion but significantly increased uPAR release, mainly from normal cells. In conclusion, during direct interactions of tumor cells with normal cells, MMPs and the uPA/uPAR system play an important role in the degradation of ECM and tumor development, but as we found, there is a reverse relationship between the concentration and the

  12. Investigation of Prolactin Receptor Activation and Blockade Using Time-Resolved Fluorescence Resonance Energy Transfer

    Directory of Open Access Journals (Sweden)

    Estelle eTallet

    2011-09-01

    Full Text Available The prolactin receptor (PRLR is emerging as a therapeutic target in oncology. Knowledge-based drug design led to the development of a pure PRLR antagonist (Del1-9-G129R-hPRL that was recently shown to prevent PRL-induced mouse prostate tumorogenesis. In humans, the first gain-of-function mutation of the PRLR (PRLRI146L was recently identified in breast tumor patients. At the molecular level, the actual mechanism of action of these two novel players in the PRL system remains elusive. In this study, we addressed whether constitutive PRLR activation (PRLRI146L or PRLR blockade (antagonist involved alteration of receptor oligomerization and/or of inter-chain distances compared to unstimulated and PRL-stimulated PRLR. Using a combination of various biochemical and spectroscopic approaches (co-IP, blue-native electrophoresis, BRET1, we demonstrated that preformed PRLR homodimers are altered neither by PRL- or I146L-induced receptor triggering, nor by antagonist-mediated blockade. These findings were confirmed using a novel time-resolved fluorescence resonance energy transfer (TR-FRET technology that allows monitoring distance changes between cell-surface tagged receptors. This technology revealed that PRLR blockade or activation did not involve detectable distance changes between extracellular domains of receptor chains within the dimer. This study merges with our previous structural investigations suggesting that the mechanism of PRLR activation solely involves intermolecular contact adaptations leading to subtle intramolecular rearrangements.

  13. Toxoplasma gondii infection regulates the balance of activating and inhibitory receptors on decidual natural killer cells.

    Directory of Open Access Journals (Sweden)

    Xiaoyan Xu

    Full Text Available Inhibitory receptors and activating receptor expressed on decidual natural killer (dNK cells are generally believed to be important in abnormal pregnancy outcomes and induced adverse pregnancy. However, if Toxoplasma gondii (T. gondii infection induced abnormal pregnancy was related to dNK cells changes is not clear. In this study, we used human dNK cells co-cultured with human extravillous cytotrophoblast (EVT cells following YFP-Toxoplasma gondii (YFP-T. gondii infection in vitro and established animal pregnant infection model. Levels of inhibitory receptors KIR2DL4 and ILT-2, their ligand HLA-G, and activating receptor NKG2D in human decidua, and NKG2A and its ligand Qa-1 and NKG2D in mice uterine were analyzed by real-time PCR and flow cytometry with levels of NKG2D significantly higher than those of KIR2DL4 and ILT-2 in vitro and in invo. The level of NKG2D was positively correlated with cytotoxic activity of dNK cells in vitro. Numbers of abnormal pregnancies were significantly greater in the infected group than in the control group. This result demonstrated that the increased NKG2D expression and imbalance between inhibitory receptors of dNK cells and HLA-G may contribute to abnormal pregnancy outcomes observed upon maternal infection with T. gondii.

  14. Diabetic Nephropathy Is Accelerated by Farnesoid X Receptor Deficiency and Inhibited by Farnesoid X Receptor Activation in a Type 1 Diabetes Model

    OpenAIRE

    Wang, Xiaoxin X.; Jiang, Tao; Shen, Yan; Caldas, Yupanqui; Miyazaki-Anzai, Shinobu; Santamaria, Hannah; Urbanek, Cydney; Solis, Nathaniel; Scherzer, Pnina; Lewis, Linda; Gonzalez, Frank J.; Adorini, Luciano; Pruzanski, Mark; Jeffrey B Kopp; Verlander, Jill W.

    2010-01-01

    OBJECTIVE The pathogenesis of diabetic nephropathy is complex and involves activation of multiple pathways leading to kidney damage. An important role for altered lipid metabolism via sterol regulatory element binding proteins (SREBPs) has been recently recognized in diabetic kidney disease. Our previous studies have shown that the farnesoid X receptor (FXR), a bile acid-activated nuclear hormone receptor, modulates renal SREBP-1 expression. The purpose of the present study was then to determ...

  15. Inhibition of testicular embryonal carcinoma cell tumorigenicity by peroxisome proliferator-activated receptor-β/δ- and retinoic acid receptor-dependent mechanisms

    OpenAIRE

    Yao, Pei-Li; Chen, Li Ping; Dobrzański, Tomasz P.; Phillips, Dylan A.; Zhu, Bokai; Kang, Boo-Hyon; Frank J. Gonzalez; Peters, Jeffrey M.

    2015-01-01

    Peroxisome proliferator-activated receptor-β/δ (PPARβ/δ) has important physiological functions in control of cell growth, lipid and glucose homeostasis, differentiation and inflammation. To investigate the role of PPARβ/δ in cancer, stable human testicular embryonal carcinoma cell lines were developed that constitutively express PPARβ/δ. Expression of PPARβ/δ caused enhanced activation of the receptor, and this significantly decreased proliferation, migration, invasion, anchorage-independent ...

  16. A Molecular Mechanism for Sequential Activation of a G Protein-Coupled Receptor

    DEFF Research Database (Denmark)

    Grundmann, Manuel; Tikhonova, Irina G; Hudson, Brian D;

    2016-01-01

    , as the first sequential activator and corroborate its two-step activation in living cells by tracking integrated responses with innovative label-free biosensors that visualize multiple signaling inputs in real time. We validate this unique pharmacology with traditional cellular readouts, including mutational...... and pharmacological perturbations along with computational methods, and propose a kinetic model applicable to the analysis of sequential receptor activation. We envision this form of dynamic agonism as a common principle of nature to spatiotemporally encode cellular information....

  17. Activation of the unliganded estrogen receptor by EGF involves the MAP kinase pathway and direct phosphorylation.

    OpenAIRE

    Bunone, G; Briand, P A; Miksicek, R J; Picard, D.

    1996-01-01

    The estrogen receptor (ER) can be activated as a transcription factor either by binding of cognate estrogenic ligand or, indirectly, by a variety of other extracellular signals. As a first step towards elucidating the mechanism of 'steroid-independent activation' of the ER by the epidermal growth factor (EGF), we have mapped the ER target domain and determined the signaling pathway. We show that the N-terminal transcriptional activation function AF-1, but not the C-terminal AF-2, is necessary...

  18. Adenosine activates brown adipose tissue and recruits beige adipocytes via A2A receptors

    DEFF Research Database (Denmark)

    Gnad, Thorsten; Scheibler, Saskia; von Kügelgen, Ivar;

    2014-01-01

    Brown adipose tissue (BAT) is specialized in energy expenditure, making it a potential target for anti-obesity therapies. Following exposure to cold, BAT is activated by the sympathetic nervous system with concomitant release of catecholamines and activation of β-adrenergic receptors. Because BAT...... that adenosine-A2A signalling plays an unexpected physiological role in sympathetic BAT activation and protects mice from diet-induced obesity. Those findings reveal new possibilities for developing novel obesity therapies....

  19. GABAA receptors in visual and auditory cortex and neural activity changes during basic visual stimulation

    Directory of Open Access Journals (Sweden)

    Pengmin eQin

    2012-12-01

    Full Text Available Recent imaging studies have demonstrated that levels of resting GABA in the visual cortex predict the degree of stimulus-induced activity in the same region. These studies have used the presentation of discrete visual stimulus; the change from closed eyes to open also represents a simple visual stimulus, however, and has been shown to induce changes in local brain activity and in functional connectivity between regions. We thus aimed to investigate the role of the GABA system, specifically GABAA receptors, in the changes in brain activity between the eyes closed (EC and eyes open (EO state in order to provide detail at the receptor level to complement previous studies of GABA concentrations. We conducted an fMRI study involving two different modes of the change from EC to EO: An EO and EC block design, allowing the modelling of the haemodynamic response, followed by longer periods of EC and EO to allow the measuring of functional connectivity. The same subjects also underwent [18F]Flumazenil PET measure GABAA receptor binding potentials. It was demonstrated that the local-to-global ratio of GABAA receptor binding potential in the visual cortex predicted the degree of changes in neural activity from EC to EO. This same relationship was also shown in the auditory cortex. Furthermore, the local-to-global ratio of GABAA receptor binding potential in the visual cortex also predicts the change of functional connectivity between visual and auditory cortex from EC to EO. These findings contribute to our understanding of the role of GABAA receptors in stimulus-induced neural activity in local regions and in inter-regional functional connectivity.

