Sample records for calcium-sensing receptor structural

  1. Structural mechanism of ligand activation in human calcium-sensing receptor

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    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.


    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.

  2. Familial hypocalciuric hypercalcemia and calcium sensing receptor

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    Mrgan, Monija; Nielsen, Sanne; Brixen, Kim


    Familial hypocalciuric hypercalcemia (FHH) is a lifelong, benign autosomal dominant disease characterized by hypercalcemia, normal to increased parathyroid hormone level, and a relatively low renal calcium excretion. Inactivation of the calcium-sensing receptor in heterozygous patients results in...

  3. Extracellular calcium-sensing receptor: structural and functional features and association with diseases

    Directory of Open Access Journals (Sweden)

    Hauache O.M.


    Full Text Available The recently cloned extracellular calcium-sensing receptor (CaR is a G protein-coupled receptor that plays an essential role in the regulation of extracellular calcium homeostasis. This receptor is expressed in all tissues related to this control (parathyroid glands, thyroid C-cells, kidneys, intestine and bones and also in tissues with apparently no role in the maintenance of extracellular calcium levels, such as brain, skin and pancreas. The CaR amino acid sequence is compatible with three major domains: a long and hydrophilic aminoterminal extracellular domain, where most of the activating and inactivating mutations described to date are located and where the dimerization process occurs, and the agonist-binding site is located, a hydrophobic transmembrane domain involved in the signal transduction mechanism from the extracellular domain to its respective G protein, and a carboxyterminal intracellular tail, with a well-established role for cell surface CaR expression and for signal transduction. CaR cloning was immediately followed by the association of genetic human diseases with inactivating and activating CaR mutations: familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism are caused by CaR-inactivating mutations, whereas autosomal dominant hypoparathyroidism is secondary to CaR-activating mutations. Finally, we will comment on the development of drugs that modulate CaR function by either activating (calcimimetic drugs or antagonizing it (calcilytic drugs, and on their potential therapeutic implications, such as medical control of specific cases of primary and uremic hyperparathyroidism with calcimimetic drugs and a potential treatment for osteoporosis with a calcilytic drug.

  4. Calcium-sensing receptor in breast physiology and cancer


    Wonnam Kim; Wysolmerski, John J.


    The calcium-sensing receptor (CaSR) is expressed in normal breast epithelial cells and in breast cancer cells. During lactation, activation of the CaSR in mammary epithelial cells increases calcium transport into milk and inhibits parathyroid hormone-related protein (PTHrP) secretion into milk and into the circulation. The ability to sense changes in extracellular calcium allows the lactating breast to actively participate in the regulation of systemic calcium and bone metabolism, and to coor...

  5. Biased agonism of the calcium-sensing receptor

    DEFF Research Database (Denmark)

    Thomsen, Alex Rojas Bie; Hvidtfeldt, Maja; Bräuner-Osborne, Hans


    After the discovery of molecules modulating G protein-coupled receptors (GPCRs) that are able to selectively affect one signaling pathway over others for a specific GPCR, thereby "biasing" the signaling, it has become obvious that the original model of GPCRs existing in either an "on" or "off...... of the calcium-sensing receptor (CaSR), by looking at 12 well-known orthosteric CaSR agonists in 3 different CaSR signaling pathways: G(q/11) protein, G(i/o) protein, and extracellular signal-regulated kinases 1 and 2 (ERK1/2). Here we show that apart from G(q/11) and G(i/o) signaling, ERK1/2 is activated...

  6. Extracellular calcium sensing receptor in human pancreatic cells (United States)

    Rácz, G Z; Kittel, Á; Riccardi, D; Case, R M; Elliott, A C; Varga, G


    Background and aims: The extracellular calcium sensing receptor (CaR) plays a key role in the calcium homeostatic system and is therefore widely expressed in tissues involved in calcium metabolism. However, the CaR has also been identified in other tissues where its role is less clear. We have investigated the presence of the CaR in the human pancreas. Methods: Messenger RNA for the CaR was detected by reverse transcription-polymerase chain reaction and the protein was localised by immunostaining. CaR function was assayed in Capan-1 cells by measuring intracellular calcium and [3H] thymidine incorporation. Results: The receptor was highly expressed in human pancreatic ducts. It was also expressed in exocrine acinar cells, in islets of Langerhans, and in intrapancreatic nerves and blood vessels. The CaR was expressed in both normal and neoplastic human tissue samples but was detected in only one of five ductal adenocarcinoma cells lines examined. Experiments on the CaR expressing adenocarcinoma cell line Capan-1 showed that the CaR was functional and was linked to mobilisation of intracellular calcium. Stimulation of the CaR reduced Capan-1 cell proliferation. Conclusions: We propose that the CaR may play multiple functional roles in the human pancreas. In particular, the CaR on the duct luminal membrane may monitor and regulate the Ca2+ concentration in pancreatic juice by triggering ductal electrolyte and fluid secretion. This could help to prevent precipitation of calcium salts in the duct lumen. The CaR may also help to regulate the proliferation of pancreatic ductal cells. PMID:12377811

  7. Penta-substituted benzimidazoles as potent antagonists of the calcium-sensing receptor (CaSR-antagonists). (United States)

    Gerspacher, Marc; Altmann, Eva; Beerli, René; Buhl, Thomas; Endres, Ralf; Gamse, Rainer; Kameni-Tcheudji, Jacques; Kneissel, Michaela; Krawinkler, Karl Heinz; Missbach, Martin; Schmidt, Alfred; Seuwen, Klaus; Weiler, Sven; Widler, Leo


    A series of novel benzimidazole derivatives has been designed via a scaffold morphing approach based on known calcilytics chemotypes. Subsequent lead optimisation led to the discovery of penta-substituted benzimidazoles that exhibit attractive in vitro and in vivo calcium-sensing receptor (CaSR) inhibitory profiles. In addition, synthesis and structure-activity relationship data are provided.

  8. Calcium-sensing receptor in breast physiology and cancer

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    Wonnam Kim


    Full Text Available The calcium-sensing receptor (CaSR is expressed in normal breast epithelial cells and in breast cancer cells. During lactation, activation of the CaSR in mammary epithelial cells increases calcium transport into milk and inhibits parathyroid hormone-related protein (PTHrP secretion into milk and into the circulation. The ability to sense changes in extracellular calcium allows the lactating breast to actively participate in the regulation of systemic calcium and bone metabolism, and to coordinate calcium usage with calcium availability during milk production. Interestingly, as compared to normal breast cells, in breast cancer cells, the regulation of PTHrP secretion by the CaSR becomes rewired due to a switch in its G-protein usage such that activation of the CaSR increases instead of decreases PTHrP production. In normal cells the CaSR couples to Gi to inhibit cAMP and PTHrP production, whereas in breast cancer cells, it couples to Gs to stimulate cAMP and PTHrP production. Activation of the CaSR on breast cancer cells regulates breast cancer cell proliferation, death and migration, in part, by stimulating PTHrP production. In this article, we discuss the biology of the CaSR in the normal breast and in breast cancer, and review recent findings suggesting that the CaSR activates a nuclear pathway of PTHrP action that stimulates cellular proliferation and inhibits cell death, helping cancer cells adapt to elevated extracellular calcium levels. Understanding the diverse actions mediated by the CaSR may help us better understand lactation physiology, breast cancer progression and osteolytic bone metastases.

  9. NFAT regulates calcium-sensing receptor-mediated TNF production

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    abdullah, huda ismail; Pedraza, Paulina L.; Hao, Shoujin; Rodland, Karin D.; McGiff, John C.; Ferreri, Nicholas R.


    Because nuclear factor of activated T cells (NFAT) has been implicated in TNF production as well as osmoregulation and salt and water homeostasis, we addressed whether calcium-sensing receptor (CaR)-mediated TNF production in medullary thick ascending limb (mTAL) cells was NFAT dependent. TNF production in response to addition of extracellular Ca2+ (1.2 mM) was abolished in mTAL cells transiently transfected with a dominant-negative CaR construct (R796W) or pretreated with the phosphatidylinositol phospholipase C (PI-PLC) inhibitor U-73122. Cyclosporine A (CsA), an inhibitor of the serine/threonine phosphatase calcineurin, and a peptide ligand, VIVIT, that selectively inhibits calcineurin-NFAT signaling, also prevented CaR-mediated TNF production. Increases in calcineurin activity in cells challenged with Ca2+ were inhibited after pretreatment with U-73122 and CsA, suggesting that CaR activation increases calcineurin activity in a PI-PLC-dependent manner. Moreover, U-73122, CsA, and VIVIT inhibited CaR-dependent activity of an NFAT construct that drives expression of firefly luciferase in transiently transfected mTAL cells. Collectively, these data verify the role of calcineurin and NFAT in CaR-mediated TNF production by mTAL cells. Activation of the CaR also increased the binding of NFAT to a consensus oligonucleotide, an effect that was blocked by U-73122 and CsA, suggesting that a calcineurin- and NFAT-dependent pathway increases TNF production in mTAL cells. This mechanism likely regulates TNF gene transcription as U-73122, CsA, and VIVIT blocked CaR-dependent activity of a TNF promoter construct. Elucidating CaR-mediated signaling pathways that regulate TNF production in the mTAL will be crucial to understanding mechanisms that regulate extracellular fluid volume and salt balance.

  10. NFAT regulates calcium-sensing receptor-mediated TNF production. (United States)

    Abdullah, Huda Ismail; Pedraza, Paulina L; Hao, Shoujin; Rodland, Karin D; McGiff, John C; Ferreri, Nicholas R


    Because nuclear factor of activated T cells (NFAT) has been implicated in TNF production as well as osmoregulation and salt and water homeostasis, we addressed whether calcium-sensing receptor (CaR)-mediated TNF production in medullary thick ascending limb (mTAL) cells was NFAT dependent. TNF production in response to addition of extracellular Ca(2+) (1.2 mM) was abolished in mTAL cells transiently transfected with a dominant-negative CaR construct (R796W) or pretreated with the phosphatidylinositol phospholipase C (PI-PLC) inhibitor U-73122. Cyclosporine A (CsA), an inhibitor of the serine/threonine phosphatase calcineurin, and a peptide ligand, VIVIT, that selectively inhibits calcineurin-NFAT signaling, also prevented CaR-mediated TNF production. Increases in calcineurin activity in cells challenged with Ca(2+) were inhibited after pretreatment with U-73122 and CsA, suggesting that CaR activation increases calcineurin activity in a PI-PLC-dependent manner. Moreover, U-73122, CsA, and VIVIT inhibited CaR-dependent activity of an NFAT construct that drives expression of firefly luciferase in transiently transfected mTAL cells. Collectively, these data verify the role of calcineurin and NFAT in CaR-mediated TNF production by mTAL cells. Activation of the CaR also increased the binding of NFAT to a consensus oligonucleotide, an effect that was blocked by U-73122 and CsA, suggesting that a calcineurin- and NFAT-dependent pathway increases TNF production in mTAL cells. This mechanism likely regulates TNF gene transcription as U-73122, CsA, and VIVIT blocked CaR-dependent activity of a TNF promoter construct. Elucidating CaR-mediated signaling pathways that regulate TNF production in the mTAL will be crucial to understanding mechanisms that regulate extracellular fluid volume and salt balance.

  11. The calcium-sensing receptor and the reproductive system

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    Isabella Ellinger


    Full Text Available Active placental transport of maternal serum calcium (Ca2+ to the offspring is pivotal for proper development of the fetal skeleton as well as various organ systems. Moreover, extracellular Ca2+ levels impact on distinct processes in mammalian reproduction. The calcium-sensing receptor (CaSR translates changes in extracellular Ca2+-concentrations into cellular reactions. This review summarizes current knowledge on the expression of CaSR and its putative functions in reproductive organs. CaSR was detected in placental cells mediating materno-fetal Ca2+-transport such as the the murine intraplacental yolk sac and the human syncytiotrophoblast. As shown in casr knock-out mice, ablation of CaSR downregulates transplacental Ca2+-transport. Receptor expression was reported in human and rat ovarian surface epithelial cells, where CaSR activation stimulates cell proliferation. In follicles of various species a role of CaSR activation in oocyte maturation was suggested. Based on studies in avian follicles, the activation of CaSR expressed in granulosa cells may support the survival of follicles after their selection. CaSR in rat and equine sperms was functionally linked to sperm motility and sperm capacitation. Implantation involves complex interactions between the blastocyst and the uterine epithelium. During early pregnancy, CaSR expression at the implantation site as well as in decidual cells indicates that CaSR is important for blastocyst implantation and decidualization in the rat uterus. Localization of CaSR in human extravillous cytotrophoblasts suggests a role of CaSR in placentation. Overall, evidence for functional involvement of CaSR in physiologic mammalian reproductive processes exists. Moreover, several studies reported altered expression of CaSR in cells of reproductive tissues under pathologic conditions. However, in many tissues we still lack knowledge on physiological ligands activating CaSR, CaSR-linked G-proteins, activated

  12. Interaction of CPCCOEt with a chimeric mGlu1b and calcium sensing receptor

    DEFF Research Database (Denmark)

    Bräuner-Osborne, H; Jensen, Anders A.; Krogsgaard-Larsen, P


    7-Hydroxyiminocyclopropan[b]chromen-1a-carboxylic acid ethyl ester (CPCCOEt) has previously been shown to be a selective non-competitive antagonist at the metabotropic glutamate (mGlu) receptor subtype 1. In this study we have tested the effect of CPCCOEt on mGlu1b, the calcium sensing receptor (...

  13. Activation of the calcium sensing receptor with cinacalcet increases serum gastrin levels in healthy older subjects (United States)

    Gastric acidity is postulated to enhance calcium absorption since calcium is better dissolved at low pH. Extracellular calcium stimulates gastrin and gastric acid secretion in humans. Ex vivo studies indicate that the calcium sensing receptor (CaR), which is expressed on the surface of human G cells...

  14. Vitamin D Receptor and Calcium Sensing Receptor Polymorphisms and the Risk of Colorectal Cancer in European Populations

    NARCIS (Netherlands)

    Jenab, Mazda; McKay, James; Bueno-de-Mesquita, Hendrik B.; van Duijnhoven, Franzel J. B.; Ferrari, Pietro; Slimani, Nadia; Jansen, Eugene H. J. M.; Pischon, Tobias; Rinaldi, Sabina; Tjonneland, Anne; Olsen, Anja; Overvad, Kim; Boutron-Ruault, Marie-Christine; Clavel-Chapelon, Francoise; Engel, Pierre; Kaaks, Rudolf; Linseisen, Jakob; Boeing, Heiner; Fisher, Eva; Trichopoulou, Antonia; Dilis, Vardis; Oustoglou, Erifili; Berrino, Franco; Vineis, Paolo; Mattiello, Amalia; Masala, Giovanna; Tumino, Rosario; Vrieling, Alina; van Gils, Carla H.; Peeters, Petra H.; Brustad, Magritt; Lund, Eiliv; Chirlaque, Maria-Dolores; Barricarte, Aurelio; Rodriguez Suarez, Laudina; Molina, Esther; Dorronsoro, Miren; Sala, Nuria; Hallmans, Goran; Palmqvist, Richard; Roddam, Andrew; Key, Timothy J.; Khaw, Kay-Tee; Bingham, Sheila; Boffetta, Paolo; Autier, Philippe; Byrnes, Graham; Norat, Teresa; Riboli, Elio


    Increased levels of vitamin D and calcium may play a protective role in colorectal cancer (CRC) risk. It has been suggested that these effects may be mediated by genetic variants of the vitamin D receptor (VDR) and the calcium sensing receptor (CASR). However, current epidemiologic evidence from Eur

  15. Calcium sensing receptors and calcium oscillations: calcium as a first messenger. (United States)

    Breitwieser, Gerda E


    Calcium sensing receptors (CaR) are unique among G-protein-coupled receptors (GPCRs) since both the first (extracellular) and second (intracellular) messengers are Ca(2+). CaR serves to translate small fluctuations in extracellular Ca(2+) into intracellular Ca(2+) oscillations. In many cells and tissues, CaR also acts as a coincidence detector, sensing both changes in extracellular Ca(2+) plus the presence of various allosteric activators including amino acids, polyamines, and/or peptides. CaR oscillations are uniquely shaped by the activating agonist, that is, Ca(2+) triggers sinusoidal oscillations while Ca(2+) plus phenylalanine trigger transient oscillations of lower frequency. The distinct oscillation patterns generated by Ca(2+)versus Ca(2+) plus phenylalanine are the results of activation of distinct signal transduction pathways. CaR is a member of Family C GPCRs, having a large extracellular agonist binding domain, and functioning as a disulfide-linked dimer. The CaR dimer likely can be driven to distinct active conformations by various Ca(2+) plus modulator combinations, which can drive preferential coupling to divergent signaling pathways. Such plasticity with respect to both agonist and signaling outcomes allows CaR to uniquely contribute to the physiology of organs and tissues where it is expressed. This chapter will examine the structural features of CaR, which contribute to its unique properties, the nature of CaR-induced intracellular Ca(2+) signals and the potential role(s) for CaR in development and differentiation.

  16. [Chronic hypocalcemia due to anti-calcium sensing receptor antibodies]. (United States)

    Marques, Pedro; Santos, Rita; Cavaco, Branca; Leite, Valeriano


    Introdução: O hipoparatiroidismo cursa com hipocalcemia e é mais frequentemente registado após cirurgia cervical. A etiologia autoimune é mais rara e difícil de diagnosticar. Caso clínico: Mulher, 52 anos, sem antecedentes pessoais, medicamentosos ou familiares relevantes, referenciada por hipocalcemia e calcificação dos núcleos da base, detetados no decurso de investigação de quadro de mialgias. Além de hipocalcemia (4,6 mg/dL), foi verificada hiperfosfatemia (8,7 mg/dL), hormona paratiroideia indetetável, calciúria, fosfatúria e magnesúria baixas. A análise molecular do gene CaSR excluiu mutações germinais. A pesquisa de anticorpos anti-receptor sensível do cálcio (anti-CaSR) foi positiva. Atualmente está assintomática e normocalcémica sob terapêutica com cálcio e vitamina D. Discussão: Embora rara, a hipocalcemia por hipoparatiroidismo autoimune deve ponderar-se em adultos sem antecedentes de cirurgia cervical, medicação hipocalcemiante, história familiar ou fenótipo sugestivo de doença genética. Hormona paratiroideia diminuída ou indetetável exclui pseudohipoparatiroidismo e a positividade para anti-CaSR confirma o diagnóstico.

  17. Calcium-Sensing Receptor: Trafficking, Endocytosis, Recycling, and Importance of Interacting Proteins. (United States)

    Ray, Kausik


    The cloning of the extracellular calcium-sensing receptor (CaSR) provided a new paradigm in G-protein-coupled receptor (GPCR) signaling in which principal physiological ligand is a cation, namely, extracellular calcium (Ca(o)(2+)). A wealth of information has accumulated in the past two decades about the CaSR's structure and function, its contribution to pathology in disorders of calcium in humans, and CaSR-based therapeutics. The CaSR unlike many other GPCRs must function in the presence of its ligand, thus understanding the mechanisms such as anterograde trafficking and endocytic pathways of this receptor are complex and fallen behind other classical GPCRs. Factors controlling CaSR signaling include various proteins affecting the expression of the CaSR as well as modulation of its trafficking to and from the cell surface. The dimeric cell-surface CaSR links to various heterotrimeric G-proteins (G(q/11), G(i/o), G(12/13), and G(s)) to regulate intracellular second messengers, lipid kinases, various protein kinases, and transcription factors that are part of the machinery enabling the receptor to modulate the functions of the wide variety of cells in which it is expressed. This chapter describes key features of CaSR structure and function and discusses novel mechanisms by which the level of cell-surface receptor expression can be regulated including forward trafficking during biosynthesis, desensitization, internalization and recycling from the cell surface, and degradation. These processes are impacted by its interactions with several proteins in addition to signaling molecules per se (i.e., G-proteins, protein kinases, inositol phosphates, etc.) and include small molecular weight G-proteins (Sar1, Rabs, ARF, P24A, RAMPs, filamin A, 14-3-3 proteins, calmodulin, and caveolin-1). Moreover, CaSR signaling seems compartmentalized in cell-type-specific manner, and caveolin and filamin A likely act as scaffolds that bind signaling components and other key cellular

  18. The calcium-sensing receptor and calcimimetics in blood pressure modulation

    DEFF Research Database (Denmark)

    Smajilovic, Sanela; Yano, Shozo; Jabbari, Reza;


    Calcium is a crucial second messenger in the cardiovascular system. However, calcium may also be an extracellular first messenger through a G-protein-coupled receptor that senses extracellular concentration (Ca(2+)(o)), the calcium-sensing receptor (CaR). The most prominent physiological function......R (calcimimetics) are the first drugs in their class to become available for clinical use and have been shown to successfully treat certain forms of primary and secondary hyperparathyroidism. In addition, several studies suggest beneficial effects of calcimimetics on cardiovascular risk factors associated...... with hyperparathyroidism. Although a plethora of studies demonstrated the CaR in heart and blood vessels, exact roles of the receptor in the cardiovascular system still remain to be elucidated. However, several studies point toward a possibility that the CaR might be involved in the regulation of vascular tone...

  19. Novel strategies in drug discovery of the calcium-sensing receptor based on biased signaling

    DEFF Research Database (Denmark)

    Thomsen, Alex Rojas Bie; Smajilovic, Sanela; Bräuner-Osborne, Hans


    A hallmark of chronic kidney disease is hyperphosphatemia due to renal phosphate retention. Prolonged parathyroid gland exposure to hyperphosphatemia leads to secondary hyperparathyroidism characterized by hyperplasia of the glands and excessive secretion of parathyroid hormone (PTH), which causes...... renal osteodystrophy. PTH secretion from the parathyroid glands is controlled by the calcium-sensing receptor (CaSR) that senses extracellular calcium. High extracellular calcium activates the CaSR causing inhibition of PTH secretion through multiple signaling pathways. Cinacalcet is the first drug...... targeting the CaSR and can be used to effectively control and reduce PTH secretion in PTH-related diseases. Cinacalcet is a positive allosteric modulator of the CaSR and affects PTH secretion from parathyroid glands by shifting the calcium-PTH concentration-response curve to the left. One major disadvantage...

  20. Calcium-sensing receptor: a key target for extracellular calcium signaling in neurons

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    Brian L Jones


    Full Text Available Though both clinicians and scientists have long recognized the influence of extracellular calcium on the function of muscle and nervous tissue, recent insights reveal that the mechanisms allowing changes in extracellular calcium to alter cellular excitability have been incompletely understood. For many years the effects of calcium on neuronal signaling were explained only in terms of calcium entry through voltage-gated calcium channels and biophysical charge screening. More recently however, it has been recognized that the calcium-sensing receptor is prevalent in the nervous system and regulates synaptic transmission and neuronal activity via multiple signaling pathways. Here we review the multiplicity of mechanisms by which changes in extracellular calcium alter neuronal signaling and propose that multiple mechanisms are required to describe the full range of experimental observations.

  1. Activation of calcium-sensing receptor increases TRPC3 expression in rat cardiomyocytes

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    Feng, Shan-Li [Department of Clinical Laboratory, The Second Affiliated Hospital of Harbin Medical University, Harbin 150086 (China); Sun, Ming-Rui [Department of Pharmacology, Qiqihaer Medical College, Qiqihaer 160001 (China); Li, Ting-Ting; Yin, Xin [Department of Clinical Laboratory, The Second Affiliated Hospital of Harbin Medical University, Harbin 150086 (China); Xu, Chang-Qing [Department of Pathophysiology, Harbin Medical University, Harbin 150086 (China); Sun, Yi-Hua, E-mail: [Department of Clinical Laboratory, The Second Affiliated Hospital of Harbin Medical University, Harbin 150086 (China)


    Research highlights: {yields} Calcium-sensing receptor (CaR) activation stimulates TRP channels. {yields} CaR promoted transient receptor potential C3 (TRPC3) expression. {yields} Adult rat ventricular myocytes display capacitative calcium entry (CCE), which was operated by TRPCs. {yields} TRPC channels activation induced by CaR activator sustained the increased [Ca{sup 2+}]{sub i} to evoke cardiomyocytes apoptosis. -- Abstract: Transient receptor potential (TRP) channels are expressed in cardiomyocytes, which gate a type of influx of extracellular calcium, the capacitative calcium entry. TRP channels play a role in mediating Ca{sup 2+} overload in the heart. Calcium-sensing receptors (CaR) are also expressed in rat cardiac tissue and promote the apoptosis of cardiomyocytes by Ca{sup 2+} overload. However, data about the link between CaR and TRP channels in rat heart are few. In this study, reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting were used to examine the expression of the TRP canonical proteins TRPC1 and TRPC3 in adult and neonatal rat cardiomyocytes. Laser scan confocal microscopy was used to detect intracellular [Ca{sup 2+}]{sub i} levels in isolated adult rat ventricular myocytes. The results showed that, in adult rat cardiomyocytes, the depletion of Ca{sup 2+} stores in the endoplasmic/sarcoplasmic reticulum (ER/SR) by thapsigargin induced a transient increase in [Ca{sup 2+}]{sub i} in the absence of [Ca{sup 2+}]{sub o} and the subsequent restoration of [Ca{sup 2+}]{sub o} sustained the increased [Ca{sup 2+}]{sub i} for a few minutes, whereas, the persisting elevation of [Ca{sup 2+}]{sub i} was reduced in the presence of the TRPC inhibitor SKF96365. The stimulation of CaR by its activator gadolinium chloride (GdCl{sub 3}) or spermine also resulted in the same effect and the duration of [Ca{sup 2+}]{sub i} increase was also shortened in the absence of [Ca{sup 2+}]{sub o}. In adult and neonatal rat cardiomyocytes, GdCl{sub 3

  2. The functional expression of extracellular calcium-sensing receptor in rat pulmonary artery smooth muscle cells

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    Zhang Wei-hua


    Full Text Available Abstract Background The extracellular calcium-sensing receptor (CaSR belongs to family C of the G protein coupled receptors. Whether the CaSR is expressed in the pulmonary artery (PA is unknown. Methods The expression and distribution of CaSR were detected by RT-PCR, Western blotting and immunofluorescence. PA tension was detected by the pulmonary arterial ring technique, and the intracellular calcium concentration ([Ca2+]i was detected by a laser-scanning confocal microscope. Results The expressions of CaSR mRNA and protein were found in both rat pulmonary artery smooth muscle cells (PASMCs and PAs. Increased levels of [Ca2+]o (extracellular calcium concentration or Gd3+ (an agonist of CaSR induced an increase of [Ca2+]i and PAs constriction in a concentration-dependent manner. In addition, the above-mentioned effects of Ca2+ and Gd3+ were inhibited by U73122 (specific inhibitor of PLC, 2-APB (specific antagonist of IP3 receptor, and thapsigargin (blocker of sarcoplasmic reticulum calcium ATPase. Conclusions CaSR is expressed in rat PASMCs, and is involved in regulation of PA tension by increasing [Ca2+]i through G-PLC-IP3 pathway.

  3. Involvement of the calcium-sensing receptor in human taste perception. (United States)

    Ohsu, Takeaki; Amino, Yusuke; Nagasaki, Hiroaki; Yamanaka, Tomohiko; Takeshita, Sen; Hatanaka, Toshihiro; Maruyama, Yutaka; Miyamura, Naohiro; Eto, Yuzuru


    By human sensory analyses, we found that various extracellular calcium-sensing receptor (CaSR) agonists enhance sweet, salty, and umami tastes, although they have no taste themselves. These characteristics are known as "kokumi taste" and often appear in traditional Japanese cuisine. Although GSH is a typical kokumi taste substance (taste enhancer), its mode of action is poorly understood. Here, we demonstrate how the kokumi taste is enhanced by the CaSR, a close relative of the class C G-protein-coupled receptors T1R1, T1R2, and T1R3 (sweet and umami receptors). We identified a large number of CaSR agonist gamma-glutamyl peptides, including GSH (gamma-Glu-Cys-Gly) and gamma-Glu-Val-Gly, and showed that these peptides elicit the kokumi taste. Further analyses revealed that some known CaSR agonists such as Ca(2+), protamine, polylysine, L-histidine, and cinacalcet (a calcium-mimetic drug) also elicit the kokumi taste and that the CaSR-specific antagonist, NPS-2143, significantly suppresses the kokumi taste. This is the first report indicating a distinct function of the CaSR in human taste perception.

  4. Comparative expression of the extracellular calcium-sensing receptor in the mouse, rat, and human kidney. (United States)

    Graca, J A Z; Schepelmann, M; Brennan, S C; Reens, J; Chang, W; Yan, P; Toka, H; Riccardi, D; Price, S A


    The calcium-sensing receptor (CaSR) was cloned over 20 years ago and functionally demonstrated to regulate circulating levels of parathyroid hormone by maintaining physiological serum ionized calcium concentration ([Ca(2+)]). The receptor is highly expressed in the kidney; however, intrarenal and intraspecies distribution remains controversial. Recently, additional functions of the CaSR receptor in the kidney have emerged, including parathyroid hormone-independent effects. It is therefore critical to establish unequivocally the localization of the CaSR in the kidney to relate this to its proposed physiological roles. In this study, we determined CaSR expression in mouse, rat, and human kidneys using in situ hybridization, immunohistochemistry (using 8 different commercially available and custom-made antibodies), and proximity ligation assays. Negative results in mice with kidney-specific CaSR ablation confirmed the specificity of the immunohistochemistry signal. Both in situ hybridization and immunohistochemistry showed CaSR expression in the thick ascending limb, distal tubule, and collecting duct of all species, with the thick ascending limb showing the highest levels. Within the collecting ducts, there was significant heterogeneity of expression between cell types. In the proximal tubule, lower levels of immunoreactivity were detected by immunohistochemistry and proximity ligation assays. Proximity ligation assays were the only technique to demonstrate expression within glomeruli. This study demonstrated CaSR expression throughout the kidney with minimal discrepancy between species but with significant variation in the levels of expression between cell and tubule types. These findings clarify the intrarenal distribution of the CaSR and enable elucidation of the full physiological roles of the receptor within this organ.

  5. Activation of the MAP Kinase Cascade by Exogenous Calcium-Sensing Receptor

    Energy Technology Data Exchange (ETDEWEB)

    Hobson, Susan A.; Wright, Jay W.; Lee, Fred; Mcneil, Scott; Bilderback, Tim R.; Rodland, Karin D.


    In Rat-1 fibroblasts and ovarian surface epithelial cells, extracellular calcium induces a proliferative response which appears to be mediated by the G-protein coupled Calcium-sensing Receptor (CaR), as expression of the non-functional CaR-R795W mutant inhibits both thymidine incorporation and activation of the extracellular-regulated kinase (ERK) in response to calcium. In this report we utilized CaR-transfected HEK293 cells to demonstrate that functional CaR is necessary and sufficient for calcium-induced ERK activation. CaR-dependent ERK activation was blocked by co-expression of the Ras dominant-negative mutant, Ras N17, and by exposure to the phosphatidyl inositol 3' kinase inhibitors wortmannin and LY294002. In contrast to Rat-1 fibroblasts, CaR-mediated in vitro kinase activity of ERK2 was unaffected by tyrosine kinase inhibitor herbimycin in CaR-transfected HEK293 cells. These results suggest that usage of distinct pathways downstream of the CaR varies in a cell-type specific manner, suggesting a potential mechanism by which activation of the CaR could couple to distinct calcium-dependent responses.

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

    Energy Technology Data Exchange (ETDEWEB)

    Magno, Aaron L. [Western Australian Institute for Medical Research and Centre for Medical Research, University of Western Australia, Nedlands, Western Australia 6009 (Australia); Department of Endocrinology and Diabetes, Sir Charles Gairdner Hospital, Hospital Avenue, Nedlands, Western Australia 6009 (Australia); Ingley, Evan [Western Australian Institute for Medical Research and Centre for Medical Research, University of Western Australia, Nedlands, Western Australia 6009 (Australia); Brown, Suzanne J. [Department of Endocrinology and Diabetes, Sir Charles Gairdner Hospital, Hospital Avenue, Nedlands, Western Australia 6009 (Australia); Conigrave, Arthur D. [School of Molecular Bioscience, University of Sydney, New South Wales 2000 (Australia); Ratajczak, Thomas [Western Australian Institute for Medical Research and Centre for Medical Research, University of Western Australia, Nedlands, Western Australia 6009 (Australia); Department of Endocrinology and Diabetes, Sir Charles Gairdner Hospital, Hospital Avenue, Nedlands, Western Australia 6009 (Australia); Ward, Bryan K., E-mail: [Western Australian Institute for Medical Research and Centre for Medical Research, University of Western Australia, Nedlands, Western Australia 6009 (Australia); Department of Endocrinology and Diabetes, Sir Charles Gairdner Hospital, Hospital Avenue, Nedlands, Western Australia 6009 (Australia)


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

  7. The calcium-sensing receptor regulates mammary gland parathyroid hormone–related protein production and calcium transport


    VanHouten, Joshua; Dann, Pamela; McGeoch, Grace; Brown, Edward M.; Krapcho, Karen; Neville, Margaret; Wysolmerski, John J


    The transfer of calcium from mother to milk during lactation is poorly understood. In this report, we demonstrate that parathyroid hormone–related protein (PTHrP) production and calcium transport in mammary epithelial cells are regulated by extracellular calcium acting through the calcium-sensing receptor (CaR). The CaR becomes expressed on mammary epithelial cells at the transition from pregnancy to lactation. Increasing concentrations of calcium, neomycin, and a calcimimetic compound suppre...