  20. Receptor-mediated regional sympathetic nerve activation by leptin.

    OpenAIRE

    Haynes, W G; Morgan, D A; Walsh, S A; Mark, A L; Sivitz, W I

    1997-01-01

    Leptin is a peptide hormone produced by adipose tissue which acts centrally to decrease appetite and increase energy expenditure. Although leptin increases norepinephrine turnover in thermogenic tissues, the effects of leptin on directly measured sympathetic nerve activity to thermogenic and other tissues are not known. We examined the effects of intravenous leptin and vehicle on sympathetic nerve activity to brown adipose tissue, kidney, hindlimb, and adrenal gland in anesthetized Sprague-Da...

  1. Activation of mu opioid receptors sensitizes transient receptor potential vanilloid type 1 (TRPV1 via β-arrestin-2-mediated cross-talk.

    Directory of Open Access Journals (Sweden)

    Matthew P Rowan

    Full Text Available The transient receptor potential family V1 channel (TRPV1 is activated by multiple stimuli, including capsaicin, acid, endovanilloids, and heat (>42C. Post-translational modifications to TRPV1 result in dynamic changes to the sensitivity of receptor activation. We have previously demonstrated that β-arrestin2 actively participates in a scaffolding mechanism to inhibit TRPV1 phosphorylation, thereby reducing TRPV1 sensitivity. In this study, we evaluated the effect of β-arrestin2 sequestration by G-protein coupled receptors (GPCRs on thermal and chemical activation of TRPV1. Here we report that activation of mu opioid receptor by either morphine or DAMGO results in β-arrestin2 recruitment to mu opioid receptor in sensory neurons, while activation by herkinorin does not. Furthermore, treatment of sensory neurons with morphine or DAMGO stimulates β-arrestin2 dissociation from TRPV1 and increased sensitivity of the receptor. Conversely, herkinorin treatment has no effect on TRPV1 sensitivity. Additional behavioral studies indicate that GPCR-driven β-arrestin2 sequestration plays an important peripheral role in the development of thermal sensitivity. Taken together, the reported data identify a novel cross-talk mechanism between GPCRs and TRPV1 that may contribute to multiple clinical conditions.

  2. Interaction between Calpain 5, Peroxisome proliferator-activated receptor-gamma and Peroxisome proliferator-activated receptor-delta genes: a polygenic approach to obesity

    Directory of Open Access Journals (Sweden)

    Ruiz Agustín

    2008-07-01

    Full Text Available Abstract Context Obesity is a multifactorial disorder, that is, a disease determined by the combined effect of genes and environment. In this context, polygenic approaches are needed. Objective To investigate the possibility of the existence of a crosstalk between the CALPAIN 10 homologue CALPAIN 5 and nuclear receptors of the peroxisome proliferator-activated receptors family. Design Cross-sectional, genetic association study and gene-gene interaction analysis. Subjects The study sample comprise 1953 individuals, 725 obese (defined as body mass index ≥ 30 and 1228 non obese subjects. Results In the monogenic analysis, only the peroxisome proliferator-activated receptor delta (PPARD gene was associated with obesity (OR = 1.43 [1.04–1.97], p = 0.027. In addition, we have found a significant interaction between CAPN5 and PPARD genes (p = 0.038 that reduces the risk for obesity in a 55%. Conclusion Our results suggest that CAPN5 and PPARD gene products may also interact in vivo.

  3. Protease-activated receptor 2 activation of myeloid dendritic cells regulates allergic airway inflammation

    Directory of Open Access Journals (Sweden)

    Dienger Krista

    2011-09-01

    Full Text Available Abstract Background A common characteristic of allergens is that they contain proteases that can activate protease-activated receptor (PAR-2; however the mechanism by which PAR-2 regulates allergic airway inflammation is unclear. Methods Mice (wild type and PAR-2-deficient were sensitized using German cockroach (GC feces (frass, the isolated protease from GC frass, or through adoptive transfer of GC frass-treated bone marrow-derived dendritic cells (BMDC and measurements of airway inflammation (cellular infiltration, cytokine expression, and mucin production, serum IgE levels and airway hyperresponsiveness (AHR were assessed. BMDC were cultured, treated with GC frass and assessed for cytokine production. PAR-2 expression on pulmonary mDCs was determined by flow cytometry. Results Exposure to GC frass induced AHR and airway inflammation in wild type mice; however PAR-2-deficient mice had significantly attenuated responses. To directly investigate the role of the protease, we isolated the protease from GC frass and administered the endotoxin-free protease into the airways of mice in the presence of OVA. GC frass proteases were sufficient to promote the development of AHR, serum IgE, and Th2 cytokine production. PAR-2 expression on mDC was upregulated following GC frass exposure, but the presence of a functional PAR-2 did not alter antigen uptake. To determine if PAR-2 activation led to differential cytokine production, we cultured BMDC in the presence of GM-CSF and treated these cells ex vivo with GC frass. PAR-2-deficient BMDC released significantly less IL-6, IL-23 and TNFα compared to BMDC from wild type mice, suggesting PAR-2 activation was important in Th2/Th17 skewing cytokine production. To determine the role for PAR-2 on mDCs on the initiation of allergic airway inflammation, BMDCs from wild type and PAR-2-deficient mice were treated in the presence or absence of GC frass and then adoptively transferred into the airway of wild type mice

  4. Exercise reduces adipose tissue via cannabinoid receptor type 1 which is regulated by peroxisome proliferator-activated receptor-delta.

    Science.gov (United States)

    Yan, Zhen Cheng; Liu, Dao Yan; Zhang, Li Li; Shen, Chen Yi; Ma, Qun Li; Cao, Ting Bing; Wang, Li Juan; Nie, Hai; Zidek, Walter; Tepel, Martin; Zhu, Zhi Ming

    2007-03-01

    Obesity is one major cardiovascular risk factor. We tested effects of endurance exercise on cannabinoid receptor type 1 (CB1) and peroxisome proliferator-activated receptor-delta (PPAR-delta)-dependent pathways in adipose tissue. Male Wistar rats were randomly assigned to standard laboratory chow or a high-fat diet without and with regular endurance exercise. Exercise in rats on high-fat diet significantly reduced visceral fat mass, blood pressure, and adipocyte size (each pAdipocyte hypertrophy induced by high-fat diet was accompanied by increased CB1 expression in adipose tissue, whereas exercise significantly reduced CB1 expression (each padipocyte differentiation were directly regulated by PPAR-delta. Adipocyte hypertrophy induced by high-fat diet was accompanied by reduced PPAR-delta. Furthermore, selective silencing of PPAR-delta by RNA interference in 3T3-L1-preadipocyte cells significantly increased CB1 expression from 1.00+/-0.06 (n=3) to 1.91+/-0.06 (n=3; padipocyte differentiation, whereas adenovirus-mediated overexpression of PPAR-delta significantly reduced CB1 expression to 0.39+/-0.03 (n=3; padipocyte differentiation. In the presence of the CB1 antagonist rimonabant adipocyte differentiation in stimulated 3T3 L1 preadipocyte cells was significantly reduced. The study indicates that high-fat diet-induced hypertrophy of adipocytes is associated with increased CB1 receptor expression which is directly regulated by PPAR-delta. Both CB1 and PPAR-delta are intimately involved in therapeutic interventions against a most important cardiovascular risk factor. PMID:17223076

  5. Structure-dependent binding and activation of perfluorinated compounds on human peroxisome proliferator-activated receptor γ

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Lianying [State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, P.O. Box 2871, 18 Shuangqing Road, Beijing 100085 (China); College of Life Science, Dezhou University, Dezhou 253023 (China); Ren, Xiao-Min; Wan, Bin [State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, P.O. Box 2871, 18 Shuangqing Road, Beijing 100085 (China); Guo, Liang-Hong, E-mail: LHGuo@rcees.ac.cn [State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, P.O. Box 2871, 18 Shuangqing Road, Beijing 100085 (China)