  8. Cytoskeletal changes induced by allosteric modulators of calcium-sensing receptor in esophageal epithelial cells. (United States)

    Abdulnour-Nakhoul, Solange; Brown, Karen L; Rabon, Edd C; Al-Tawil, Youhanna; Islam, Mohammed T; Schmieg, John J; Nakhoul, Nazih L


    The calcium-sensing receptor (CaSR), a G-protein-coupled receptor, plays a role in glandular and fluid secretion in the gastrointestinal tract, and regulates differentiation and proliferation of epithelial cells. We examined the expression of CaSR in normal and pathological conditions of human esophagus and investigated the effect of a CaSR agonist, cinacalcet (CCT), and antagonist, calhex (CHX), on cell growth and cell-cell junctional proteins in primary cultures of porcine stratified squamous esophageal epithelium. We used immunohistochemistry and Western analysis to monitor expression of CaSR and cell-cell adhesion molecules, and MTT assay to monitor cell proliferation in cultured esophageal cells. CCT treatment significantly reduced proliferation, changed the cell shape from polygonal to spindle-like, and caused redistribution of E-cadherin and β-catenin from the cell membrane to the cytoplasm. Furthermore, it reduced expression of β-catenin by 35% (P < 0.02) and increased expression of a proteolysis cleavage fragment of E-cadherin, Ecad/CFT2, by 2.3 folds (P < 0.01). On the other hand, CHX treatment enhanced cell proliferation by 27% (P < 0.01), increased the expression of p120-catenin by 24% (P < 0.04), and of Rho, a GTPase involved in cytoskeleton remodeling, by 18% (P < 0.03). In conclusion, CaSR is expressed in normal esophagus as well as in Barrett's, esophageal adenocarcinoma, squamous cell carcinoma, and eosinophilic esophagitis. Long-term activation of CaSR with CCT disrupted the cadherin-catenin complex, induced cytoskeletal remodeling, actin fiber formation, and redistribution of CaSR to the nuclear area. These changes indicate a significant and complex role of CaSR in epithelial remodeling and barrier function of esophageal cells.

  9. Calcium-sensing receptor expression and parathyroid hormone secretion in hyperplastic parathyroid glands from humans. (United States)

    Cañadillas, Sagrario; Canalejo, Antonio; Santamaría, Rafael; Rodríguez, Maria E; Estepa, Jose C; Martín-Malo, Alejandro; Bravo, Juan; Ramos, Blanca; Aguilera-Tejero, Escolastico; Rodríguez, Mariano; Almadén, Yolanda


    In uremic patients, severe parathyroid hyperplasia is associated with reduced parathyroid calcium-sensing receptor (CaR) expression. Thus, in these patients, a high serum Ca concentration may be required to inhibit parathyroid hormone (PTH) secretion. This study compares the magnitude of reduction in CaR expression and the degree of the abnormality in Ca-regulated PTH release in vitro. A total of 50 glands from 23 hemodialysis patients with refractory hyperparathyroidism were studied. Tissue slices were incubated in vitro to evaluate (1) the PTH secretory output in a normal Ca concentration (1.25 mM) and (2) the PTH secretory response to high (1.5 mM) and low (0.6 mM) Ca concentration. Tissue aliquots were processed for determination of CaRmRNA expression. The results showed that, corrected for DNA, parathyroid tissue with lowest CaR expression secreted more PTH than that with relatively high CaR expression (146 +/- 23 versus 60 +/- 2 pg/microg DNA; P < 0.01). Furthermore, glands with low CaR expression demonstrated a blunted PTH secretory response to both the inhibitory effect of high Ca and the stimulatory effect of low Ca. The study also showed that the larger the gland, the lower the CaRmRNA expression. Thus, large parathyroid glands produce a large amount of PTH not only as a result of the increased gland size but also because the parathyroid tissue secretory output is increased. These abnormalities in PTH regulation are related to low CaR expression.

  10. The calcium-sensing receptor participates in testicular damage in streptozotocin-induced diabetic rats. (United States)

    Kong, Wei-Yuan; Tong, Li-Quan; Zhang, Hai-Jun; Cao, Yong-Gang; Wang, Gong-Chen; Zhu, Jin-Zhi; Zhang, Feng; Sun, Xue-Ying; Zhang, Tie-Hui; Zhang, Lin-Lin


    Male infertility caused by testicular damage is one of the complications of diabetes mellitus. The calcium-sensing receptor (CaSR) is expressed in testicular tissues and plays a pivotal role in calcium homeostasis by activating cellular signaling pathways, but its role in testicular damage induced by diabetes remains unclear. A diabetic model was established by a single intraperitoneal injection of streptozotocin (STZ, 40 mg kg-1 ) in Wistar rats. Animals then received GdCl 3 (an agonist of CaSR, 8.67 mg kg-1 ), NPS-2390 (an antagonist of CaSR, 0.20 g kg-1 ), or a combination of both 2 months after STZ injection. Diabetic rats had significantly lower testes weights and serum levels of testosterone compared to healthy rats, indicating testicular damage and dysfunction in STZ-induced diabetic rats. Compared with healthy controls, the testicular tissues of diabetic rats overexpressed the CaSR protein and had higher levels of malondialdehyde (MDA), lower superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activity, and higher numbers of apoptotic germ cells. The testicular tissues from diabetic rats also expressed lower levels of Bcl-2 and higher levels of Bax and cleaved caspase-3 in addition to higher phosphorylation rates of c-Jun NH 2 -terminal protein kinase (JNK), p38, and extracellular signaling-regulated kinase (ERK) 1/2. The above parameters could be further increased or aggravated by the administration of GdCl 3 , but could be attenuated by injection of NPS-2390. In conclusion, the present results indicate that CaSR activation participates in diabetes-induced testicular damage, implying CaSR may be a potential target for protective strategies against diabetes-induced testicular damage and could help to prevent infertility in diabetic men.

  11. The calcium-sensing receptor participates in testicular damage in streptozotocin-induced diabetic rats

    Directory of Open Access Journals (Sweden)

    Wei-Yuan Kong


    Full Text Available Male infertility caused by testicular damage is one of the complications of diabetes mellitus. The calcium-sensing receptor (CaSR is expressed in testicular tissues and plays a pivotal role in calcium homeostasis by activating cellular signaling pathways, but its role in testicular damage induced by diabetes remains unclear. A diabetic model was established by a single intraperitoneal injection of streptozotocin (STZ, 40 mg kg−1 in Wistar rats. Animals then received GdCl 3 (an agonist of CaSR, 8.67 mg kg−1 , NPS-2390 (an antagonist of CaSR, 0.20 g kg−1 , or a combination of both 2 months after STZ injection. Diabetic rats had significantly lower testes weights and serum levels of testosterone compared to healthy rats, indicating testicular damage and dysfunction in STZ-induced diabetic rats. Compared with healthy controls, the testicular tissues of diabetic rats overexpressed the CaSR protein and had higher levels of malondialdehyde (MDA, lower superoxide dismutase (SOD and glutathione peroxidase (GSH-Px activity, and higher numbers of apoptotic germ cells. The testicular tissues from diabetic rats also expressed lower levels of Bcl-2 and higher levels of Bax and cleaved caspase-3 in addition to higher phosphorylation rates of c-Jun NH 2 -terminal protein kinase (JNK, p38, and extracellular signaling-regulated kinase (ERK 1/2. The above parameters could be further increased or aggravated by the administration of GdCl 3 , but could be attenuated by injection of NPS-2390. In conclusion, the present results indicate that CaSR activation participates in diabetes-induced testicular damage, implying CaSR may be a potential target for protective strategies against diabetes-induced testicular damage and could help to prevent infertility in diabetic men.


    Directory of Open Access Journals (Sweden)

    Anna eChiarini


    Full Text Available In aged subjects, late-onset Alzheimer’s disease (LOAD starts in the lateral entorhinal allocortex where a failure of clearance mechanisms triggers an accumulation neurotoxic of amyloid-β42 oligomers (Aβ42-os. In neurons and astrocytes, Aβ42-os enhance the transcription of Aβ precursor protein (APP and β-secretase/BACE1 genes. Thus, by acting together with γ-secretase, the surpluses of APP and BACE1 amplify the endogenous production of Aβ42-os which pile up, damage mitochondria, and are oversecreted. At the plasmalemma, exogenous Aβ42-os bind neurons' and astrocytes' calcium-sensing receptors (CaSRs activating a set of intracellular signalling pathways which upkeep Aβ42-os intracellular accumulation and oversecretion by hindering Aβ42-os proteolysis. In addition, Aβ42-os accumulating in the extracellular milieu spread and reach mounting numbers of adjacent and remoter teams of neurons and astrocytes which in turn are recruited, again via Aβ42-osCaSR-governed mechanisms, to produce and release additional Aβ42-os amounts. This relentless self-sustaining mechanism drives AD progression towards upper cortical areas. Later on accumulating Aβ42-os elicit the advent of hyperphosphorylated (p-Tau oligomers which acting together with Aβ42-os and other glial neurotoxins cooperatively destroy wider and wider cognition-related cortical areas. In parallel, Aβ42-osCaSR signals also elicit an excess production and secretion of nitric oxide and vascular endothelial growth factor-A from astrocytes, of Aβ42-os and myelin basic protein from oligodendrocytes, and of proinflammatory cytokines, nitric oxide and (likely Aβ42-os from microglia. Activated astrocytes and microglia survive the toxic onslaught, whereas neurons and oligodendrocytes increasingly die. However, we have shown that highly selective allosteric CaSR antagonists (calcilytics, like NPS 2143 and NPS 89626, efficiently suppress all the neurotoxic effects Aβ42-osCaSR signalling

  13. Cross-talk between the calcium-sensing receptor and the epidermal growth factor receptor in Rat-1 fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Tomlins, Scott A.; Bollinger, Nikki; Creim, Jeffrey A.; Rodland, Karin D.


    The calcium-sensing receptor (CaR) is a G-protein coupled receptor that is activated by extracellular calcium (Ca2+o). Rat-1 fibroblasts have been shown to proliferate and increase ERK activity in response to elevation of [Ca2+]o, and these responses are dependent on functional CaR expression. In this report, we examined the role of cross-talk between the CaR and the epidermal growth factor receptor (EGFR) in mediating these responses in Rat-1 cells. This report shows that AG1478, a specific inhibitor of the EGFR kinase, significantly inhibits the increase in proliferation induced by elevated Ca2+o. Further, we show that AG1478 acts downstream or separately from G-protein subunit activation of phospholipase C. AG1478 significantly inhibits Ca2+o-stimulated ERK phosphorylation and in vitro kinase activity. A similar inhibition of ERK phosphorylation was observed in response to the inhibitor AG494. In addition, treatment with inhibitors of metalloproteases involved in shedding of membrane anchored EGF family ligands substantially inhibited the increase in ERK activation in response to elevated Ca2+o. This is consistent with the known expression of TGFa by Rat-1 cells. These results indicate that EGFR transactivation is an important component of the CaR mediated response to increased Ca2+o in Rat-1 fibroblasts, and most likely involves CaR-mediated induction of regulated proteolysis and ligand shedding.

  14. Calcium, calcium-sensing receptor and growth control in the colonic mucosa


    Varani, James


    A role for calcium in epithelial growth control is well-established in the colon and other tissues. In the colon, Ca2+ “drives” the differentiation process. This results in sequestration of ß-catenin in the cell surface / cytoskeletal complex, leaving ß-catenin unavailable to serve as a growth-promoting transcription enhancer in the nucleus. The signaling events that lead from Ca2+ stimulation to differentiation are not fully understood. A critical role for the extracellular calcium-sensing r...

  15. Normalization of serum calcium by cinacalcet in a patient with hypercalcaemia due to a de novo inactivating mutation of the calcium-sensing receptor.

    NARCIS (Netherlands)

    Timmers, H.J.L.M.; Karperien, M.; Hamdy, N.A.; Boer, H. de; Hermus, A.R.M.M.


    Familial benign hypocalciuric hypercalcaemia (FHH) results from a heterozygous inactivating mutation of the calcium-sensing receptor (CaR) and is characterized by hypercalcaemia, hypocalciuria and inappropriately normal plasma levels of parathyroid hormone. In a minority of patients, a loss of funct

  16. A novel mutation in the calcium-sensing receptor gene in an Irish pedigree showing familial hypocalciuric hypercalcemia: a case report.

    LENUS (Irish Health Repository)

    Elamin, Wael F


    Familial hypocalciuric hypercalcemia is a rare autosomal dominant disorder characterized by asymptomatic and non-progressive hypercalcemia due to mutations of the calcium-sensing receptor gene. Disorders of calcium metabolism are very common in the elderly, and they can coexist with familial hypocalciuric hypercalcemia in affected families.

  17. Molecular Basis of the Extracellular Ligands Mediated Signaling by the Calcium Sensing Receptor

    Directory of Open Access Journals (Sweden)

    Chen Zhang


    Full Text Available Ca2+-sensing receptors (CaSRs play a central role in regulating extracellular calcium concentration ([Ca2+]o homeostasis and many (pathophysiological processes in multiple organs. This regulation is orchestrated by a cooperative response to extracellular stimuli such as small changes in Ca2+, Mg2+, amino acids and other ligands. In addition, CaSR is a pleiotropic receptor regulating several intracellular signaling pathways, including calcium mobilization and intracellular calcium oscillation. Nearly 200 mutations and polymorphisms have been found in CaSR in relation to a variety of human disorders associated with abnormal Ca2+ homeostasis. In this review, we summarize efforts directed at identifying binding sites for calcium and amino acids. Both homotropic cooperativity among multiple calcium binding sites and heterotropic cooperativity between calcium and amino acid were revealed using computational modeling, predictions, and site-directed mutagenesis coupled with functional assays. The hinge region of the bilobed Venus flytrap (VFT domain of CaSR plays a pivotal role in coordinating multiple extracellular stimuli, leading to cooperative responses from the receptor. We further highlight the extensive number of disease-associated mutations that have also been shown to affect CaSR’s cooperative action via several types of mechanisms. These results provide insights into the molecular bases of the structure and functional cooperativity of this receptor and other members of family C of the G protein-coupled receptors (cGPCRs in health and disease states, and may assist in the prospective development of novel receptor-based therapeutics.

  18. [Pharmacological characteristics of drugs targeted on calcium-sensing receptor.-properties of cinacalcet hydrochloride as allosteric modulator]. (United States)

    Nagano, Nobuo; Tsutsui, Takaaki


    Calcimimetics act as positive allosteric modulators of the calcium-sensing receptor (CaSR), thereby decreasing parathyroid hormone (PTH) secretion from the parathyroid glands. On the other hand, negative allosteric modulators of the CaSR with stimulatory effect on PTH secretion are termed calcilytics. The calcimimetic cinacalcet hydrochloride (cinacalcet) is the world's first allosteric modulator of G protein-coupled receptor to enter the clinical market. Cinacalcet just tunes the physiological effects of Ca(2+), an endogenous ligand, therefore, shows high selectivity and low side effects. Calcimimetics also increase cell surface CaSR expression by acting as pharmacological chaperones (pharmacoperones). It is considered that the cinacalcet-induced upper gastrointestinal problems are resulted from enhanced physiological responses to Ca(2+) and amino acids via increased sensitivity of digestive tract CaSR by cinacalcet. While clinical developments of calcilytics for osteoporosis were unfortunately halted or terminated due to paucity of efficacy, it is expected that calcilytics may be useful for the treatment of patients with activating CaSR mutations, asthma, and idiopathic pulmonary artery hypertension.

  19. Increased Calcium-Sensing Receptor Immunoreactivity in the Hippocampus of a Triple Transgenic Mouse Model of Alzheimer's Disease (United States)

    Gardenal, Emanuela; Chiarini, Anna; Armato, Ubaldo; Dal Prà, Ilaria; Verkhratsky, Alexei; Rodríguez, José J.


    The Calcium-Sensing Receptor (CaSR) is a G-protein coupled, 7-transmembrane domain receptor ubiquitously expressed throughout the body, brain including. The role of CaSR in the CNS is not well understood; its expression is increasing during development, which has been implicated in memory formation and consolidation, and CaSR localization in nerve terminals has been related to synaptic plasticity and neurotransmission. There is an emerging evidence of CaSR involvement in neurodegenerative disorders and Alzheimer's disease (AD) in particular, where the over-production of β-amyloid peptides was reported to activate CaSR. In the present study, we performed CaSR immunohistochemical and densitometry analysis in the triple transgenic mouse model of AD (3xTg-AD). We found an increase in the expression of CaSR in hippocampal CA1 area and in dentate gyrus in the 3xTg-AD mice when compared to non-transgenic control animals. This increase was significant at 9 months of age and further increased at 12 and 18 months of age. This increase paralleled the accumulation of β-amyloid plaques with age. Increased expression of CaSR favors β-amyloidogenic pathway following direct interactions between β-amyloid and CaSR and hence may contribute to the pathological evolution of the AD. In the framework of this paradigm CaSR may represent a novel therapeutic target.

  20. The Calcium-Sensing Receptor Is Necessary for the Rapid Development of Hypercalcemia in Human Lung Squamous Cell Carcinoma

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    Gwendolen Lorch


    Full Text Available The calcium-sensing receptor (CaR is responsible for the regulation of extracellular calcium (Ca2+o homeostasis. CaR activation has been shown to increase proliferation in several cancer cell lines; however, its presence or function has never been documented in lung cancer. We report that Ca2+o-activated CaR results in MAPK-mediated stimulation of parathyroid hormone-related protein (PTHrP production in human lung squamous cell carcinoma (SCC lines and humoral hypercalcemia of malignancy (HHM in vivo. Furthermore, a single nucleotide polymorphism in CaR identified from a hypercalcemia-inducing lung SCC reduced the receptor's activation threshold leading to increased PTHrP expression and secretion. Increasing the expression of either wild-type CaR or a CaR variant with a single nucleotide polymorphism in the cytoplasmic domain was both necessary and sufficient for lung SCC to induce HHM. Because lung cancer patients who frequently develop HHM and PTHrP expression in lung cancer has been only partially explained, the significance of our findings indicates that CaR variants may provide a positive feedback between PTHrP and calcium and result in the syndrome of HHM.

  1. Calcium sensing receptor suppresses human pancreatic tumorigenesis through a novel NCX1/Ca(2+)/β-catenin signaling pathway. (United States)

    Tang, Bo; Chow, Jimmy Y C; Dong, Tobias Xiao; Yang, Shi-Ming; Lu, De-Sheng; Carethers, John M; Dong, Hui


    The calcium sensing receptor (CaSR) is functionally expressed in normal human pancreases, but its pathological role in pancreatic tumorigenesis is currently unknown. We sought to investigate the role of CaSR in pancreatic cancer (PC) and the underlying molecular mechanisms. We revealed that the expression of CaSR was consistently downregulated in the primary cancer tissues from PC patients, which was correlated with tumor size, differentiation and poor survival of the patients. CaSR activation markedly suppressed pancreatic tumorigenesis in vitro and in vivo likely through the Ca(2+) entry mode of Na(+)/Ca(2+) exchanger 1 (NCX1) to induce Ca(2+) entry into PC cells. Moreover, NCX1-mediated Ca(2+) entry resulted in Ca(2+)-dependent inhibition of β-catenin signaling in PC cells, eventually leading to the inhibition of pancreatic tumorigenesis. Collectively, we demonstrate for the first time that CaSR exerts a suppressive function in pancreatic tumorigenesis through a novel NCX1/Ca(2+)/β-catenin signaling pathway. Targeting this specific signaling pathway could be a potential therapeutic strategy for PC.

  2. Cross talk between the calcium-sensing receptor and the vitamin D system in prevention of cancer

    Directory of Open Access Journals (Sweden)

    Enikö Kallay


    Full Text Available There is epidemiological evidence for the cancer preventive effect of dietary calcium (Ca2+ and vitamin D. This effect is strongest in colorectal cancer (CRC. The active vitamin D metabolite, 1,25-dihydroxyvitamin D3 (1,25D3, bound to its receptor, the vitamin D receptor (VDR regulates the expression of hundreds of different genes in a cell- and tissue-specific manner. While Ca2+ acts through multiple mechanisms and pathways, some of its effects are mediated by the calcium-sensing receptor (CaSR. The joint action of Ca2+ and 1,25D3 is due to the fact that both regulate some of the main processes involved in the development of various cancers, such as proliferation, differentiation, apoptosis, migration, and inflammation. Moreover, 1,25D3, bound to VDR can induce translation of the CaSR, while the amount and activity of the CaSR affects 1,25D3 signalling. However, the complexity of the cross-talk between the CaSR and the vitamin D system goes beyond regulating similar pathways and affecting each other’s expression. Our aim was to review some of the mechanisms that drive the cross-talk between the vitamin D system and the CaSR with a special focus on the interaction in colorectal cancer cells. We evaluated the molecular evidence that supports the epidemiological observation that both vitamin D and calcium are needed for protection against malignant transformation of the colon and that their effect is modulated by the presence of a functional CaSR.

  3. Mammary-Specific Ablation of the Calcium-Sensing Receptor During Lactation Alters Maternal Calcium Metabolism, Milk Calcium Transport, and Neonatal Calcium Accrual


    Mamillapalli, Ramanaiah; VanHouten, Joshua; Dann, Pamela; Bikle, Daniel; Chang, Wenhan; Brown, Edward; Wysolmerski, John


    To meet the demands for milk calcium, the lactating mother adjusts systemic calcium and bone metabolism by increasing dietary calcium intake, increasing bone resorption, and reducing renal calcium excretion. As part of this adaptation, the lactating mammary gland secretes PTHrP into the maternal circulation to increase bone turnover and mobilize skeletal calcium stores. Previous data have suggested that, during lactation, the breast relies on the calcium-sensing receptor (CaSR) to coordinate ...

  4. The calcium-sensing receptor promotes urinary acidification to prevent nephrolithiasis.

    NARCIS (Netherlands)

    Renkema, K.Y.; Velic, A.; Dijkman, H.B.; Verkaart, S.A.J.; Kemp, J.W.C.M. van der; Nowik, M.; Timmermans, K.; Doucet, A.; Wagner, C.A.; Bindels, R.J.M.; Hoenderop, J.G.J.


    Hypercalciuria increases the risk for urolithiasis, but renal adaptive mechanisms reduce this risk. For example, transient receptor potential vanilloid 5 knockout (TPRV5(-/-)) mice lack kidney stones despite urinary calcium (Ca(2+)) wasting and hyperphosphaturia, perhaps as a result of their signifi

  5. Calcium-sensing receptors regulate cardiomyocyte Ca2+ signaling via the sarcoplasmic reticulum-mitochondrion interface during hypoxia/reoxygenation

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    Lu Fang-hao


    Full Text Available Abstract Communication between the SR (sarcoplasmic reticulum, SR and mitochondria is important for cell survival and apoptosis. The SR supplies Ca2+ directly to mitochondria via inositol 1,4,5-trisphosphate receptors (IP3Rs at close contacts between the two organelles referred to as mitochondrion-associated ER membrane (MAM. Although it has been demonstrated that CaR (calcium sensing receptor activation is involved in intracellular calcium overload during hypoxia/reoxygenation (H/Re, the role of CaR activation in the cardiomyocyte apoptotic pathway remains unclear. We postulated that CaR activation plays a role in the regulation of SR-mitochondrial inter-organelle Ca2+ signaling, causing apoptosis during H/Re. To investigate the above hypothesis, cultured cardiomyocytes were subjected to H/Re. We examined the distribution of IP3Rs in cardiomyocytes via immunofluorescence and Western blotting and found that type 3 IP3Rs were located in the SR. [Ca2+]i, [Ca2+]m and [Ca2+]SR were determined using Fluo-4, x-rhod-1 and Fluo 5N, respectively, and the mitochondrial membrane potential was detected with JC-1 during reoxygenation using laser confocal microscopy. We found that activation of CaR reduced [Ca2+]SR, increased [Ca2+]i and [Ca2+]m and decreased the mitochondrial membrane potential during reoxygenation. We found that the activation of CaR caused the cleavage of BAP31, thus generating the pro-apoptotic p20 fragment, which induced the release of cytochrome c from mitochondria and the translocation of bak/bax to mitochondria. Taken together, these results reveal that CaR activation causes Ca2+ release from the SR into the mitochondria through IP3Rs and induces cardiomyocyte apoptosis during hypoxia/reoxygenation.

  6. Calcium-sensing receptors regulate cardiomyocyte Ca2+ signaling via the sarcoplasmic reticulum-mitochondrion interface during hypoxia/reoxygenation. (United States)

    Lu, Fang-hao; Tian, Zhiliang; Zhang, Wei-hua; Zhao, Ya-jun; Li, Hu-lun; Ren, Huan; Zheng, Hui-shuang; Liu, Chong; Hu, Guang-xia; Tian, Ye; Yang, Bao-feng; Wang, Rui; Xu, Chang-qing


    Communication between the SR (sarcoplasmic reticulum, SR) and mitochondria is important for cell survival and apoptosis. The SR supplies Ca2+ directly to mitochondria via inositol 1,4,5-trisphosphate receptors (IP3Rs) at close contacts between the two organelles referred to as mitochondrion-associated ER membrane (MAM). Although it has been demonstrated that CaR (calcium sensing receptor) activation is involved in intracellular calcium overload during hypoxia/reoxygenation (H/Re), the role of CaR activation in the cardiomyocyte apoptotic pathway remains unclear. We postulated that CaR activation plays a role in the regulation of SR-mitochondrial inter-organelle Ca2+ signaling, causing apoptosis during H/Re. To investigate the above hypothesis, cultured cardiomyocytes were subjected to H/Re. We examined the distribution of IP3Rs in cardiomyocytes via immunofluorescence and Western blotting and found that type 3 IP3Rs were located in the SR. [Ca2+]i, [Ca2+]m and [Ca2+]SR were determined using Fluo-4, x-rhod-1 and Fluo 5N, respectively, and the mitochondrial membrane potential was detected with JC-1 during reoxygenation using laser confocal microscopy. We found that activation of CaR reduced [Ca2+]SR, increased [Ca2+]i and [Ca2+]m and decreased the mitochondrial membrane potential during reoxygenation. We found that the activation of CaR caused the cleavage of BAP31, thus generating the pro-apoptotic p20 fragment, which induced the release of cytochrome c from mitochondria and the translocation of bak/bax to mitochondria. Taken together, these results reveal that CaR activation causes Ca2+ release from the SR into the mitochondria through IP3Rs and induces cardiomyocyte apoptosis during hypoxia/reoxygenation.

  7. Enterotoxin preconditioning restores calcium-sensing receptor-mediated cytostasis in colon cancer cells


    Pitari, Giovanni M.; Lin, Jieru E.; Shah, Fawad J.; Lubbe, Wilhelm J.; Zuzga, David S.; Li, Peng; Schulz, Stephanie; Waldman, Scott A.


    Guanylyl cyclase C (GCC), the receptor for diarrheagenic bacterial heat-stable enterotoxins (STs), inhibits colorectal cancer cell proliferation by co-opting Ca2+ as the intracellular messenger. Similarly, extracellular Ca2+ (Ca2+o) opposes proliferation and induces terminal differentiation in intestinal epithelial cells. In that context, human colon cancer cells develop a phenotype characterized by insensitivity to cytostasis imposed by Ca2+o. Here, preconditioning with ST, mediated by GCC s...

  8. Isolation and characterization of calcium sensing receptor null cells: a highly malignant and drug resistant phenotype of colon cancer. (United States)

    Singh, Navneet; Liu, Guangming; Chakrabarty, Subhas


    The expression of calcium sensing receptor (CaSR) in the human colonic crypt epithelium is linked to cellular differentiation while its lack of expression is associated with undifferentiated and invasive colon carcinoma. Human colon carcinoma cell lines contain small subpopulations (10-20%) that do not express CaSR (termed CaSR null cells). Here, we report on the isolation, propagation, maintenance and characterization of CaSR null cells from the CBS and HCT116 human colon carcinoma cell lines. CaSR null cells grew as three-dimensional non-adherent spherical clusters with increased propensity for anchorage independent growth, cellular proliferation and invasion of matrigels. CaSR null cells were highly resistant to fluorouracil and expressed abundant amount of thymidylate synthase and survivin. Molecular profiling by real time reverse transcription-polymerase chain reaction (RT-PCR) and Western blots showed a high level of expression of the previously reported cancer stem cell markers CD133, CD44 and Nanog in CaSR null cells. A significant increase in the expression of epithelial-mesenchymal transitional molecules and transcription factors was also observed. These include N-cadherin, β-catenin, vimentin, fibronectin, Snail1, Snail2, Twist and FOXC2. The expression of the tumor suppressive E-cadherin and miR145, on the other hand, was greatly reduced while expression of the oncogenic microRNAs: miR21, miR135a and miR135b was significantly up-regulated. CaSR null cells possess a myriad of cellular and molecular features that drive and sustain the malignant phenotype. We conclude that CaSR null constitutes a highly malignant and drug resistant phenotype of colon cancer.

  9. Calcium-sensing receptor activation contributed to apoptosis stimulates TRPC6 channel in rat neonatal ventricular myocytes

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Yi-hua [Department of Clinical Laboratory, The Second Affiliated Hospital of Harbin Medical University, Harbin 150086 (China); Li, Yong-quan [Harbin Medical University, Harbin 150086 (China); Feng, Shan-li [Department of Clinical Laboratory, The Second Affiliated Hospital of Harbin Medical University, Harbin 150086 (China); Li, Bao-xin; Pan, Zhen-wei [Department of Pharmacology, Harbin Medical University, Harbin 150086 (China); Xu, Chang-qing [Department of Pathophysiology, Harbin Medical University, Harbin 150086 (China); Li, Ting-ting [Department of Clinical Laboratory, The Second Affiliated Hospital of Harbin Medical University, Harbin 150086 (China); Yang, Bao-feng, E-mail: [Department of Pharmacology, Harbin Medical University, Harbin 150086 (China)


    Capacitative calcium entry (CCE) refers to the influx of calcium through plasma membrane channels activated on depletion of endoplasmic sarcoplasmic/reticulum (ER/SR) Ca{sup 2+} stores, which is performed mainly by the transient receptor potential (TRP) channels. TRP channels are expressed in cardiomyocytes. Calcium-sensing receptor (CaR) is also expressed in rat cardiac tissue and plays an important role in mediating cardiomyocyte apoptosis. However, there are no data regarding the link between CaR and TRP channels in rat heart. In this study, in rat neonatal myocytes, by Ca{sup 2+} imaging, we found that the depletion of ER/SR Ca{sup 2+} stores by thapsigargin (TG) elicited a transient rise in cytoplasmic Ca{sup 2+} ([Ca{sup 2+}]{sub i}), followed by sustained increase depending on extracellular Ca{sup 2+}. But, TRP channels inhibitor (SKF96365), not L-type channels or the Na{sup +}/Ca{sup 2+} exchanger inhibitors, inhibited [Ca{sup 2+}]{sub i} relatively high. Then, we found that the stimulation of CaR with its activator gadolinium chloride (GdCl{sub 3}) or by an increased extracellular Ca{sup 2+}([Ca{sup 2+}]{sub o}) increased the concentration of intracelluar Ca{sup 2+}, whereas, the sustained elevation of [Ca{sup 2+}]{sub i} was reduced in the presence of SKF96365. Similarly, the duration of [Ca{sup 2+}]{sub i} increase was also shortened in the absence of extracellular Ca{sup 2+}. Western blot analysis showed that GdCl{sub 3} increased the expression of TRPC6, which was reversed by SKF96365. Additionally, SKF96365 reduced cardiomyocyte apoptosis induced by GdCl{sub 3}. Our results suggested that CCE exhibited in rat neonatal myocytes and CaR activation induced Ca{sup 2+}-permeable cationic channels TRPCs to gate the CCE, for which TRPC6 was one of the most likely candidates. TRPC6 channel was functionally coupled with CaR to enhance the cardiomyocyte apoptosis.