    2014-09-15

    Perfluorinated compounds (PFCs) have been shown to disrupt lipid metabolism and even induce cancer in rodents through activation of peroxisome proliferator-activated receptors (PPARs). Lines of evidence showed that PPARα was activated by PFCs. However, the information on the binding interactions between PPARγ and PFCs and subsequent alteration of PPARγ activity is still limited and sometimes inconsistent. In the present study, in vitro binding of 16 PFCs to human PPARγ ligand binding domain (hPPARγ-LBD) and their activity on the receptor in cells were investigated. The results showed that the binding affinity was strongly dependent on their carbon number and functional group. For the eleven perfluorinated carboxylic acids (PFCAs), the binding affinity increased with their carbon number from 4 to 11, and then decreased slightly. The binding affinity of the three perfluorinated sulfonic acids (PFSAs) was stronger than their PFCA counterparts. No binding was detected for the two fluorotelomer alcohols (FTOHs). Circular dichroim spectroscopy showed that PFC binding induced distinctive structural change of the receptor. In dual luciferase reporter assays using transiently transfected Hep G2 cells, PFCs acted as hPPARγ agonists, and their potency correlated with their binding affinity with hPPARγ-LBD. Molecular docking showed that PFCs with different chain length bind with the receptor in different geometry, which may contribute to their differences in binding affinity and transcriptional activity. - Highlights: • Binding affinity between PFCs and PPARγ was evaluated for the first time. • The binding strength was dependent on fluorinated carbon chain and functional group. • PFC binding induced distinctive structural change of the receptor. • PFCs could act as hPPARγ agonists in Hep G2 cells.

  6. Insulin receptor activation in the nucleus accumbens reflects nutritive value of a recently ingested meal.

    Science.gov (United States)

    Woods, C A; Guttman, Z R; Huang, D; Kolaric, R A; Rabinowitsch, A I; Jones, K T; Cabeza de Vaca, S; Sclafani, A; Carr, K D

    2016-05-15

    With respect to feeding, insulin is typically thought of as a satiety hormone, acting in the hypothalamus to limit ingestive behavior. However, accumulating evidence suggests that insulin also has the ability to alter dopamine release in the striatum and influence food preferences. With increased access to high calorie foods, Western societies have a high prevalence of obesity, accompanied by insulin insensitivity. Little is known about how insulin is trafficked into the brain following food consumption and whether insulin insensitivity in the periphery is mirrored in the central nervous system. We investigated insulin receptor activation in the ventral striatum of rats receiving water or 16% glucose either orally or intragastrically. We also investigated whether glucose-induced insulin receptor activation was altered in food-restricted (FR) or diet-induced obesity (OB) rat models. Lastly, we examined whether insulin plays a significant role in flavor-nutrient preference learning. Glucose intake stimulated a rapid increase in insulin receptor activity in the ventral striatum of FR and ad libitum (AL) fed rats, but not OB rats. Similarly, both AL and FR, but not OB rats demonstrated significant flavor-nutrient preferences. However AL rats receiving brief inhibition of insulin activity during conditioning failed to acquire a significant flavor-nutrient preference. These findings suggest that impaired insulin receptor activation in the ventral striatum may result in inaccurate valuation of nutritive foods, which could lead to overconsumption of food or the selection of foods that don't accurately meet the body's current physiological needs. PMID:26988281

  7. Distinct pathways of ERK1/2 activation by hydroxy-carboxylic acid receptor-1.

    Directory of Open Access Journals (Sweden)

    Guo Li

    Full Text Available Mechanistic investigations have shown that, upon agonist activation, hydroxy-carboxylic acid receptor-1(HCA1 couples to a Gi protein and inhibits adenylate cyclase activity, leading to inhibition of liberation of free fatty acid. However, the underlying molecular mechanisms for HCA1 signaling remain largely unknown. Using CHO-K1 cells stably expressing HCA1, and L6 cells, which endogenously express rat HCA1 receptors, we found that activation of ERK1/2 by HCA1 was rapid, peaking at 5 min, and was significantly blocked by pertussis toxin. Furthermore, time course experiments with different kinase inhibitors demonstrated that HCA1 induced ERK1/2 activation via the extracellular Ca2+, PKC and IGF-I receptor transactivation-dependent pathways. In addition, we observed that pretreated the cells with M119K, an inhibitor of Gβγ subunit-dependent signaling, effectively attenuated the ERK1/2 activation triggered by HCA1, suggesting a critical role for βγ-subunits in HCA1-activated ERK1/2 phosphorylation. Furthermore, the present results also indicated that the arrestin2/3 were not required for ERK1/2 activation. In conclusion, our findings demonstrate that upon binding to agonist, HCA1 receptors initially activate Gi, leading to dissociation of the Gβγ subunit from activated Gi, and subsequently induce ERK1/2 activation via two distinct pathways: one PKC-dependent pathway and the other IGF-IR transactivation-dependent pathway. Our results provide the first in-depth evidence that defines the molecular mechanism of HCA1-mediated ERK1/2 activation.

  8. Haemolysis induced by α-toxin from Staphylococcus aureus requires P2X receptor activation

    DEFF Research Database (Denmark)

    Skals, Marianne Gerberg; Leipziger, Jens Georg; Prætorius, Helle

    2011-01-01

    -forming bacterial toxins. In this context, it is essential to know whether this is specific to HlyA-induced cell damage or if other bacterial pore-forming toxins involve purinergic signals to orchestrate haemolysis. Here, we investigate if the haemolysis produced by α-toxin from Staphylococcus aureus involves P2...... receptor activation. We observed that α-toxin-induced haemolysis is completely blocked by the unselective P2 receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid. Moreover, several selective blockers of P2X1 and P2X7 ionotropic receptors abolished haemolysis in murine and equine...... erythrocytes. Inhibitors of pannexin channels partially reduced the α-toxin induced lysis. Thus, we conclude that α-toxin, similar to HlyA from E. coli produces cell damage by specific activation of a purinergic signalling cascade. These data indicate that pore-forming toxins in general require purinergic...

  9. Sch proteins are localized on endoplasmic reticulum membranes and are redistributed after tyrosine kinase receptor activation

    DEFF Research Database (Denmark)

    Lotti, L V; Lanfrancone, L; Migliaccio, E;

    1996-01-01

    area of the cell and mostly associated with the cytosolic side of rough endoplasmic reticulum membranes. Upon epidermal growth factor treatment and receptor tyrosine kinase activation, the immunolabeling became peripheral and was found to be associated with the cytosolic surface of the plasma membrane......The intracellular localization of Shc proteins was analyzed by immunofluorescence and immunoelectron microscopy in normal cells and cells expressing the epidermal growth factor receptor or the EGFR/erbB2 chimera. In unstimulated cells, the immunolabeling was localized in the central perinuclear....... The rough endoplasmic reticulum localization of Shc proteins in unstimulated cells and their massive recruitment to the plasma membrane, endocytic structures, and peripheral cytosol following receptor tyrosine kinase activation could account for multiple putative functions of the adaptor protein....

  10. Synthesis and biological activity of novel small peptides with aminophosphonates moiety as NOP receptor ligands.

    Science.gov (United States)

    Naydenova, Emilia D; Todorov, Petar T; Mateeva, Polina I; Zamfirova, Rositza N; Pavlov, Nikola D; Todorov, Simeon B

    2010-11-01

    The aim of the present study was the synthesis and the biological screening of new analogs of Ac-RYYRWK-NH2, modified at the N-terminal with 1-[(methoxyphosphono)methylamino]cycloalkanecarboxylic acids. The four newly synthesized ligands for the nociceptin/orphanin FQ (N/OFQ) receptor (NOP) have been prepared by solid-phase peptide synthesis--Fmoc-strategy. These compounds were tested for agonistic activity in vitro on electrically stimulated smooth-muscle preparations isolated from vas deferens of Wistar rats. Our data showed that substitution of Arg at position 1 with aminophosphonates moiety decreased significantly the affinity of ligands to the NOP receptor. Furthermore, the enlargement of the cycle (with 5-8 carbon atoms) additionally diminished both the activity and the selectivity for NOP-receptor.

  11. Internalization mechanisms of the epidermal growth factor receptor after activation with different ligands.

    Directory of Open Access Journals (Sweden)

    Lasse Henriksen

    Full Text Available The epidermal growth factor receptor (EGFR regulates normal growth and differentiation, but dysregulation of the receptor or one of the EGFR ligands is involved in the pathogenesis of many cancers. There are eight ligands for EGFR, however most of the research into trafficking of the receptor after ligand activation focuses on the effect of epidermal growth factor (EGF and transforming growth factor-α (TGF-α. For a long time it was believed that clathrin-mediated endocytosis was the major pathway for internalization of the receptor, but recent work suggests that different pathways exist. Here we show that clathrin ablation completely inhibits internalization of EGF- and TGF-α-stimulated receptor, however the inhibition of receptor internalization in cells treated with heparin-binding EGF-like growth factor (HB-EGF or betacellulin (BTC was only partial. In contrast, clathrin knockdown fully inhibits EGFR degradation after all ligands tested. Furthermore, inhibition of dynamin function blocked EGFR internalization after stimulation with all ligands. Knocking out a number of clathrin-independent dynamin-dependent pathways of internalization had no effect on the ligand-induced endocytosis of the EGFR. We suggest that EGF and TGF-α lead to EGFR endocytosis mainly via the clathrin-mediated pathway. Furthermore, we suggest that HB-EGF and BTC also lead to EGFR endocytosis via a clathrin-mediated pathway, but can additionally use an unidentified internalization pathway or better recruit the small amount of clathrin remaining after clathrin knockdown.

  12. Activation of GABA(B) receptors inhibits protein kinase B/glycogen synthase kinase 3 signaling.

    Science.gov (United States)

    Lu, Frances Fangjia; Su, Ping; Liu, Fang; Daskalakis, Zafiris J

    2012-11-28

    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 GABA(B) receptors significantly inhibits Akt/GSK-3 signaling in a β-arrestin-dependent pathway. Agonist stimulation of GABA(B) 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 GABA(B) receptors and rat hippocampal slices. Furthermore, knocking down the expression of β-arrestin2 using siRNA abolishes the GABA(B) receptor-mediated modulation of GSK-3 signaling. Our data may help to identify potentially novel targets through which GABA(B) receptor agents may exert therapeutic effects in the treatment of schizophrenia.

  13. Dopamine D2 receptor-mediated Akt/PKB signalling: initiation by the D2S receptor and role in quinpirole-induced behavioural activation.

    Science.gov (United States)

    Chen, Han-Ting; Ruan, Nan-Yu; Chen, Jin-Chung; Lin, Tzu-Yung

    2012-09-24

    The short and long isoforms of the dopamine D2 receptor (D2S and D2L respectively) are highly expressed in the striatum. Functional D2 receptors activate an intracellular signalling pathway that includes a cAMP-independent route involving Akt/GSK3 (glycogen synthase kinase 3). To investigate the Akt/GSK3 response to the seldom-studied D2S receptor, we established a rat D2S receptor-expressing cell line [HEK (human embryonic kidney)-293/rD2S]. We found that in HEK-293/rD2S cells, the D2/D3 agonists bromocriptine and quinpirole significantly induced Akt and GSK3 phosphorylation, as well as ERK1/2 (extracellular-signal-regulated kinase 1/2) activation. The D2S receptor-induced Akt signals were profoundly inhibited by the internalization blockers monodansyl cadaverine and concanavalin A. Activation of the D2S receptor in HEK-293/rD2S cells appeared to trigger Akt/phospho-Akt translocation to the cell membrane. In addition to our cell culture experiments, we studied D2 receptor-dependent Akt in vivo by systemic administration of the D2/D3 agonist quinpirole. The results show that quinpirole evoked Akt-Ser473 phosphorylation in the ventral striatum. Furthermore, intra-accumbens administration of wortmannin, a PI3K (phosphoinositide 3-kinase) inhibitor, significantly suppressed the quinpirole-evoked behavioural activation. Overall, we demonstrate that activation of the dopamine D2S receptor stimulates Akt/GSK3 signalling. In addition, in vivo Akt activity in the ventral striatum appears to play an important role in systemic D2/D3 agonist-induced behavioural activation.

  14. Dopamine D2 receptor-mediated Akt/PKB signalling: initiation by the D2S receptor and role in quinpirole-induced behavioural activation

    Directory of Open Access Journals (Sweden)

    Jin‑Chung Chen

    2012-09-01

    Full Text Available The short and long isoforms of the dopamine D2 receptor (D2S and D2L respectively are highly expressed in the striatum. Functional D2 receptors activate an intracellular signalling pathway that includes a cAMP-independent route involving Akt/GSK3 (glycogen synthase kinase 3. To investigate the Akt/GSK3 response to the seldom-studied D2S receptor, we established a rat D2S receptor-expressing cell line [HEK (human embryonic kidney-293/rD2S]. We found that in HEK-293/rD2S cells, the D2/D3 agonists bromocriptine and quinpirole significantly induced Akt and GSK3 phosphorylation, as well as ERK1/2 (extracellular-signal-regulated kinase 1/2 activation. The D2S receptor-induced Akt signals were profoundly inhibited by the internalization blockers monodansyl cadaverine and concanavalin A. Activation of the D2S receptor in HEK-293/rD2S cells appeared to trigger Akt/phospho-Akt translocation to the cell membrane. In addition to our cell culture experiments, we studied D2 receptor-dependent Akt in vivo by systemic administration of the D2/D3 agonist quinpirole. The results show that quinpirole evoked Akt-Ser473 phosphorylation in the ventral striatum. Furthermore, intra-accumbens administration of wortmannin, a PI3K (phosphoinositide 3-kinase inhibitor, significantly suppressed the quinpirole-evoked behavioural activation. Overall, we demonstrate that activation of the dopamine D2S receptor stimulates Akt/GSK3 signalling. In addition, in vivo Akt activity in the ventral striatum appears to play an important role in systemic D2/D3 agonist-induced behavioural activation.

  15. Multiplicity of nuclear receptor activation by PFOA and PFOS in primary human and rodent hepatocytes

    International Nuclear Information System (INIS)

    Perfluorooctanoate (PFOA) and perfluorooctanesulfonate (PFOS) are surface active fluorochemicals that, due to their exceptional stability to degradation, are persistent in the environment. Both PFOA and PFOS are eliminated slowly in humans, with geometric mean serum elimination half-lives estimated at 3.5 and 4.8 years, respectively. The biological activity of PFOA and PFOS in rodents is attributed primarily to transactivation of the nuclear receptor peroxisome proliferator activated receptor alpha (PPARA), which is an important regulator of lipid and carbohydrate metabolism. However, there are significant species-specific differences in the response to PFOA and PFOS exposure; non-rodent species, including humans, are refractory to several but not all of these effects. Many of the metabolic effects have been attributed to the activation of PPARA; however, recent studies using PPARα knockout mice demonstrate residual PPARA-independent effects, some of which may involve the activation of alternate nuclear receptors, including NR1I2 (PXR), NR1I3 (CAR), NR1H3 (LXRA), and NR1H4 (FXR). The objective of this investigation was to characterize the activation of multiple nuclear receptors and modulation of metabolic pathways associated with exposure to PFOA and PFOS, and to compare and contrast the effects between rat and human primary liver cells using quantitative reverse transcription PCR (RT-qPCR). Our results demonstrate that multiple nuclear receptors participate in the metabolic response to PFOA and PFOS exposure resulting in a substantial shift from carbohydrate metabolism to fatty acid oxidation and hepatic triglyceride accumulation in rat liver cells. This shift in intermediary metabolism was more pronounced for PFOA than PFOS. Furthermore, while there is some similarity in the activation of metabolic pathways between rat and humans, particularly in PPARA regulated responses; the changes in primary human cells were more subtle and possibly reflect an adaptive

  16. Suppression of pancreatic carcinoma growth by activating peroxisome proliferator-activated receptor γ involves angiogenesis inhibition

    Institute of Scientific and Technical Information of China (English)