  10. Molecular genetic analysis of the calcium sensing receptor gene in patients clinically suspected to have familial hypocalciuric hypercalcemia: phenotypic variation and mutation spectrum in a Danish population

    DEFF Research Database (Denmark)

    Nissen, Peter H; Christensen, Signe E; Heickendorff, Lene


    hyperparathyroidism and is caused by inactivating mutations in the calcium sensing receptor (CASR) gene. OBJECTIVE: We sought to define the mutation spectrum of the CASR gene in a Danish FHH population and to establish genotype-phenotype relationships regarding the different mutations. DESIGN AND PARTICIPANTS...... calcium concentrations moderately above the upper reference limit, to calcium levels more than 20% above the upper reference limit. Furthermore, the mean plasma PTH concentration was within the normal range in eight of 11 studied mutations, but mild to moderately elevated in families with the mutations p...

  11. Expression of calcium-sensing receptor in papillary thyroid carcinoma%钙敏感受体在甲状腺乳头状癌中的表达

    Institute of Scientific and Technical Information of China (English)

    游振辉; 陈刚; 张保根


    目的 探讨钙敏感受体在甲状腺乳头状癌、甲状腺良性肿瘤、正常甲状腺组织中的表达特点,分析其与甲状腺乳头状癌的的关系.方法 甲状腺手术后标本70例(甲状腺乳头状癌组织40例、甲状腺良性肿瘤20例、甲状腺正常组织10例),应用MaxvisionTM2/HRP免疫组化二步法染色对甲状腺乳头状癌组织、甲状腺良性肿瘤及甲状腺正常组织的钙敏感受体进行检测.所有资料均采用SPSS 17.0进行统计分析,采用非参数检验,P<0.05为差异有统计学意义.结果 钙敏感受体在甲状腺乳头状癌与甲状腺良性肿瘤、甲状腺正常组织间的表达差异有统计学意义(分别为90%,40%,0%;P<0.05).甲状腺乳头状癌钙化组中钙敏感受体的阳性表达高于甲状腺癌无钙化组,且高于甲状腺良性肿瘤钙化组,差异有统计学意义(P<0.05).结论 钙敏感受体与甲状腺乳头状癌有着密切的关系.钙敏感受体可能在甲状腺乳头状癌组织的钙化起到重要作用.%Objective To explore the relationship between calcium-sensing receptor's expression and papillary thyroid carcinoma.Methods Seventy cases of postoperative papillary thyroid carcinoma were selected.Immunohistochemical technique was used to detect expression of calcium-sensing receptor in papillary thyroid carcinoma,thyroid benign lesions,and normal thyroid tissue,respectively.SPSS 17.0 statistical analysis was used with non parametric test,P<0.05 indicated significant difference.Results The expression of calcium-sensing receptor in papillary thyroid carcinoma,benign thyroid,and normal thyroid were significantly different (90%,40%,0%,respectively; P < 0.05).The expression of the calcium-sensing receptor in the group of papillary thyroid carcinoma with calcification was significantly higher than that in thyroid cancer without calcification (P < 0.05).Conclusions This study suggests that the calcium-sensing receptor may be associated

  12. Potential roles for calcium-sensing receptor (CaSR) and transient receptor potential ankyrin-1 (TRPA1) in murine anorectic response to deoxynivalenol (vomitoxin). (United States)

    Wu, Wenda; Zhou, Hui-Ren; Pestka, James J


    Food contamination by the trichothecene mycotoxin deoxynivalenol (DON, vomitoxin) has the potential to adversely affect animal and human health by suppressing food intake and impairing growth. In mice, the DON-induced anorectic response results from aberrant satiety hormone secretion by enteroendocrine cells (EECs) of the gastrointestinal tract. Recent in vitro studies in the murine STC-1 EEC model have linked DON-induced satiety hormone secretion to activation of calcium-sensing receptor (CaSR), a G-coupled protein receptor, and transient receptor potential ankyrin-1 (TRPA1), a TRP channel. However, it is unknown whether similar mechanisms mediate DON's anorectic effects in vivo. Here, we tested the hypothesis that DON-induced food refusal and satiety hormone release in the mouse are linked to activation of CaSR and TRPA1. Oral treatment with selective agonists for CaSR (R-568) or TRPA1 (allyl isothiocyanate (AITC)) suppressed food intake in mice, and the agonist's effects were suppressed by pretreatment with corresponding antagonists NPS-2143 or ruthenium red (RR), respectively. Importantly, NPS-2143 or RR inhibited both DON-induced food refusal and plasma elevations of the satiety hormones cholecystokinin (CCK) and peptide YY3-36 (PYY3-36); cotreatment with both antagonists additively suppressed both anorectic and hormone responses to DON. Taken together, these in vivo data along with prior in vitro findings support the contention that activation of CaSR and TRPA1 contributes to DON-induced food refusal by mediating satiety hormone exocytosis from EEC.

  13. Role of receptor activity modifying protein 1 in function of the calcium sensing receptor in the human TT thyroid carcinoma cell line.

    Directory of Open Access Journals (Sweden)

    Aditya J Desai

    Full Text Available The Calcium Sensing Receptor (CaSR plays a role in calcium homeostasis by sensing minute changes in serum Ca(2+ and modulating secretion of calciotropic hormones. It has been shown in transfected cells that accessory proteins known as Receptor Activity Modifying Proteins (RAMPs, specifically RAMPs 1 and 3, are required for cell-surface trafficking of the CaSR. These effects have only been demonstrated in transfected cells, so their physiological relevance is unclear. Here we explored CaSR/RAMP interactions in detail, and showed that in thyroid human carcinoma cells, RAMP1 is required for trafficking of the CaSR. Furthermore, we show that normal RAMP1 function is required for intracellular responses to ligands. Specifically, to confirm earlier studies with tagged constructs, and to provide the additional benefit of quantitative stoichiometric analysis, we used fluorescence resonance energy transfer to show equal abilities of RAMP1 and 3 to chaperone CaSR to the cell surface, though RAMP3 interacted more efficiently with the receptor. Furthermore, a higher fraction of RAMP3 than RAMP1 was observed in CaSR-complexes on the cell-surface, suggesting different ratios of RAMPs to CaSR. In order to determine relevance of these findings in an endogenous expression system we assessed the effect of RAMP1 siRNA knock-down in medullary thyroid carcinoma TT cells, (which express RAMP1, but not RAMP3 constitutively and measured a significant 50% attenuation of signalling in response to CaSR ligands Cinacalcet and neomycin. Blockade of RAMP1 using specific antibodies induced a concentration-dependent reduction in CaSR-mediated signalling in response to Cinacalcet in TT cells, suggesting a novel functional role for RAMP1 in regulation of CaSR signalling in addition to its known role in receptor trafficking. These data provide evidence that RAMPs traffic the CaSR as higher-level oligomers and play a role in CaSR signalling even after cell surface localisation has

  14. Analysis of α-Klotho, Fibroblast Growth Factor-, Vitamin-D and Calcium-Sensing Receptor in 70 Patients with Secondary Hyperparathyroidism

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    Joerg Latus


    Full Text Available Background/Aims: Secondary hyperparathyroidism (sHPT is known as a very common complication in patients with chronic kidney disease, and G-protein-coupled calcium-sensing receptor (CaSR, Vitamin D receptor (VDR and Fibroblast growth factor receptor (FGFR/Klotho complexes seem to be involved in its development. Methods: Hyperplastic parathyroid glands from 70 sHPT patients and normal parathyroid tissue from 7 patients were obtained during parathyroidectomy. Conventional morphological and immunohistochemical analysis of parathyroid glands was performed after dividing each slide in a 3x3 array. Results: The presence of lipocytes in the normal parathyroid gland and tissue architecture (nodal in patients with sHPT allows for discrimination between normal parathyroid glands and parathyroid glands of patients with sHPT. Protein expression of Klotho, FGFR, CaSR and VDR was higher in the normal parathyroid glands compared to the sHPT group (p0.05. Conclusions: CaSR, VDR and an impaired Klotho-FGFR-axis seem to be the major players in the development of sHPT. Whether the detected correlation between FGFR and VDR and the shift to a more mixed nuclear/cytoplasmic staining of VDR will yield new insights into the pathogenesis of the disease has to be evaluated in further studies.

  15. Functional importance of the Ala(116)-Pro(136) region in the calcium-sensing receptor. Constitutive activity and inverse agonism in a family C G-protein-coupled receptor

    DEFF Research Database (Denmark)

    Jensen, Anders A.; Spalding, T A; Burstein, E S


    The calcium-sensing receptor (CaR) belongs to family C of the G-protein-coupled receptor superfamily. To date 14 activating mutations in CaR showing increased sensitivity to Ca(2+) have been identified in humans with autosomal dominant hypocalcemia. Four of these activating mutations are found......, suppressed the elevated basal response of the constitutively activated Ca/1a mutants demonstrating inverse agonist activity of CPCCOEt. Taken together, our results demonstrate that the Ala(116)-Pro(136) region is of key importance for the maintenance of the inactive conformation of CaR....

  16. Review article: loss of the calcium-sensing receptor in colonic epithelium is a key event in the pathogenesis of colon cancer.

    LENUS (Irish Health Repository)

    Rogers, Ailín C


    The calcium-sensing receptor (CaSR) is expressed abundantly in normal colonic epithelium and lost in colon cancer, but its exact role on a molecular level and within the carcinogenesis pathway is yet to be described. Epidemiologic studies show that inadequate dietary calcium predisposes to colon cancer; this may be due to the ability of calcium to bind and upregulate the CaSR. Loss of CaSR expression does not seem to be an early event in carcinogenesis; indeed it is associated with late stage, poorly differentiated, chemo-resistant tumors. Induction of CaSR expression in neoplastic colonocytes arrests tumor progression and deems tumors more sensitive to chemotherapy; hence CaSR may be an important target in colon cancer treatment. The CaSR has a complex role in colon cancer; however, more investigation is required on a molecular level to clarify its exact function in carcinogenesis. This review describes the mechanisms by which the CaSR is currently implicated in colon cancer and identifies areas where further study is needed.

  17. JTT-305, an orally active calcium-sensing receptor antagonist, stimulates transient parathyroid hormone release and bone formation in ovariectomized rats. (United States)

    Kimura, Shuichi; Nakagawa, Takashi; Matsuo, Yushi; Ishida, Yuji; Okamoto, Yoshihisa; Hayashi, Mikio


    Intermittent administration of parathyroid hormone (PTH) has a potent anabolic effect on bone in humans and animals. Calcium-sensing receptor (CaSR) antagonists stimulate endogenous PTH secretion through CaSR on the surface of parathyroid cells and thereby may be anabolic agents for osteoporosis. JTT-305 is a potent oral short-acting CaSR antagonist and transiently stimulates endogenous PTH secretion. The objective of the present study was to investigate the effects of JTT-305 on PTH secretion and bone in ovariectomized rats. Female rats, immediately after ovariectomy (OVX), were orally administered vehicle or JTT-305 (0.3, 1, or 3 mg/kg) for 12 weeks. The serum PTH concentrations were transiently elevated with increasing doses of JTT-305. In the proximal tibia, JTT-305 prevented OVX-induced decreases in both the cancellous and total bone mineral density (BMD) except for the 0.3mg/kg dose. At the 3mg/kg dose, JTT-305 increased the mineralizing surface and bone formation rate in histomorphometry. The efficacy of JTT-305 at the 3mg/kg dose on the BMD corresponded to that of exogenous rat PTH1-84 injection at doses between 3 and 10 μg/kg. In conclusion, JTT-305 stimulated endogenous transient PTH secretion and bone formation, and consequently prevented bone loss in OVX rats. These results suggest that JTT-305 is orally active and has the potential to be an anabolic agent for the treatment of osteoporosis.

  18. The Intron 4 Polymorphism in the Calcium-Sensing Receptor Gene in Diabetes Mellitus and its Chronic Complications, Diabetic Nephropathy and Non-Diabetic Renal Disease

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    Viera Železníková


    Full Text Available Background/Aims: Calcium-Sensing Receptor (CaSR significantly affects calcium-phosphate metabolism in kidneys, and it is implicated in the pathogenesis of diabetes mellitus (DM due to its expression in pancreatic F-cells. The role of CaSR as one of the players in pathogenesis of chronic kidney disease (CKD has been speculated. Methods: 158 Type 2 diabetic patients divided into three groups according to occurrence and type of kidney complications, 66 nondiabetic patients CKD, and 93 healthy subjects were enrolled into the study to analyze the role of two CaSR polymorphisms (in the codon 990 and in the intron 4 in ethiopathogenesis of DM and CKD. The Type 2 diabetic groups consisted of 48 patients without any kidney abnormalities, 58 patients with diabetic nephropathy (DN, and 52 patients with nondiabetic renal disease (NDRD. The distribution of genotype and allele frequencies was studied using PCR with the TaqMan Discrimination Assay or followed by the Restriction Fragment Length Polymorphism method, respectively. Results: We have found that the intron 4 polymorphism is a risk factor for the development of DM and CKD, except DN, while the codon 990 does not show any disease association. Conclusion: We conclude that CaSR is a general factor in pancreas and kidney pathologies. i 2014 S. Karger AG, Basel

  19. Genome-wide meta-analysis for serum calcium identifies significantly associated SNPs near the calcium-sensing receptor (CASR gene.

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    Karen Kapur


    Full Text Available Calcium has a pivotal role in biological functions, and serum calcium levels have been associated with numerous disorders of bone and mineral metabolism, as well as with cardiovascular mortality. Here we report results from a genome-wide association study of serum calcium, integrating data from four independent cohorts including a total of 12,865 individuals of European and Indian Asian descent. Our meta-analysis shows that serum calcium is associated with SNPs in or near the calcium-sensing receptor (CASR gene on 3q13. The top hit with a p-value of 6.3 x 10(-37 is rs1801725, a missense variant, explaining 1.26% of the variance in serum calcium. This SNP had the strongest association in individuals of European descent, while for individuals of Indian Asian descent the top hit was rs17251221 (p = 1.1 x 10(-21, a SNP in strong linkage disequilibrium with rs1801725. The strongest locus in CASR was shown to replicate in an independent Icelandic cohort of 4,126 individuals (p = 1.02 x 10(-4. This genome-wide meta-analysis shows that common CASR variants modulate serum calcium levels in the adult general population, which confirms previous results in some candidate gene studies of the CASR locus. This study highlights the key role of CASR in calcium regulation.

  20. The extracellular calcium-sensing receptor is required for cholecystokinin secretion in response to L-phenylalanine in acutely isolated intestinal I cells. (United States)

    Liou, Alice P; Sei, Yoshitatsu; Zhao, Xilin; Feng, Jianying; Lu, Xinping; Thomas, Craig; Pechhold, Susanne; Raybould, Helen E; Wank, Stephen A


    The extracellular calcium-sensing receptor (CaSR) has recently been recognized as an L-amino acid sensor and has been implicated in mediating cholecystokinin (CCK) secretion in response to aromatic amino acids. We investigated whether direct detection of L-phenylalanine (L-Phe) by CaSR results in CCK secretion in the native I cell. Fluorescence-activated cell sorting of duodenal I cells from CCK-enhanced green fluorescent protein (eGFP) transgenic mice demonstrated CaSR gene expression. Immunostaining of fixed and fresh duodenal tissue sections confirmed CaSR protein expression. Intracellular calcium fluxes were CaSR dependent, stereoselective for L-Phe over D-Phe, and responsive to type II calcimimetic cinacalcet in CCK-eGFP cells. Additionally, CCK secretion by an isolated I cell population was increased by 30 and 62% in response to L-Phe in the presence of physiological (1.26 mM) and superphysiological (2.5 mM) extracellular calcium concentrations, respectively. While the deletion of CaSR from CCK-eGFP cells did not affect basal CCK secretion, the effect of L-Phe or cinacalcet on intracellular calcium flux was lost. In fact, both secretagogues, as well as superphysiological Ca(2+), evoked an unexpected 20-30% decrease in CCK secretion compared with basal secretion in CaSR(-/-) CCK-eGFP cells. CCK secretion in response to KCl or tryptone was unaffected by the absence of CaSR. The present data suggest that CaSR is required for hormone secretion in the specific response to L-Phe by the native I cell, and that a receptor-mediated mechanism may inhibit hormone secretion in the absence of a fully functional CaSR.

  1. Extracellular Ca2+ is a danger signal activating the NLRP3 inflammasome through G protein-coupled calcium sensing receptors

    DEFF Research Database (Denmark)

    Rossol, Manuela; Pierer, Matthias; Raulien, Nora;


    Activation of the NLRP3 inflammasome enables monocytes and macrophages to release high levels of interleukin-1ß during inflammatory responses. Concentrations of extracellular calcium can increase at sites of infection, inflammation or cell activation. Here we show that increased extracellular cal......, and this effect was inhibited in GPRC6A(-/-) mice. Our results demonstrate that G-protein-coupled receptors can activate the inflammasome, and indicate that increased extracellular calcium has a role as a danger signal and amplifier of inflammation....