    Yu-Wei Dong; Xing-Peng Wang; Kai Wu

    2009-01-01

    AIM: To study the possible actions and mechanisms of peroxisome proliferator-activated receptor γ (PPARγ), a ligand-activated transcription factor, in pancreatic carcinogenesis,especially in angiogenesis.METHODS: Expressions of PPARγ and retinoid acid receptor (RXRα) were examined by reverse-transcription polymerase chain reaction (RT-PCR) with immunocytochemical staining. Pancreatic carcinoma cells, PANC-1,were treated either with 9-cis-RA, a ligand of RXRα,or with 15-deoxy-Δ12,14 prostaglandin J2(15d-PGJ2), a ligand of PPARγ, or both. Antiproliferative effect was evaluated by cell viability using methyltetrazolium (MTT) assay. A pancreatic carcinoma xenograft tumor model of nude mice was established by inoculating PANC-1 cells subcutaneously. Rosiglitazone, a specific ligand of PPARγ, was administered via water drinking in experimental group of nude mice. After 75 d, all mice were sacrificed. Expression of proliferating cell nuclear antigen (PCNA) in tumor tissue was examined with immunohistochemical staining. Expression of vascular endothelial growth factor (VEGF) mRNA in PANC-1 cells, which were treated with 15d-PGJ2 or 9-cis-RA at variousconcentrations or different duration, was detected by semi-quantitative RT-PCR. Effects of Rosiglitazone on changes of microvascular density (MVD) and VEGF expression were investigated in xenograft tumor tissue. Neovasculature was detected with immunohistochemistry staining labeled with anti-Ⅳ collagen antibody, and indicated by MVD.RESULTS: RT-PCR and immunocytochemical staining showed that PPARγ and RXRα were expressed in PANC-1 cells at both transcription level and translation level. MTT assay demonstrated that 15d-PGJ2, 9-cis-RA and their combination inhibited the growth of PANC-1 cells in a dose-dependent manner. 9-cis-RA had a combined inhibiting action with 15d-PGJ2 on the growth of pancreatic carcinoma. In vivo studies revealed that Rosiglitazone significantly suppressed the growth of pancreatic carcinoma

  17. GABA/sub B/ receptor activation inhibits Ca2+-activated potassium channels in synaptosomes: involvement of G-proteins

    International Nuclear Information System (INIS)

    86Rb-efflux assay from preloaded synaptosomes of rat cerebral cortex was developed to study the effect of GABA/sub B/ receptor agonist baclofen on Ca2+-activated K+-channels. Depolarization of 86Rb-loaded synaptosomes in physiological buffer increased Ca2+-activated 86Rb-efflux by 400%. The 86Rb-efflux was blocked by quinine sulfate, tetraethylammonium, and La3+ indicating the involvement of Ca2+-activated K+-channels. (-)Baclofen inhibited Ca2+-activated 86Rb-efflux in a stereospecific manner. The inhibitory effect of (-)baclofen was mediated by GABA/sub B/ receptor activation, since it was blocked by GABA/sub B/ antagonist phaclofen, but not by bicuculline. Further, pertussis toxin also blocked the ability of baclofen or depolarizing action to affect Ca2+-activated K+-channels. These results suggest that baclofen inhibits Ca2+-activated K+-channels in synaptosomes and these channels are regulated by G-proteins. This assay may provide an ideal in vitro model to study GABA/sub B/ receptor pharmacology

  18. Cannabinoid receptor-2 selective antagonist negatively regulates receptor activator of nuclear factor kappa B ligand mediated osteoclastogenesis

    Institute of Scientific and Technical Information of China (English)

    GENG De-chun; XU Yao-zeng; YANG Hui-lin; ZHU Guang-ming; WANG Xian-bin; ZHU Xue-song

    2011-01-01

    Background The cannabinoid receptor-2 (CB2) is important for bone remodeling. In this study, we investigated the effects of CB2 selective antagonist (AM630) on receptor activator of nuclear factor kappa B (RANK) ligand (RANKL)induced osteoclast differentiation and the underlying signaling pathway using a monocyte-macrophage cell line-RAW264.7.Methods RAW264.7 was cultured with RANKL for 6 days and then treated with AM630 for 24 hours. Mature osteoclasts were measured by tartrate-resistant acid phosphatase (TRAP) staining using a commercial kit. Total ribonucleic acid (RNA)was isolated and real-time reverse transcriptase-polymerase chain reaction (RT-PCR) was done to examine the expression of RANK, cathepsin K (CPK) and nuclear factor kappa B (NF-κB). The extracellular signal-regulated kinase (ERK),phosphorylation of ERK (P-ERK) and NF-κB production were tested by Western blotting. The effect of AM630 on RAW264.7 viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay.Results AM630 did not affect the viability of RAW264.7. However, this CB2 selective antagonist markedly inhibited osteoclast formation and the inhibition rate was dose-dependent. The dose of >100 nmol/L could reduce TRAP positive cells to the levels that were significantly lower than the control. AM630 suppressed the expression of genes associated with osteoclast differentiation and activation, such as RANK and CPK. An analysis of a signaling pathway showed that AM630 inhibited the RANKL-induced activation of ERK, but not NF-κB.Conclusion AM630 could inhibit the osteoclastogenesis from RAW264.7 induced with RANKL.

  19. Constitutive Activity among Orphan Class-A G Protein Coupled Receptors.

    Directory of Open Access Journals (Sweden)

    Adam L Martin

    Full Text Available The purpose of this study was to evaluate the extent of constitutive activity among orphan class-A G protein coupled receptors within the cAMP signaling pathway. Constitutive signaling was revealed by changes in gene expression under control of the cAMP response element. Gene expression was measured in Chinese hamster ovary cells transiently co-transfected with plasmids containing a luciferase reporter and orphan receptor. Criteria adopted for defining constitutive activation were: 1 200% elevation over baseline reporter gene expression; 2 40% inhibition of baseline expression; and 3 40% inhibition of expression stimulated by 3 μM forskolin. Five patterns of activity were noted: 1 inhibition under both baseline and forskolin stimulated expression (GPR15, GPR17, GPR18, GPR20, GPR25, GPR27, GPR31, GPR32, GPR45, GPR57, GPR68, GPR83, GPR84, GPR132, GPR150, GPR176; 2 no effect on baseline expression, but inhibition of forskolin stimulated expression (GPR4, GPR26, GPR61, GPR62, GPR78, GPR101, GPR119; 3 elevation of baseline signaling coupled with inhibition of forskolin stimulated expression (GPR6, GPR12; 4 elevation of baseline signaling without inhibition of forskolin stimulated expression (GPR3, GPR21, GPR52, GPR65; and 5 no effect on expression (GPR1, GPR19, GPR22, GPR34, GPR35, GPR39, GPR63, GPR82, GPR85, GPR87. Constitutive activity was observed in 75% of the orphan class-A receptors examined (30 of 40. This constitutive signaling cannot be explained by simple overexpression of the receptor. Inhibition of cAMP mediated expression was far more common (65% than stimulation of expression (15%. Orphan receptors that were closely related based on amino acid homology tended to have similar effects on gene expression. These results suggest that identification of inverse agonists may be a fruitful approach for categorizing these orphan receptors and targeting them for pharmacological intervention.

  20. Constitutive Activity among Orphan Class-A G Protein Coupled Receptors.

    Science.gov (United States)

    Martin, Adam L; Steurer, Michael A; Aronstam, Robert S

    2015-01-01

    The purpose of this study was to evaluate the extent of constitutive activity among orphan class-A G protein coupled receptors within the cAMP signaling pathway. Constitutive signaling was revealed by changes in gene expression under control of the cAMP response element. Gene expression was measured in Chinese hamster ovary cells transiently co-transfected with plasmids containing a luciferase reporter and orphan receptor. Criteria adopted for defining constitutive activation were: 1) 200% elevation over baseline reporter gene expression; 2) 40% inhibition of baseline expression; and 3) 40% inhibition of expression stimulated by 3 μM forskolin. Five patterns of activity were noted: 1) inhibition under both baseline and forskolin stimulated expression (GPR15, GPR17, GPR18, GPR20, GPR25, GPR27, GPR31, GPR32, GPR45, GPR57, GPR68, GPR83, GPR84, GPR132, GPR150, GPR176); 2) no effect on baseline expression, but inhibition of forskolin stimulated expression (GPR4, GPR26, GPR61, GPR62, GPR78, GPR101, GPR119); 3) elevation of baseline signaling coupled with inhibition of forskolin stimulated expression (GPR6, GPR12); 4) elevation of baseline signaling without inhibition of forskolin stimulated expression (GPR3, GPR21, GPR52, GPR65); and 5) no effect on expression (GPR1, GPR19, GPR22, GPR34, GPR35, GPR39, GPR63, GPR82, GPR85, GPR87). Constitutive activity was observed in 75% of the orphan class-A receptors examined (30 of 40). This constitutive signaling cannot be explained by simple overexpression of the receptor. Inhibition of cAMP mediated expression was far more common (65%) than stimulation of expression (15%). Orphan receptors that were closely related based on amino acid homology tended to have similar effects on gene expression. These results suggest that identification of inverse agonists may be a fruitful approach for categorizing these orphan receptors and targeting them for pharmacological intervention. PMID:26384023

  1. Rearrangement of the Extracellular Domain/Extracellular Loop 1 Interface Is Critical for Thyrotropin Receptor Activation.

    Science.gov (United States)

    Schaarschmidt, Joerg; Nagel, Marcus B M; Huth, Sandra; Jaeschke, Holger; Moretti, Rocco; Hintze, Vera; von Bergen, Martin; Kalkhof, Stefan; Meiler, Jens; Paschke, Ralf

    2016-07-01

    The thyroid stimulating hormone receptor (TSHR) is a G protein-coupled receptor (GPCR) with a characteristic large extracellular domain (ECD). TSHR activation is initiated by binding of the hormone ligand TSH to the ECD. How the extracellular binding event triggers the conformational changes in the transmembrane domain (TMD) necessary for intracellular G protein activation is poorly understood. To gain insight in this process, the knowledge on the relative positioning of ECD and TMD and the conformation of the linker region at the interface of ECD and TMD are of particular importance. To generate a structural model for the TSHR we applied an integrated structural biology approach combining computational techniques with experimental data. Chemical cross-linking followed by mass spectrometry yielded 17 unique distance restraints within the ECD of the TSHR, its ligand TSH, and the hormone-receptor complex. These structural restraints generally confirm the expected binding mode of TSH to the ECD as well as the general fold of the domains and were used to guide homology modeling of the ECD. Functional characterization of TSHR mutants confirms the previously suggested close proximity of Ser-281 and Ile-486 within the TSHR. Rigidifying this contact permanently with a disulfide bridge disrupts ligand-induced receptor activation and indicates that rearrangement of the ECD/extracellular loop 1 (ECL1) interface is a critical step in receptor activation. The experimentally verified contact of Ser-281 (ECD) and Ile-486 (TMD) was subsequently utilized in docking homology models of the ECD and the TMD to create a full-length model of a glycoprotein hormone receptor. PMID:27129207

  2. Receptor-G Protein Interaction Studied by Bioluminescence Resonance Energy Transfer: Lessons From Protease-Activated Receptor 1

    Directory of Open Access Journals (Sweden)

    Mohammed Akli eAYOUB

    2012-06-01

    Full Text Available Since its development, the bioluminescence resonance energy transfer (BRET approach has been extensively applied to study G protein-coupled receptors (GPCRs in real time and in live cells. One of the major aspects of GPCRs investigated in considerable details is their physical coupling to the heterotrimeric G proteins. As a result, new concepts have emerged, but few questions are still a matter of debate illustrating the complexity of GPCR-G protein interactions and coupling. Here, we summarized the recent advances on our understanding of GPCR-G protein coupling based on BRET approaches and supported by other FRET-based studies. We essentially focused on our recent studies in which we addressed the concept of preassembly versus the agonist-dependent interaction between the protease-activated receptor 1 (PAR1 and its cognate G proteins. We discussed the concept of agonist-induced conformational changes within the preassembled PAR1-G protein complexes as well as the critical question how the multiple coupling of PAR1 with two different G proteins, Gi1 and G12, but also -arrestin 1, can be regulated.

  3. Retinoids induce integrin-independent lymphocyte adhesion through RAR-α nuclear receptor activity

    Energy Technology Data Exchange (ETDEWEB)

    Whelan, Jarrett T.; Wang, Lei; Chen, Jianming; Metts, Meagan E.; Nasser, Taj A.; McGoldrick, Liam J. [Department of Biochemistry and Molecular Biology, The Brody School of Medicine at East Carolina University, Greenville, NC 27834 (United States); Bridges, Lance C., E-mail: bridgesl@ecu.edu [Department of Biochemistry and Molecular Biology, The Brody School of Medicine at East Carolina University, Greenville, NC 27834 (United States); East Carolina Diabetes and Obesity Institute, The Brody School of Medicine at East Carolina University, Greenville, NC 27834 (United States)

    2014-11-28

    Highlights: • Transcription and translation are required for retinoid-induced lymphocyte adhesion. • RAR activation is sufficient to induced lymphocyte cell adhesion. • Vitamin D derivatives inhibit RAR-prompted lymphocyte adhesion. • Adhesion occurs through a novel binding site within ADAM disintegrin domains. • RARα is a key nuclear receptor for retinoid-dependent lymphocyte cell adhesion. - Abstract: Oxidative metabolites of vitamin A, in particular all-trans-retinoic acid (atRA), have emerged as key factors in immunity by specifying the localization of immune cells to the gut. Although it is appreciated that isomers of retinoic acid activate the retinoic acid receptor (RAR) and retinoid X receptor (RXR) family of nuclear receptors to elicit cellular changes, the molecular details of retinoic acid action remain poorly defined in immune processes. Here we employ a battery of agonists and antagonists to delineate the specific nuclear receptors utilized by retinoids to evoke lymphocyte cell adhesion to ADAM (adisintegrin and metalloprotease) protein family members. We report that RAR agonism is sufficient to promote immune cell adhesion in both immortal and primary immune cells. Interestingly, adhesion occurs independent of integrin function, and mutant studies demonstrate that atRA-induced adhesion to ADAM members required a distinct binding interface(s) as compared to integrin recognition. Anti-inflammatory corticosteroids as well as 1,25-(OH){sub 2}D{sub 3}, a vitamin D metabolite that prompts immune cell trafficking to the skin, potently inhibited the observed adhesion. Finally, our data establish that induced adhesion was specifically attributable to the RAR-α receptor isotype. The current study provides novel molecular resolution as to which nuclear receptors transduce retinoid exposure into immune cell adhesion.

  4. Activation of muscarinic acetylcholine receptors elicits pigment granule dispersion in retinal pigment epithelium isolated from bluegill

    Directory of Open Access Journals (Sweden)

    Crittenden Elizabeth L

    2004-07-01

    Full Text Available Abstract Background In fish, melanin pigment granules in the retinal pigment epithelium disperse into apical projections as part of the suite of responses the eye makes to bright light conditions. This pigment granule dispersion serves to reduce photobleaching and occurs in response to neurochemicals secreted by the retina. Previous work has shown that acetylcholine may be involved in inducing light-adaptive pigment dispersion. Acetylcholine receptors are of two main types, nicotinic and muscarinic. Muscarinic receptors are in the G-protein coupled receptor superfamily, and five different muscarinic receptors have been molecularly cloned in human. These receptors are coupled to adenylyl cyclase, calcium mobilization and ion channel activation. To determine the receptor pathway involved in eliciting pigment granule migration, we isolated retinal pigment epithelium from bluegill and subjected it to a battery of cholinergic agents. Results The general cholinergic agonist carbachol induces pigment granule dispersion in isolated retinal pigment epithelium. Carbachol-induced pigment granule dispersion is blocked by the muscarinic antagonist atropine, by the M1 antagonist pirenzepine, and by the M3 antagonist 4-DAMP. Pigment granule dispersion was also induced by the M1 agonist 4-[N-(4-chlorophenyl carbamoyloxy]-4-pent-2-ammonium iodide. In contrast the M2 antagonist AF-DX 116 and the M4 antagonist tropicamide failed to block carbachol-induced dispersion, and the M2 agonist arecaidine but-2-ynyl ester tosylate failed to elicit dispersion. Conclusions Our results suggest that carbachol-mediated pigment granule dispersion occurs through the activation of Modd muscarinic receptors, which in other systems couple to phosphoinositide hydrolysis and elevation of intracellular calcium. This conclusion must be corroborated by molecular studies, but suggests Ca2+-dependent pathways may be involved in light-adaptive pigment dispersion.

  5. Influence of metabotropic glutamate receptor agonists on the inhibitory effects of adenosine A1 receptor activation in the rat hippocampus.