  2. Disorders of the calcium-sensing receptor and partner proteins: insights into the molecular basis of calcium homeostasis (United States)

    Hannan, Fadil M; Babinsky, Valerie N


    The extracellular calcium (Ca2+o)-sensing receptor (CaSR) is a family C G protein-coupled receptor, which detects alterations in Ca2+o concentrations and modulates parathyroid hormone secretion and urinary calcium excretion. The central role of the CaSR in Ca2+o homeostasis has been highlighted by the identification of mutations affecting the CASR gene on chromosome 3q21.1. Loss-of-function CASR mutations cause familial hypocalciuric hypercalcaemia (FHH), whereas gain-of-function mutations lead to autosomal dominant hypocalcaemia (ADH). However, CASR mutations are only detected in ≤70% of FHH and ADH cases, referred to as FHH type 1 and ADH type 1, respectively, and studies in other FHH and ADH kindreds have revealed these disorders to be genetically heterogeneous. Thus, loss- and gain-of-function mutations of the GNA11 gene on chromosome 19p13.3, which encodes the G-protein α-11 (Gα11) subunit, lead to FHH type 2 and ADH type 2, respectively; whilst loss-of-function mutations of AP2S1 on chromosome 19q13.3, which encodes the adaptor-related protein complex 2 sigma (AP2σ) subunit, cause FHH type 3. These studies have demonstrated Gα11 to be a key mediator of downstream CaSR signal transduction, and also revealed a role for AP2σ, which is involved in clathrin-mediated endocytosis, in CaSR signalling and trafficking. Moreover, FHH type 3 has been demonstrated to represent a more severe FHH variant that may lead to symptomatic hypercalcaemia, low bone mineral density and cognitive dysfunction. In addition, calcimimetic and calcilytic drugs, which are positive and negative CaSR allosteric modulators, respectively, have been shown to be of potential benefit for these FHH and ADH disorders. PMID:27647839

  3. Calcilytic Ameliorates Abnormalities of Mutant Calcium-Sensing Receptor (CaSR) Knock-In Mice Mimicking Autosomal Dominant Hypocalcemia (ADH). (United States)

    Dong, Bingzi; Endo, Itsuro; Ohnishi, Yukiyo; Kondo, Takeshi; Hasegawa, Tomoka; Amizuka, Norio; Kiyonari, Hiroshi; Shioi, Go; Abe, Masahiro; Fukumoto, Seiji; Matsumoto, Toshio


    Activating mutations of calcium-sensing receptor (CaSR) cause autosomal dominant hypocalcemia (ADH). ADH patients develop hypocalcemia, hyperphosphatemia, and hypercalciuria, similar to the clinical features of hypoparathyroidism. The current treatment of ADH is similar to the other forms of hypoparathyroidism, using active vitamin D3 or parathyroid hormone (PTH). However, these treatments aggravate hypercalciuria and renal calcification. Thus, new therapeutic strategies for ADH are needed. Calcilytics are allosteric antagonists of CaSR, and may be effective for the treatment of ADH caused by activating mutations of CaSR. In order to examine the effect of calcilytic JTT-305/MK-5442 on CaSR harboring activating mutations in the extracellular and transmembrane domains in vitro, we first transfected a mutated CaSR gene into HEK cells. JTT-305/MK-5442 suppressed the hypersensitivity to extracellular Ca(2+) of HEK cells transfected with the CaSR gene with activating mutations in the extracellular and transmembrane domains. We then selected two activating mutations locating in the extracellular (C129S) and transmembrane (A843E) domains, and generated two strains of CaSR knock-in mice to build an ADH mouse model. Both mutant mice mimicked almost all the clinical features of human ADH. JTT-305/MK-5442 treatment in vivo increased urinary cAMP excretion, improved serum and urinary calcium and phosphate levels by stimulating endogenous PTH secretion, and prevented renal calcification. In contrast, PTH(1-34) treatment normalized serum calcium and phosphate but could not reduce hypercalciuria or renal calcification. CaSR knock-in mice exhibited low bone turnover due to the deficiency of PTH, and JTT-305/MK-5442 as well as PTH(1-34) increased bone turnover and bone mineral density (BMD) in these mice. These results demonstrate that calcilytics can reverse almost all the phenotypes of ADH including hypercalciuria and renal calcification, and suggest that calcilytics can become a

  4. Association between calcium sensing receptor gene polymorphisms and chronic pancreatitis in a US population: Role of serine protease inhibitor Kazal 1type and alcohol

    Institute of Scientific and Technical Information of China (English)

    Venkata Muddana; David C Whitcomb; Janette Lamb; Julia B Greer; Beth Elinoff; Robert H Hawes; Peter B cotton; Michelle A Anderson; Randall E Brand; Adam Slivka


    AIM: To test the hypothesis that calcium sensing receptor (CASR) polymorphisms are associated with chronic pancreatitis (CP), and to determine whether serine protease inhibitor Kazal 1type (SPfNK1) N34S or alcohol are necessary co-factors in its etiology.METHODS: Initially, 115 subjects with pancreatitis and 66 controls were evaluated, of whom 57 patients and 21 controls were predetermined to carry the high-risk SP/NK1 N34S polymorphism. We sequenced CASR gene exons 2, 3, 4, 5 and 7, areas containing the majority of reported polymorphisms and novel mutations. Based on the initial results, we added 223 patients and 239 controls to analyze three common nonsynonymous single nucleotide polymorphisrns (SNPs) in exon 7 (A986S, R990G, and Q1011E).RESULTS: The CASR exon 7 R990G polymorphism was significantly associated with CP (OR, 2.01; 95% CI, 1.12-3.59; P = 0.015). The association between CASR R990G and CP was stronger in subjects who reported moderate or heavy alcohol consumption (OR,3.12; 95% CI, 1.14-9.13; P = 0.018). There was no association between the various CASR genotypes and SPINK1 N34S in pancreatitis. None of the novel CASR polymorphisms reported from Germany and India was detected.CONCLUSION: Our United States-based study confirmed an association of CASR and CP and for the first time demonstrated that CASR R990G is a significant risk factor for CP. We also conclude that the risk of CP with CASR R990G is increased in subjects with moderate to heavy alcohol consumption.

  5. Elevation of extracellular Ca2+ induces store-operated calcium entry via calcium-sensing receptors: a pathway contributes to the proliferation of osteoblasts.

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    Fen Hu

    Full Text Available AIMS: The local concentration of extracellular Ca(2+ ([Ca(2+]o in bone microenvironment is accumulated during bone remodeling. In the present study we investigated whether elevating [Ca(2+]o induced store-operated calcium entry (SOCE in primary rat calvarial osteoblasts and further examined the contribution of elevating [Ca(2+]o to osteoblastic proliferation. METHODS: Cytosolic Ca(2+ concentration ([Ca(2+]c of primary cultured rat osteoblasts was detected by fluorescence imaging using calcium-sensitive probe fura-2/AM. Osteoblastic proliferation was estimated by cell counting, MTS assay and ATP assay. Agonists and antagonists of calcium-sensing receptors (CaSR as well as inhibitors of phospholipase C (PLC, SOCE and voltage-gated calcium (Cav channels were applied to study the mechanism in detail. RESULTS: Our data showed that elevating [Ca(2+]o evoked a sustained increase of [Ca(2+]c in a dose-dependent manner. This [Ca(2+]c increase was blocked by TMB-8 (Ca(2+ release inhibitor, 2-APB and BTP-2 (both SOCE blockers, respectively, whereas not affected by Cav channels blockers nifedipine and verapamil. Furthermore, NPS2143 (a CaSR antagonist or U73122 (a PLC inhibitor strongly reduced the [Ca(2+]o-induced [Ca(2+]c increase. The similar responses were observed when cells were stimulated with CaSR agonist spermine. These data indicated that elevating [Ca(2+]o resulted in SOCE depending on the activation of CaSR and PLC in osteoblasts. In addition, high [Ca(2+]o significantly promoted osteoblastic proliferation, which was notably reversed by BAPTA-AM (an intracellular calcium chelator, 2-APB, BTP-2, TMB-8, NPS2143 and U73122, respectively, but not affected by Cav channels antagonists. CONCLUSIONS: Elevating [Ca(2+]o induced SOCE by triggering the activation of CaSR and PLC. This process was involved in osteoblastic proliferation induced by high level of extracellular Ca(2+ concentration.

  6. Enhanced Proliferation of Porcine Bone Marrow Mesenchymal Stem Cells Induced by Extracellular Calcium is Associated with the Activation of the Calcium-Sensing Receptor and ERK Signaling Pathway

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    Jingjing Ye


    Full Text Available Porcine bone marrow mesenchymal stem cells (pBMSCs have the potential for application in regenerative medicine. This study aims to investigate the effects of extracellular calcium (Ca2+o on pBMSCs proliferation and to explore the possible underlying mechanisms. The results demonstrated that 4 mM Ca2+o significantly promoted pBMSCs proliferation by reducing the G0/G1 phase cell percentage and by increasing the S phase cell proportion and the proliferation index of pBMSCs. Accordingly, Ca2+o stimulated the expression levels of proliferative genes such as cyclin A2, cyclin D1/3, cyclin E2, and PCNA and inhibited the expression of p21. In addition, Ca2+o resulted in a significant elevation of intracellular calcium and an increased ratio of p-ERK/ERK. However, inhibition of calcium-sensing receptor (CaSR by its antagonist NPS2143 abolished the aforementioned effects of Ca2+o. Moreover, Ca2+o-induced promotion of pBMSCs proliferation, the changes of proliferative genes expression levels, and the activation of ERK1/2 signaling pathway were effectively blocked by U0126, a selective ERK kinase inhibitor. In conclusion, our findings provided evidence that the enhanced pBMSCs proliferation in response to Ca2+o was associated with the activation of CaSR and ERK1/2 signaling pathway, which may be useful for the application of pBMSCs in future clinical studies aimed at tissue regeneration and repair.

  7. Induction of calcium sensing receptor in human colon cancer cells by calcium, vitamin D and aquamin: Promotion of a more differentiated, less malignant and indolent phenotype. (United States)

    Singh, Navneet; Aslam, Muhammad N; Varani, James; Chakrabarty, Subhas


    The calcium sensing receptor (CaSR) is a robust promoter of differentiation in colonic epithelial cells and functions as a tumor suppressor. Cancer cells that do not express CaSR (termed CaSR null) are highly malignant while acquisition of CaSR expression in these cells circumvents the malignant phenotype. We hypothesize that chemopreventive agents mediate their action through the induction of CaSR. Here, we compare the effectiveness of Ca(2+), vitamin D, and Aquamin (a marine algae product containing Ca(2+), magnesium and detectable levels of 72 additional minerals) on the induction of CaSR in the CBS and HCT116 human colon carcinoma cell lines and the corresponding CaSR null cells isolated from these lines. All three agonists induced CaSR mRNA and protein expression and inhibited cellular proliferation in the parental and CaSR null cells. Aquamin was found to be most potent in this regard. Induction of CaSR expression by these agonists resulted in demethylation of the CaSR gene promoter with a concurrent increase in CaSR promoter reporter activity. However, demethylation per se did not induce CaSR transcription. Induction of CaSR expression resulted in a down-regulated expression of tumor inducers and up-regulated expression of tumor suppressors. Again, Aquamin was found to be most potent in these biologic effects. This study provides a rationale for the use of a multi-mineral approach in the chemoprevention of colon cancer and suggests that induction of CaSR may be a measure of the effectiveness of chemopreventive agents.

  8. Calcium and calcium sensing receptor modulates the expression of thymidylate synthase, NAD(P)H:quinone oxidoreductase 1 and survivin in human colon carcinoma cells: promotion of cytotoxic response to mitomycin C and fluorouracil


    Liu, Guangming; Hu, Xin; Varani, James; Chakrabarty, Subhas


    Ca2+ and the cell-surface calcium sensing receptor (CaSR) constitute a novel and robust ligand/receptor system in regulating the proliferation and differentiation of colonic epithelial cells. Here we show that activation of CaSR by extracellular Ca2+ (or CaSR agonists) enhanced the sensitivity of human colon carcinoma cells to mitomycin C (MMC) and fluorouracil (5-FU). Activation of CaSR up-regulated the expression of MMC activating enzyme, NAD(P)H:quinone oxidoreductase 1 (NQO-1) and down-re...

  9. Post-conditioning protects cardiomyocytes from apoptosis via PKC(epsilon)-interacting with calcium-sensing receptors to inhibit endo(sarco)plasmic reticulum-mitochondria crosstalk. (United States)

    Dong, Shiyun; Teng, Zongyan; Lu, Fang-Hao; Zhao, Ya-Jun; Li, Hulun; Ren, Huan; Chen, He; Pan, Zhen-Wei; Lv, Yan-Jie; Yang, Bao-Feng; Tian, Ye; Xu, Chang-Qing; Zhang, Wei-Hua


    The intracellular Ca(2+) concentration ([Ca(2+)](i)) is increased during cardiac ischemia/reperfusion injury (IRI), leading to endo(sarco)plasmic reticulum (ER) stress. Persistent ER stress, such as with the accumulation of [Ca(2+)](i), results in apoptosis. Ischemic post-conditioning (PC) can protect cardiomyocytes from IRI by reducing the [Ca(2+)](i) via protein kinase C (PKC). The calcium-sensing receptor (CaR), a G protein-coupled receptor, causes the production of inositol phosphate (IP(3)) to increase the release of intracellular Ca(2+) from the ER. This process can be negatively regulated by PKC through the phosphorylation of Thr-888 of the CaR. This study tested the hypothesis that PC prevents cardiomyocyte apoptosis by reducing the [Ca(2+)](i) through an interaction of PKC with CaR to alleviate [Ca(2+)](ER) depletion and [Ca(2+)](m) elevation by the ER-mitochondrial associated membrane (MAM). Cardiomyocytes were post-conditioned after 3 h of ischemia by three cycles of 5 min of reperfusion and 5 min of re-ischemia before 6 h of reperfusion. During PC, PKC(epsilon) translocated to the cell membrane and interacted with CaR. While PC led to a significant decrease in [Ca(2+)](i), the [Ca(2+)](ER) was not reduced and [Ca(2+)](m) was not increased in the PC and GdCl(3)-PC groups. Furthermore, there was no evident psi(m) collapse during PC compared with ischemia/reperfusion (I/R) or PKC inhibitor groups, as evaluated by laser confocal scanning microscopy. The apoptotic rates detected by TUNEL and Hoechst33342 were lower in PC and GdCl(3)-PC groups than those in I/R and PKC inhibitor groups. Apoptotic proteins, including m-calpain, BAP31, and caspase-12, were significantly increased in the I/R and PKC inhibitor groups. These results suggested that PKC(epsilon) interacting with CaR protected post-conditioned cardiomyocytes from programmed cell death by inhibiting disruption of the mitochondria by the ER as well as preventing calcium-induced signaling of the

  10. Calcium-sensing receptor stimulates Cl(-)- and SCFA-dependent but inhibits cAMP-dependent HCO3(-) secretion in colon. (United States)

    Tang, Lieqi; Peng, Minzhi; Liu, Li; Chang, Wenhan; Binder, Henry J; Cheng, Sam X


    Colonic bicarbonate (HCO3(-)) secretion is a well-established physiological process that is closely linked to overall fluid and electrolyte movement in the mammalian colon. These present studies show that extracellular calcium-sensing receptor (CaSR), a fundamental mechanism for sensing and regulating ionic and nutrient compositions of extracellular milieu in the small and large intestine, regulates HCO3(-) secretion. Basal and induced HCO3(-) secretory responses to CaSR agonists were determined by pH stat techniques used in conjunction with short-circuit current measurements in mucosa from rat distal colon mounted in Ussing chambers. R568, a specific CaSR activator, stimulated lumen Cl(-)- and short-chain fatty acid (SCFA)-dependent HCO3(-) secretion but inhibited cyclic nucleotide-activated HCO3(-) secretion. Consequently, at physiological conditions (either at basal or during lumen acid challenge) when electroneutral Cl(-)/HCO3(-) and SCFA/HCO3(-) exchangers dominate, CaSR stimulates HCO3(-) secretion; in contrast, in experimental conditions that stimulate fluid and HCO3(-) secretion, e.g., when forskolin activates electrogenic cystic fibrosis transmembrane conductance regulator-mediated HCO3(-) conductance, CaSR activation inhibits HCO3(-) secretion. Corresponding changes in JHCO3 (μeq·h(-1)·cm(-2), absence vs. presence of R568) were 0.18 ± 0.03 vs. 0.31 ± 0.08 under basal nonstimulated conditions and 1.85 ± 0.23 vs. 0.45 ± 0.06 under forskolin-stimulated conditions. Similarly, activation of CaSR by R568 stimulated Cl(-)- and SCFA-dependent HCO3(-) secretion and inhibited cAMP-dependent HCO3(-) secretion in colon mucosa of wild-type mice; such effects were abolished in CaSR-null mice. These results suggest a new paradigm for regulation of intestinal ion transport in which HCO3(-) secretion may be fine-tuned by CaSR in accordance with nutrient availability and state of digestion and absorption. The ability of CaSR agonists to inhibit secretagogue