    Science.gov (United States)

    de Mendonça, A; Ribeiro, J A

    1997-08-01

    1. Glutamate and other amino acids are the main excitatory neurotransmitters in many brain regions, including the hippocampus, by activating ion channel-coupled glutamate receptors, as well as metabotropic receptors linked to G proteins and second messenger systems. Several conditions which promote the release of glutamate, like frequency stimulation and hypoxia, also lead to an increase in the extracellular levels of the important neuromodulator, adenosine. We studied whether the activation of different subgroups of metabotropic glutamate receptors (mGluR) could modify the known inhibitory effects of a selective adenosine A1 receptor agonist on synaptic transmission in the hippocampus. The experiments were performed on hippocampal slices taken from young (12-14 days old) rats. Stimulation was delivered to the Schaffer collateral/commissural fibres, and evoked field excitatory postsynaptic potentials (fe. p.s.p.) recorded extracellularly from the stratum radiatum in the CAI area. 2. The concentration-response curve for the inhibitory effects of the selective adenosine A1 receptor agonist, N6-cyclopentyladenosine (CPA; 2-50 nM), on the fe.p.s.p. slope (EC50 = 12.5 (9.2-17.3; 95% confidence intervals)) was displaced to the right by the group I mGluR selective agonist, (R,S)-3,5-dihydroxyphenylglycine (DPHG; 10 microM) (EC50 = 27.2 (21.4-34.5) nM, n = 4). The attenuation of the inhibitory effect of CPA (10 nM) on the fe.p.s.p. slope by DHPG (10 microM) was blocked in the presence of the mGluR antagonist (which blocks group I and II mGluR), (R,S)-alpha-methyl-4-carboxyphenylglycine (MCPG; 500 microM). DHPG (10 microM) itself had an inhibitory effect of 20.1 +/- 1.9% (n = 4) on the fe.p.s.p. slope. 3. The concentration-response curves for the inhibitory effects of CPA (2-20 nM) on the fe.p.s.p. slope were not modified either in the presence of the group II mGluR selective agonist, (2S,3S,4S)-alpha-(carboxycyclopropyl)glycine (L-CCG-I; 1 microM), or in the presence of

  6. The phosphoproteome of toll-like receptor-activated macrophages

    DEFF Research Database (Denmark)

    Weintz, Gabriele; Olsen, Jesper Velgaard; Frühauf, Katja;

    2010-01-01

    of primary macrophages using stable isotope labelling with amino acids in cell culture, phosphopeptide enrichment and high-resolution mass spectrometry. In parallel, nascent RNA was profiled to link transcription factor (TF) phosphorylation to TLR4-induced transcriptional activation. We reproducibly...

  7. Melanocortin-4 receptor activation inhibits c-Jun N-terminal kinase activity and promotes insulin signaling

    OpenAIRE

    Chai, Biaoxin; Li, Ji-Yao; Zhang, Weizhen; Wang, Hui; Mulholland, Michael W.

    2009-01-01

    The melanocortin system is crucial to regulation of energy homeostasis. The melanocortin receptor type 4 (MC4R) modulates insulin signaling via effects on c-Jun N-terminal kinase (JNK). The melanocortin agonist NDP-MSH dose-dependently inhibited JNK activity in HEK293 cells stably expressing the human MC4R; effects were reversed by melanocortin receptor antagonist. NDP-MSH time- and dose-dependently inhibited IRS-1ser307 phosphorylation, effects also reversed by a specific melanocortin recept...

  8. The role of chemokines and chemokine receptors in eosinophil activation during inflammatory allergic reactions

    Directory of Open Access Journals (Sweden)

    Oliveira S.H.P.

    2003-01-01

    Full Text Available Chemokines are important chemotactic cytokines that play a fundamental role in the trafficking of leukocytes to sites of inflammation. They are also potent cell-activating factors, inducing cytokine and histamine release and free radical production, a fact that makes them particularly important in the pathogenesis of allergic inflammation. The action of chemokines is regulated at the level of agonist production and processing as well as at the level of receptor expression and coupling. Therefore, an analysis of the ligands must necessarily consider receptors. Eosinophils are target cells involved in the allergic inflammatory response since they are able to release a wide variety of mediators including CC and CXC chemokines and express their receptors. These mediators could damage the airway epithelial cells and might be important to stimulate other cells inducing an amplification of the allergic response. This review focuses on recently emerging data pertaining to the importance of chemokines and chemokine receptors in promoting eosinophil activation and migration during the allergic inflammatory process. The analysis of the function of eosinophils and their chemokine receptors during allergic inflammation might be a good approach to understanding the determinants of asthma severity and to developing novel therapies.

  9. (+)-Cannabidiol analogues which bind cannabinoid receptors but exert peripheral activity only.

    Science.gov (United States)

    Fride, Ester; Feigin, Cfir; Ponde, Datta E; Breuer, Aviva; Hanus, Lumír; Arshavsky, Nina; Mechoulam, Raphael

    2004-12-15

    Delta9-Tetrahydrocannabinol (Delta9-THC) and (-)-cannabidiol are major constituents of the Cannabis sativa plant with different pharmacological profiles: (-)-Delta9-tetrahydrocannabinol, but not (-)-cannabidiol, activates cannabinoid CB1 and CB2 receptors and induces psychoactive and peripheral effects. We have tested a series of (+)-cannabidiol derivatives, namely, (+)-cannabidiol-DMH (DMH-1,1-dimethylheptyl-), (+)-7-OH-cannabidiol-DMH, (+)-7-OH- cannabidiol, (+)-7-COOH- cannabidiol and (+)-7-COOH-cannabidiol-DMH, for central and peripheral (intestinal, antiinflammatory and peripheral pain) effects in mice. Although all (+)-cannabidiols bind to cannabinoid CB1 and CB2 receptors, only (+)-7-OH-cannabidiol-DMH was centrally active, while all (+)-cannabidiol analogues completely arrested defecation. The effects of (+)-cannabidiol-DMH and (+)-7-OH-cannabidiol-DMH were partially antagonized by the cannabinoid CB1 receptor antagonist N-(piperidiny-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (SR141716), but not by the cannabinoid CB2 receptor antagonist N-[-(1S)-endo-1,3,3-trimethil bicyclo [2.2.1] heptan-2-yl-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide (SR144528), and had no effect on CB1(-/-) receptor knockout mice. (+)-Cannabidiol-DMH inhibited the peripheral pain response and arachidonic-acid-induced inflammation of the ear. We conclude that centrally inactive (+)-cannabidiol analogues should be further developed as antidiarrheal, antiinflammatory and analgesic drugs for gastrointestinal and other peripheral conditions. PMID:15588739

  10. Caffeine stimulates locomotor activity in the mammalian spinal cord via adenosine A1 receptor-dopamine D1 receptor interaction and PKA-dependent mechanisms.

    Science.gov (United States)

    Acevedo, JeanMarie; Santana-Almansa, Alexandra; Matos-Vergara, Nikol; Marrero-Cordero, Luis René; Cabezas-Bou, Ernesto; Díaz-Ríos, Manuel

    2016-02-01

    Caffeine is a potent psychostimulant that can have significant and widely variable effects on the activity of multiple neuronal pathways. The most pronounced caffeine-induced behavioral effect seen in rodents is to increase locomotor activity which has been linked to a dose-dependent inhibition of A1 and A(2A) receptors. The effects of caffeine at the level of the lumbar spinal central pattern generator (CPG) network for hindlimb locomotion are lacking. We assessed the effects of caffeine to the locomotor function of the spinal CPG network via extracellular ventral root recordings using the isolated neonatal mouse spinal cord preparation. Addition of caffeine and of an A1 receptor antagonist significantly decreased the cycle period accelerating the ongoing locomotor rhythm, while decreasing burst duration reversibly in most preparations suggesting the role of A1 receptors as the primary target of caffeine. Caffeine and an A1 receptor antagonist failed to stimulate ongoing locomotor activity in the absence of dopamine or in the presence of a D1 receptor antagonist supporting A1/D1 receptor-dependent mechanism of action. The use of caffeine or an A1 receptor blocker failed to stimulate an ongoing locomotor rhythm in the presence of a blocker of the cAMP-dependent protein kinase (PKA) supporting the need of this intracellular pathway for the modulatory effects of caffeine to occur. These results support a stimulant effect of caffeine on the lumbar spinal network controlling hindlimb locomotion through the inhibition of A1 receptors and subsequent activation of D1 receptors via a PKA-dependent intracellular mechanism.