  11. The regulation of K- and L-cell activity by GLUT2 and the calcium-sensing receptor CasR in rat small intestine. (United States)

    Mace, Oliver J; Schindler, Marcus; Patel, Sonal


    Intestinal enteroendocrine cells (IECs) secrete gut peptides in response to both nutrients and non-nutrients. Glucose and amino acids both stimulate gut peptide secretion. Our hypothesis was that the facilitative glucose transporter, GLUT2, could act as a glucose sensor and the calcium-sensing receptor, CasR, could detect amino acids in the intestine to modify gut peptide secretion. We used isolated loops of rat small intestine to study the secretion of gluco-insulinotropic peptide (GIP), glucagon-like peptide-1 (GLP-1) and peptide tyrosine tyrosine (PYY) secretion stimulated by luminal perfusion of nutrients or bile acid. Inhibition of the sodium-dependent glucose cotransporter 1 (SGLT1) with phloridzin partially inhibited GIP, GLP-1 and PYY secretion by 45%, suggesting another glucose sensor might be involved in modulating peptide secretion. The response was completely abolished in the presence of the GLUT2 inhibitors phloretin or cytochalasin B. Given that GLUT2 modified gut peptide secretion stimulated by glucose, we investigated whether it was involved in the secretion of gut peptide by other gut peptide secretagogues. Phloretin completely abolished gut peptide secretion stimulated by artificial sweetener (sucralose), dipeptide (glycylsarcosine), lipid (oleoylethanolamine), short chain fatty acid (propionate) and major rat bile acid (taurocholate) indicating a fundamental position for GLUT2 in the gut peptide secretory mechanism. We investigated how GLUT2 was able to influence gut peptide secretion mediated by a diverse range of stimulators and discovered that GLUT2 affected membrane depolarisation through the closure of K+(ATP)-sensitive channels. In the absence of SGLT1 activity (or presence of phloridzin), the secretion of GIP, GLP-1 and PYY was sensitive to K+(ATP)-sensitive channel modulators tolbutamide and diazoxide. L-amino acids phenylalanine (Phe), tryptophan (Trp), asparagine (Asn), arginine (Arg) and glutamine (Gln) also stimulated GIP, GLP-1 and

  12. Active vitamin D potentiates the anti-neoplastic effects of calcium in the colon: A cross talk through the calcium-sensing receptor. (United States)

    Aggarwal, Abhishek; Höbaus, Julia; Tennakoon, Samawansha; Prinz-Wohlgenannt, Maximilian; Graça, João; Price, Sally A; Heffeter, Petra; Berger, Walter; Baumgartner-Parzer, Sabina; Kállay, Enikö


    Epidemiological studies suggest an inverse correlation between dietary calcium (Ca(2+)) and vitamin D intake and the risk of colorectal cancer (CRC). It has been shown in vitro that the active vitamin D metabolite, 1,25-dihydroxyvitamin D3 (1,25-D3) can upregulate expression of the calcium-sensing receptor (CaSR). In the colon, CaSR has been suggested to regulate proliferation of colonocytes. However, during tumorigenesis colonic CaSR expression is downregulated and we hypothesized that the loss of CaSR could influence the anti-tumorigenic effects of Ca(2+) and vitamin D. Our aim was to assess the impact of CaSR expression and function on the anti-neoplastic effects of 1,25-D3 in colon cancer cell lines. We demonstrated that in the healthy colon of mice, high vitamin D diet (2500 IU/kg diet) increased expression of differentiation and apoptosis markers, decreased expression of proliferation markers and significantly upregulated CaSR mRNA expression, compared with low vitamin D diet (100 IU/kg diet). To determine the role of CaSR in this process, we transfected Caco2-15 and HT29 CRC cells with wild type CaSR (CaSR-WT) or a dominant negative CaSR mutant (CaSR-DN) and treated them with 1,25-D3 alone, or in combination with CaSR activators (Ca(2+) and NPS R-568). 1,25-D3 enhanced the anti-proliferative effects of Ca(2+) and induced differentiation and apoptosis only in cells with a functional CaSR, which were further enhanced in the presence of NPS R-568, a positive allosteric modulator of CaSR. The mutant CaSR inhibited the anti-tumorigenic effects of 1,25-D3 suggesting that the anti-neoplastic effects of 1,25-D3 are, at least in part, mediated by the CaSR. Taken together, our data provides molecular evidence to support the epidemiological observation that both, vitamin D and calcium are needed for protection against malignant transformation of the colon and that their effect is modulated by the presence of a functional CaSR. This article is part of a Special Issue

  13. Role of post-translational modifications on structure, function and pharmacology of class C G protein-coupled receptors

    DEFF Research Database (Denmark)

    Nørskov-Lauritsen, Lenea; Bräuner-Osborne, Hans


    taste receptors (T1R1-3), one calcium-sensing (CaS) receptor, one GPCR, class C, group 6, subtype A (GPRC6) receptor, and seven orphan receptors. G protein-coupled receptors undergo a number of post-translational modifications, which regulate their structure, function and/or pharmacology. Here, we...

  14. Hipercalcemia hipocalciúrica debida a una mutación de novo del gen del receptor sensor de calcio Hypocalciuric hypercalcemia due to de novo mutation of the calcium sensing receptor

    Directory of Open Access Journals (Sweden)

    Marcelo Sarli


    Full Text Available El objetivo de este trabajo es presentar el inusual caso clínico de una paciente de 34 años que consultó para establecer diagnóstico de certeza y conducta terapéutica ante una hipercalcemia asintomática, detectada en un examen bioquímico de rutina. La elevación de la calcemia en ausencia de inhibición de la secreción de parathormona orientó hacia una patología paratiroidea. La persistencia de la hipercalcemia concomitante con hipocalciuria y coincidente con una relación clearance de calcio/clearance de creatinina inferior a 0.01, hicieron sospechar el diagnóstico de hipercalcemia hipocalciúrica familiar. La falta de antecedentes familiares llevó a realizar un estudio molecular de la paciente y su grupo familiar. Los resultados de los estudios nos permitieron concluir que la paciente es portadora de una mutación de novo (inactivante del gen del receptor sensor del calcio. Se incluyen los datos del estudio molecular y una breve revisión bibliográfica del tema.The aim of this paper is to refer the unusual case of a 34 years old woman who consulted because of asymptomatic hypercalcemia, detected in a biochemical routine examination. The elevated values of serum calcium without blunted parathyroid hormone secretion suggested a parathyroid pathology. The concomitance of hypocalciuria with hypercalcemia and a calcium clearance/creatinine clearance ratio less than 0.01 reverted the diagnosis of familial hypocalciuric hypercalcemia, the first option. The absence of familial background led to the molecular study of the patient and her family. The latter confirmed the diagnosis of a de novo inactivating mutation of the calcium sensing receptor. Details on the molecular study and a brief review of this subject are included.

  15. Role of Ca2+ and L-Phe in regulating functional cooperativity of disease-associated "toggle" calcium-sensing receptor mutations.

    Directory of Open Access Journals (Sweden)

    Chen Zhang

    Full Text Available The Ca(2+-sensing receptor (CaSR regulates Ca(2+ homeostasis in the body by monitoring extracellular levels of Ca(2+ ([Ca(2+]o and amino acids. Mutations at the hinge region of the N-terminal Venus flytrap domain (VFTD produce either receptor inactivation (L173P, P221Q or activation (L173F, P221L related to hypercalcemic or hypocalcemic disorders. In this paper, we report that both L173P and P221Q markedly impair the functional positive cooperativity of the CaSR as reflected by [Ca(2+]o-induced [Ca(2+]i oscillations, inositol-1-phosphate (IP1 accumulation and extracellular signal-regulated kinases (ERK1/2 activity. In contrast, L173F and P221L show enhanced responsiveness of these three functional readouts to [Ca(2+]o. Further analysis of the dynamics of the VFTD mutants using computational simulation studies supports disruption in the correlated motions in the loss-of-function CaSR mutants, while these motions are enhanced in the gain-of-function mutants. Wild type (WT CaSR was modulated by L-Phe in a heterotropic positive cooperative way, achieving an EC50 similar to those of the two activating mutations. The response of the inactivating P221Q mutant to [Ca(2+]o was partially rescued by L-Phe, illustrating the capacity of the L-Phe binding site to enhance the positive homotropic cooperativity of CaSR. L-Phe had no effect on the other inactivating mutant. Moreover, our results carried out both in silico and in intact cells indicate that residue Leu(173, which is close to residues that are part of the L-Phe-binding pocket, exhibited impaired heterotropic cooperativity in the presence of L-Phe. Thus, Pro(221 and Leu(173 are important for the positive homo- and heterotropic cooperative regulation elicited by agonist binding.

  16. Reduced Expression of the Extracellular Calcium-Sensing Receptor (CaSR Is Associated with Activation of the Renin-Angiotensin System (RAS to Promote Vascular Remodeling in the Pathogenesis of Essential Hypertension.

    Directory of Open Access Journals (Sweden)

    Yuan-Yuan Qu

    Full Text Available The proliferation of vascular smooth muscle cells (VSMCs, remodeling of the vasculature, and the renin-angiotensin system (RAS play important roles in the development of essential hypertension (EH, which is defined as high blood pressure (BP in which secondary causes, such as renovascular disease, are absent. The calcium-sensing receptor (CaSR is involved in the regulation of BP. However, the underlying mechanisms by which the CaSR regulates BP are poorly understood. In the present study, the role of the CaSR in EH was investigated using male spontaneously hypertensive rats (SHRs and rat and human plasma samples. The percentages of medial wall thickness to external diameter (WT%, total vessel wall cross-sectional area to the total area (WA% of thoracic arteries, as well as the percentage of wall area occupied by collagen to total vessel wall area (CA% were determined. Tissue protein expression and plasma concentrations of the CaSR, cyclic adenosine monophosphate (cAMP, renin, and angiotensin II (Ang II were additionally assessed. WT%, WA%, and CA% were found to increase with increasing BP, whereas the plasma concentration of CaSR was found to decrease. With increasing BP, the levels of smooth muscle actin and calponin decreased, whereas those of osteopontin and proliferating cell nuclear antigen increased. The CaSR level negatively correlated with the levels of cAMP and Ang II, but positively correlated with those of renin. Our data suggest that reduced expression of the CaSR is correlated with activation of the RAS, which induces increased vascular remodeling and VSMC proliferation, and thereby associated with EH in the SHR model and in the Han Chinese population. Our findings provide new insights into the pathogenesis of EH.

  17. Inflammatory cytokine signaling in insulin producing beta-cells enhances the colocalization correlation coefficient between L-type voltage-dependent calcium channel and calcium-sensing receptor. (United States)

    Parkash, Jai


    The immunological processes in type 1 diabetes and metabolic/inflammatory disorder in type 2 diabetes converge on common signaling pathway(s) leading to beta-cell death in these two diseases. The cytokine-mediated beta-cell death seems to be dependent on voltage-dependent calcium channel (VDCC)-mediated Ca2+ entry. The Ca2+ handling molecular networks control the homeostasis of [Ca2+]i in the beta-cell. The activity and membrane density of VDCC are regulated by several mechanisms including G protein-coupled receptors (GPCRs). CaR is a 123-kDa seven transmembrane extracellular Ca2+ sensing protein that belongs to GPCR family C. Tumor necrosis factor-alpha (TNF-alpha), is a cytokine widely known to activate nuclear factor-kappaB (NF-kappaB) transcription in beta-cells. To obtain a better understanding of TNF-alpha-induced molecular interactions between CaR and VDCC, confocal fluorescence measurements were performed on insulin-producing beta-cells exposed to varying concentrations of TNF-alpha and the results are discussed in the light of increased colocalization correlation coefficient. The insulin producing beta-cells were exposed to 5, 10, 20, 30, and 50 ng/ml TNF-alpha for 24 h at 37 degrees . The cells were then immunolabelled with antibodies directed against CaR, VDCC, and NF-kappaB. The confocal fluorescence imaging data showed enhancement in the colocalization correlation coefficient between CaR and VDCC in beta-cells exposed to TNF-alpha thereby indicating increased membrane delimited spatial interactions between these two membrane proteins. TNF-alpha-induced colocalization of VDCC with CaR was inhibited by nimodipine, an inhibitor of L-type VDCC thereby suggesting that VDCC activity is required for spatial interactions with CaR. The 3-D confocal fluorescence imaging data also demonstrated that addition of TNF-alpha to RIN cells led to the translocation of NF-kappaB from the cytoplasm to the nucleus. Such molecular interactions between CaR and VDCC in tissues

  18. Calcium-sensing receptor antagonist (calcilytic) NPS 2143 specifically blocks the increased secretion of endogenous Aβ42 prompted by exogenous fibrillary or soluble Aβ25-35 in human cortical astrocytes and neurons-therapeutic relevance to Alzheimer's disease. (United States)

    Armato, Ubaldo; Chiarini, Anna; Chakravarthy, Balu; Chioffi, Franco; Pacchiana, Raffaella; Colarusso, Enzo; Whitfield, James F; Dal Prà, Ilaria


    The "amyloid-β (Aβ) hypothesis" posits that accumulating Aβ peptides (Aβs) produced by neurons cause Alzheimer's disease (AD). However, the Aβs contribution by the more numerous astrocytes remains undetermined. Previously we showed that fibrillar (f)Aβ25-35, an Aβ42 proxy, evokes a surplus endogenous Aβ42 production/accumulation in cortical adult human astrocytes. Here, by using immunocytochemistry, immunoblotting, enzymatic assays, and highly sensitive sandwich ELISA kits, we investigated the effects of fAβ25-35 and soluble (s)Aβ25-35 on Aβ42 and Aβ40 accumulation/secretion by human cortical astrocytes and HCN-1A neurons and, since the calcium-sensing receptor (CaSR) binds Aβs, their modulation by NPS 2143, a CaSR allosteric antagonist (calcilytic). The fAβ25-35-exposed astrocytes and surviving neurons produced, accumulated, and secreted increased amounts of Aβ42, while Aβ40 also accrued but its secretion was unchanged. Accordingly, secreted Aβ42/Aβ40 ratio values rose for astrocytes and neurons. While slightly enhancing Aβ40 secretion by fAβ25-35-treated astrocytes, NPS 2143 specifically suppressed the fAβ25-35-elicited surges of endogenous Aβ42 secretion by astrocytes and neurons. Therefore, NPS 2143 addition always kept Aβ42/Aβ40 values to baseline or lower levels. Mechanistically, NPS 2143 decreased total CaSR protein complement, transiently raised proteasomal chymotrypsin activity, and blocked excess NO production without affecting the ongoing increases in BACE1/β-secretase and γ-secretase activity in fAβ25-35-treated astrocytes. Compared to fAβ25-35, sAβ25-35 also stimulated Aβ42 secretion by astrocytes and neurons and NPS 2143 specifically and wholly suppressed this effect. Therefore, since NPS 2143 thwarts any Aβ/CaSR-induced surplus secretion of endogenous Aβ42 and hence further vicious cycles of Aβ self-induction/secretion/spreading, calcilytics might effectively prevent/stop the progression to full-blown AD.