  11. Salt bridges overlapping the gonadotropin-releasing hormone receptor agonist binding site reveal a coincidence detector for G protein-coupled receptor activation.

    Science.gov (United States)

    Janovick, Jo Ann; Pogozheva, Irina D; Mosberg, Henry I; Conn, P Michael

    2011-08-01

    G protein-coupled receptors (GPCRs) play central roles in most physiological functions, and mutations in them cause heritable diseases. Whereas crystal structures provide details about the structure of GPCRs, there is little information that identifies structural features that permit receptors to pass the cellular quality control system or are involved in transition from the ground state to the ligand-activated state. The gonadotropin-releasing hormone receptor (GnRHR), because of its small size among GPCRs, is amenable to molecular biological approaches and to computer modeling. These techniques and interspecies comparisons are used to identify structural features that are important for both intracellular trafficking and GnRHR activation yet distinguish between these processes. Our model features two salt (Arg(38)-Asp(98) and Glu(90)-Lys(121)) and two disulfide (Cys(14)-Cys(200) and Cys(114)-Cys(196)) bridges, all of which are required for the human GnRHR to traffic to the plasma membrane. This study reveals that both constitutive and ligand-induced activation are associated with a "coincidence detector" that occurs when an agonist binds. The observed constitutive activation of receptors lacking Glu(90)-Lys(121), but not Arg(38)-Asp(98) ionic bridge, suggests that the role of the former connection is holding the receptor in the inactive conformation. Both the aromatic ring and hydroxyl group of Tyr(284) and the hydrogen bonding of Ser(217) are important for efficient receptor activation. Our modeling results, supported by the observed influence of Lys(191) from extracellular loop 2 (EL2) and a four-residue motif surrounding this loop on ligand binding and receptor activation, suggest that the positioning of EL2 within the seven-α-helical bundle regulates receptor stability, proper trafficking, and function. PMID:21527534

  12. CXC Chemokine Receptor 3 Alternative Splice Variants Selectively Activate Different Signaling Pathways.

    Science.gov (United States)

    Berchiche, Yamina A; Sakmar, Thomas P

    2016-10-01

    The G protein-coupled receptor (GPCR) C-X-C chemokine receptor 3 (CXCR3) is a potential drug target that mediates signaling involved in cancer metastasis and inflammatory diseases. The CXCR3 primary transcript has three potential alternative splice variants and cell-type specific expression results in receptor variants that are believed to have different functional characteristics. However, the molecular pharmacology of ligand binding to CXCR3 alternative splice variants and their downstream signaling pathways remain poorly explored. To better understand the role of the functional consequences of alternative splicing of CXCR3, we measured signaling in response to four different chemokine ligands (CXCL4, CXCL9, CXCL10, and CXCL11) with agonist activity at CXCR3. Both CXCL10 and CXCL11 activated splice variant CXCR3A. Whereas CXCL10 displayed full agonistic activity for Gαi activation and extracellular signal regulated kinase (ERK) 1/2 phosphorylation and partial agonist activity for β-arrestin recruitment, CXCL9 triggered only modest ERK1/2 phosphorylation. CXCL11 induced CXCR3B-mediated β-arrestin recruitment and little ERK phosphorylation. CXCR3Alt signaling was limited to modest ligand-induced receptor internalization and ERK1/2 phosphorylation in response to chemokines CXCL11, CXCL10, and CXCL9. These results show that CXCR3 splice variants activate different signaling pathways and that CXCR3 variant function is not redundant, suggesting a mechanism for tissue specific biased agonism. Our data show an additional layer of complexity for chemokine receptor signaling that might be exploited to target specific CXCR3 splice variants. PMID:27512119

  13. Improved Insulin Resistance and Lipid Metabolism by Cinnamon Extract through Activation of Peroxisome Proliferator-Activated Receptors

    OpenAIRE

    Xiaoyan Sheng; Yuebo Zhang; Zhenwei Gong; Cheng Huang; Ying Qin Zang

    2008-01-01

    Peroxisome proliferator-activated receptors (PPARs) are transcriptional factors involved in the regulation of insulin resistance and adipogenesis. Cinnamon, a widely used spice in food preparation and traditional antidiabetic remedy, is found to activate PPARγ and α, resulting in improved insulin resistance, reduced fasted glucose, FFA, LDL-c, and AST levels in high-caloric diet-induced obesity (DIO) and db/db mice in its water extract form. In vitro studies demonstrate that cinnamon increase...

  14. Molecular Interactions Between the Active Sites of RGD (Arg-Gly-Asp with its Receptor (Integrine

    Directory of Open Access Journals (Sweden)

    E. Jauregui

    2000-03-01

    Full Text Available A study of the molecular interactions between the active sites of RGD (Arg-Gly-Asp with it Receptor using simultaions is reported. Our calculations indicate that the guanidine-carboxylate complex is energetically favourd with respect to the guanidine-methyl tetrazole complex.

  15. Activation of the Ah receptor by extracts of dietary herbal supplements, vegetables, and fruits

    NARCIS (Netherlands)

    Jeuken, A.; Keser, B.J.G.; Khan, E.; Brouwer, A.; Koeman, J.H.; Denison, M.S.

    2003-01-01

    The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that can be activated by a structurally diverse range of synthetic and natural chemicals, and it mediates the toxic and biological effects of environmental contaminants such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).

  16. Blood coagulation-(in)dependent protease activated receptor signaling in pancreatic cancer

    NARCIS (Netherlands)

    K. Shi

    2015-01-01

    There is no doubt that pancreatic cancer is one of the most lethal diseases in general and current treatment options are limited. The studies described in this thesis pinpoint Protease activated receptors (PARs) as key players in pancreatic cancer progression. Inhibition of PAR-1 may be clinical rel

  17. Distinct neural pathways mediate alpha7 nicotinic acetylcholine receptor-dependent activation of the forebrain

    DEFF Research Database (Denmark)

    Thomsen, Morten S; Hay-Schmidt, Anders; Hansen, Henrik H;

    2010-01-01

    alpha(7) nicotinic acetylcholine receptor (nAChR) agonists are candidates for the treatment of cognitive deficits in schizophrenia. Selective alpha(7) nAChR agonists, such as SSR180711, activate neurons in the medial prefrontal cortex (mPFC) and nucleus accumbens shell (ACCshell) in rats, regions...

  18. Domains of the growth hormone receptor required for association and activation of JAK2 tyrosine kinase

    DEFF Research Database (Denmark)

    VanderKuur, J A; Wang, X; Zhang, L;

    1994-01-01

    Growth hormone (GH) has recently been shown to activate the GH receptor (GHR)-associated tyrosine kinase JAK2. In the present study, regions of the GHR required for JAK2 association with GHR were identified. GH-dependent JAK2 association with GHR was detected in Chinese hamster ovary (CHO) cells ...

  19. Proteinase-activated receptor 2 modulates neuroinflammation in experimental autoimmune encephalomyelitis and multiple sclerosis

    NARCIS (Netherlands)

    F. Noorbakhsh (Farshid); K. Tsutsui (Kazuyoshi); N. Vergnolle (Nathalie); L.A. Boven (Leonie); S.F. Shariat (Shahrokh); M. Vodjgani (Mohammed); K.G. Warren (Kenneth); P. Andrade-Gordon (Patricia); N.K. Hollenberg (Norman); C. Power (Christopher)

    2006-01-01

    textabstractThe proteinase-activated receptors (PARs) are widely recognized for their modulatory properties of inflammation and neurodegeneration. We investigated the role of PAR2 in the pathogenesis of multiple sclerosis (MS) in humans and experimental autoimmune encephalomyelitis (EAE) in mice. PA

  20. Inhibition of PKC activity blocks the increase of ETB receptor expression in cerebral arteries

    DEFF Research Database (Denmark)

    Henriksson, Marie; Vikman, Petter; Stenman, Emelie;

    2006-01-01

    BACKGROUND: Previous studies have shown that there is a time-dependent upregulation of contractile endothelin B (ETB) receptors in middle cerebral arteries (MCA) after organ culture. This upregulation is dependent on mitogen-activated protein kinases and possibly protein kinase C (PKC). The aim o...