  19. Protective mechanism of the interaction between protein kinase C and calcium sensing receptor in jschemiapreconditioning%蛋白激酶C与钙敏感受体在心肌缺血预适应中的保护作用

    Institute of Scientific and Technical Information of China (English)

    杜丽娟; 王艳丽; 孙智睿; 赵雅君; 李全凤; 王丽娜; 张伟华


    Objective To investigate the protective mechanism of protein kinase C(PKC)and calcium sensing receptor(CaR)in ischemia preconditioned rat hearts.Methods Using cell culture method,in vitro cultured inhibitor(IPC+CaRI).Apoptosis was detected using TUNEL and Hoechst33342 cell viability was detected by MTT,the protein expression of easpase-12,calpain and CaR in endochylema were detected using Wedtetm blot.ResultsIn I/R group nucleus was shrank,big blue,chromatin concentrated,apoptotle body appeared.Other groups haddifferent fluorescence intensity varying degree,IPC+PKCI+CaRS group had more big blue nucleus.Myocardialcell viability and apoptotic rate,I/R group[(62.99±0.65)%,(19.13±0.87)%],IPC group[(78.67±0.37)%,(14.21±0.74)%],IPC+PKCI group[(71.09±0.52)%,(20.46±0.81)%],IPC+PKCI+CaRS group(66.10±0.75)%,(24.89±1.43)%],IPC+CaRS group[(69.56±0.44)%,(21.64±0.77)%],IPC+CaRI group(85.81±0.60)%,(13.12±0.69)%],all had a difference(P<0.05 or<0.01)compared with C group[(100.00)%,(6.02±0. 31)%].Western blot identified that CaR expression in IPC+PKCI and IPC+CaRS,IPC+PKCI+CaRS groupswas more than that in IPC and IPC+CaRI groups;easpase-12 had more active fragment(60×103)in I/R,IPC+CaRS,IPC+PKCI+CaRS groups;ealpain expressions in I/R,IPC,IPC+PKCI,IPC+PKCI+CaRS,IPC+CaRSgroups were higher than those in C and IPC+CaRI,I/R group was the highest one,C group the second,IPC+CaRI the third.Conclusion The interaction of PKC and CaR can reduce the intracellular Ca2+ from sarcoplasmicreticulum thus provide a protection.%目的 探讨蛋白激酶C(PKC)与钙敏感受体(CaR),在心肌缺血预适应(IPC)中的保护作用.方法 采用细胞培养方法 ,体外培养大鼠乳鼠心肌细胞,模拟缺血预适应模型,实验分为7组:①正常对照组(C),②缺血再灌注组(1/R),③IPC组,④IPC+PKC抑制剂组(IPC+PKCI),⑤IPC+PKCI+CaR激动剂组(IPC+PKCI+CARS),⑥IPC+CaRS组,⑦IPC+CaR抑制剂组(IPC+CaRI).分别用TUNEL,Hoechst 33342染色法检测细胞凋亡,四甲

  20. 钙敏感受体通过 G 蛋白-PLC-IP3信号途径调节肺动脉张力%Calcium-sensing receptor modulates pulmonary artery tension through G-protein-PLC-IP3 pathways

    Institute of Scientific and Technical Information of China (English)

    李光伟; 苗宏志; 李波; 王国忠; 金莉; 林岩; 邓志会; 肖微


    AIM:To observe the role of calcium-sensing receptor (CaSR) in the regulation of pulmonary artery tension.METHODS:The intracellular calcium concentration ([Ca2+]i) was detected by laser-scanning confocal micros-copy, and the pulmonary artery tension was determined by the pulmonary arterial ring technique .RESULTS: Increased levels of [Ca2+]o or Gd3+(an agonist of CaSR) induced the increase in [Ca2+]i and pulmonary artery constriction in a concentration-dependent manner.Additionally, the effects of Ca2+and Gd3+were inhibited by U73122 and D609 (specific inhibitor of PLC), and 2-APB and heparin (specific antagonist of IP3 receptor).However, U73343 (U73122 inactive ana-logue) did not take effect.CONCLUSION: CaSR may be involved in the regulation of pulmonary artery tension by in-creasing [Ca2+]i through G-protein-PLC-IP3 pathway.%目的:探讨钙敏感受体(calcium-sensing receptor,CaSR)在肺血管张力调节中的作用及信号途径。方法:采用Ⅱ型胶原酶消化法提取大鼠肺动脉平滑肌细胞(pulmonary artery smooth muscle cells,PASMCs),激光共聚焦扫描显微镜技术观察不同条件下PASMCs中钙离子浓度的变化,组织浴槽血管环技术观察血管张力的变化。结果:CaSR激动剂(钙、钆)引起剂量依赖性的细胞内钙增加和血管张力增大, U73122和D609( PLC抑制剂)以及2-APB和肝素(IP3受体抑制剂)可减弱CaSR的作用(P<0.05),而U73343(U73122的无生物活性类似物)则无此作用(P>0.05)。结论:CaSR活化导致细胞内钙增加,进而引起肺动脉环收缩,这些过程是通过G蛋白-PLC-IP3信号转导通路实现的。

  1. Extracellular calcium sensing and extracellular calcium signaling (United States)

    Brown, E. M.; MacLeod, R. J.; O'Malley, B. W. (Principal Investigator)


    The cloning of a G protein-coupled extracellular Ca(2+) (Ca(o)(2+))-sensing receptor (CaR) has elucidated the molecular basis for many of the previously recognized effects of Ca(o)(2+) on tissues that maintain systemic Ca(o)(2+) homeostasis, especially parathyroid chief cells and several cells in the kidney. The availability of the cloned CaR enabled the development of DNA and antibody probes for identifying the CaR's mRNA and protein, respectively, within these and other tissues. It also permitted the identification of human diseases resulting from inactivating or activating mutations of the CaR gene and the subsequent generation of mice with targeted disruption of the CaR gene. The characteristic alterations in parathyroid and renal function in these patients and in the mice with "knockout" of the CaR gene have provided valuable information on the CaR's physiological roles in these tissues participating in mineral ion homeostasis. Nevertheless, relatively little is known about how the CaR regulates other tissues involved in systemic Ca(o)(2+) homeostasis, particularly bone and intestine. Moreover, there is evidence that additional Ca(o)(2+) sensors may exist in bone cells that mediate some or even all of the known effects of Ca(o)(2+) on these cells. Even more remains to be learned about the CaR's function in the rapidly growing list of cells that express it but are uninvolved in systemic Ca(o)(2+) metabolism. Available data suggest that the receptor serves numerous roles outside of systemic mineral ion homeostasis, ranging from the regulation of hormonal secretion and the activities of various ion channels to the longer term control of gene expression, programmed cell death (apoptosis), and cellular proliferation. In some cases, the CaR on these "nonhomeostatic" cells responds to local changes in Ca(o)(2+) taking place within compartments of the extracellular fluid (ECF) that communicate with the outside environment (e.g., the gastrointestinal tract). In others

  2. 继发性甲状旁腺功能亢进症患者维生素D受体和钙敏感受体的免疫组化研究%Immunohistochemistry study on vitamin D receptor and calcium-sensing receptor in parathyroid from secondary hyperparathyroidism patients

    Institute of Scientific and Technical Information of China (English)

    王文博; 张凌; 笪冀平; 卞维静; 李文歌


    Objective To investigate the relationship between the expression of vitamin D receptor (VDR) and calcium-sensing receptor receptor (CaSR) and the proliferative activity in the parathyroid tissue from secondary hyperparathyroidism (SHPT) patients.Methods The numbers of VDR- and CaSR-positive cells after immunohistochemistry staining in the parathyroid tissues of various degrees of SHPT were compared with those in the normal parathyroid tissues.Results Among the 21 proliferation parathyroid glands, one was diagnosed as diffuse hyperplasia (group B), 13 were diagnosed as nodular hyperplasia (group C), and 7 as adenoma hyperplasia (group D).(a) VDR was mostly nuclear localized in cells of normal parathyroid (group A).The expression of VDR in groups A, B, C and D was (78.0 ± 2.5)%, 57.1%, (31.0 ± 6.7)% and (23.0 ±2.1)%, respectively (P < 0.01 ).The expression level of VDR correlated with the hyperplasia degree of parathyroid glands.(b) CaSR was mostly expressed on cell membrane and in cytoplasm.CaSR expression was reduced in parathyroid samples from SHPT patients (48.0 ± 17.9%), as compared with that in group A (79.0 ± 1.0%, P < 0.01).CaSR expression was 51%, (47.0 ± 9.8)% and 29.0 ± 10.1% in group B, C and D, respectively (P <0.01).Conclusions (a) The lower expression of VDR and CaSR appears to be the cause of SHPT refractory to the calcitrol pulse therapy.(b) Attempts to increase the expression of the two receptors or to use the agonists of the two receptors may be the new approaches to SHPT therapy.%目的 通过研究维生素D受体(vitamin D receptor,VDR)和钙敏感受体(calciumsensing receptor,CaSR)在继发性甲状旁腺功能亢进症(Secondary hyperparathyroidism,SHPT)患者甲状旁腺组织中的表达,探讨VDR和CaSR与SHPT甲状旁腺组织病理改变的关系.方法 用免疫组化的方法 计数CaSR和VDR阳性细胞,比较其在不同程度SHPT中甲状旁腺组织及正常甲状旁

  3. 白细胞介素6与钙感应性受体基因多态性对青春期女童骨量增长的交互作用%Effect of interleukin-6 and calcium sensing receptor gene polymorphisms on bone mass accrual in Chinese adolescent girls

    Institute of Scientific and Technical Information of China (English)

    李星; 何国鹏; 苏宜香


    目的 探讨白细胞介素6(interleukin-6,IL-6)与钙感应性受体(calcium sensing receptor,CASR)基因多态性对青春期女童骨量增长的交互作用.方法 选择228名9-11岁半未月经初潮的健康女童进行两年追踪,采用双能X线骨密度仪检测对象追踪前后全身、左侧近端股骨(包括股骨颈、大转子、粗隆间和华氏三角区)和L1-L4腰椎骨矿含量(bone mineral content,BMC)和骨密度(bone mineral density,BMD),采用聚合酶链反应-限制性片段长度多态性技术检测IL-6-634C/G位点多态性,等位基因特异性突变分离扩增-PCR技术检测CASR A986S位点多态性.结果 176名女童完成整个研究.IL-6基因-634C/G和CASR基因A986S位点多态性与青春期女童骨量增长有关联,IL-6基因-634C/G位点CG+GG基因型女童全身和股骨大转子BMD较CC基因型分别低25.7%和20.6%,CASR基因A986S位点AS+SS基因型女童L1.L4腰椎BMC和华氏三角区BMD增长率较AA基因型分别低14.9%和51.3%,差异有统计学意义(P<0.05).交互作用分析发现,同时具有IL-6基因-634C/G位点G等位基因和CASR基因A986S位点S等位基因的女童,其股骨颈和L1-L4腰椎BMC增长率最低.结论 同时具有IL-6基因-634C/G位点G等位基因和CASR基因A986S位点S等位基因的女童,可能是低骨量增长的危险人群.%Objective To investigate the effect of interleukin-6(IL-6)and calcium sensing receptor(CASR)gene polymorphisms on bone mass accrual in Chinese adolescent girls.Methods A total of 228 premenarche Chinese girls(9-11.5 years)were recruited for a 2-year study.Bone mineral densities(BMD)and bone mineral contents(BMC)in the total body,total left hip including femoral neck,trochanter,intertrochanter and Ward's triangle area,and lumbar spine(L1-L4)were measired by DEXA.The-634C/G polymorphism of IL-6 gene was detected by PCR-restriction fragment length polymorphism and A986S polymorphism of CASR gene was detected by allele-special mutagenically separated

  4. Calcium Nutrition and Extracellular Calcium Sensing: Relevance for the Pathogenesis of Osteoporosis, Cancer and Cardiovascular Diseases

    Directory of Open Access Journals (Sweden)

    Meinrad Peterlik


    Full Text Available Through a systematic search in Pubmed for literature, on links between calcium malnutrition and risk of chronic diseases, we found the highest degree of evidence for osteoporosis, colorectal and breast cancer, as well as for hypertension, as the only major cardiovascular risk factor. Low calcium intake apparently has some impact also on cardiovascular events and disease outcome. Calcium malnutrition can causally be related to low activity of the extracellular calcium-sensing receptor (CaSR. This member of the family of 7-TM G-protein coupled receptors allows extracellular Ca2+ to function as a “first messenger” for various intracellular signaling cascades. Evidence demonstrates that Ca2+/CaSR signaling in functional linkage with vitamin D receptor (VDR-activated pathways (i promotes osteoblast differentiation and formation of mineralized bone; (ii targets downstream effectors of the canonical and non-canonical Wnt pathway to inhibit proliferation and induce differentiation of colorectal cancer cells; (iii evokes Ca2+ influx into breast cancer cells, thereby activating pro-apoptotic intracellular signaling. Furthermore, Ca2+/CaSR signaling opens Ca2+-sensitive K+ conductance channels in vascular endothelial cells, and also participates in IP3-dependent regulation of cytoplasmic Ca2+, the key intermediate of cardiomyocyte functions. Consequently, impairment of Ca2+/CaSR signaling may contribute to inadequate bone formation, tumor progression, hypertension, vascular calcification and, probably, cardiovascular disease.

  5. The calcium-sensing receptor--a driver of colon cell differentiation. (United States)

    Whitfield, J F


    Dietary Ca(2+) reduces colon cell proliferation and carcinogenesis, but it becomes ineffective or even tumor-promoting during carcinogenesis. It appears that Ca(2+) and the colon cell CaSR together brake the massive cell production in normal colon crypts. The rapid proliferation of the transit-amplifying (TA) progeny of the colon stem cells at the bases of the crypts is driven by the "Wnt" signaling mechanism that stimulates proliferogenic genes and prevents apoptogenesis. It appears that TA cell cycling stops and terminal differentiation starts when the cells reach a higher level in the crypt where there is enough external Ca(2+) to stimulate the expression of CaSRs, the signals from which stimulate the expression of E-cadherin. At this point the APC (adenomatous polyposis coli) protein appears and some of it enters the nucleus. There it removes the apoptogenesis shield and stops the beta-cateninTcf-4 complex from driving further TA cell proliferation by releasing beta-catenin from the nucleus, and delivering it to cytoplasmic APCaxinGSK-3beta complexes for ultimate proteasomal destruction. Cytoplasmic beta-catenin is prevented from returning to the nucleus by destruction in APCaxinGSK-3beta complexes or locked by the emerging E-cadherin into adherens junctions which link the cell to proliferatively shut-down functioning cells with APC-dependent cytoskeletons moving up and out of the crypt. A common first step in colon carcinogenesis is the loss of functional APC which results in the retention of proliferogenic nuclear beta-cateninTcf-4. This drives the eventual appearance of mutation accumulating, apoptosis-resistant clones the proliferation of which cannot be inhibited by external Ca(2+) because of CaSR-disabling gene mutations.

  6. Diverse roles of extracellular calcium-sensing receptor in the central nervous system

    DEFF Research Database (Denmark)

    Bandyopadhyay, Sanghamitra; Tfelt-Hansen, Jacob; Chattopadhyay, Naibedya


    membrane excitability of neurons and glia and affects myelination, olfactory and gustatory signal integration, axonal and dendritic growth, and gonadotrophin-releasing hormonal-neuronal migration. Insofar as the CaSR is a clinically important therapeutic target for parathyroid disorders, development of its...

  7. Aquaporin 2 and apical calcium-sensing receptor: new players in polyuric disorders associated with hypercalciuria.

    NARCIS (Netherlands)

    Procino, G.; Mastrofrancesco, L.; Mira, A.; Tamma, G.; Carmosino, M.; Emma, F.; Svelto, M.; Valenti, G.


    The kidney plays a critical role in regulating water homeostasis through specific proteins highly expressed in the kidney, called aquaporins, allowing water permeation at a high rate. This brief review focuses on some nephropathies associated with impaired urinary concentrating ability and in partic

  8. Lessons from crystal structures of kainate receptors. (United States)

    Møllerud, Stine; Frydenvang, Karla; Pickering, Darryl S; Kastrup, Jette Sandholm


    Kainate receptors belong to the family of ionotropic glutamate receptors. These receptors assemble from five subunits (GluK1-5) into tetrameric ion channels. Kainate receptors are located at both pre- and postsynaptic membranes in the central nervous system where they contribute to excitatory synaptic transmission and modulate network excitability by regulating neurotransmitter release. Dysfunction of kainate receptors has been implicated in several neurological disorders such as epilepsy, schizophrenia and depression. Here we provide a review on the current understanding of kainate receptor structure and how they bind agonists, antagonists and ions. The first structure of the ligand-binding domain of the GluK1 subunit was reported in 2005, seven years after publication of the crystal structure of a soluble construct of the ligand-binding domain of the AMPA-type subunit GluA2. Today, a full-length structure has been determined of GluK2 by cryo electron microscopy to 7.6 Å resolution as well as 84 high-resolution crystal structures of N-terminal domains and ligand-binding domains, including agonist and antagonist bound structures, modulatory ions and mutations. However, there are still many unanswered questions and challenges in front of us. This article is part of the Special Issue entitled 'Ionotropic glutamate receptors'.

  9. Structure of Leptin Receptor Related with Obesity

    DEFF Research Database (Denmark)

    Toleikis, Zigmantas

    The hormone leptin is central to obesity, but the molecular processes underlying the activation of the leptin receptor are unknown. To further the understanding of the system, an atomic resolution structure of this cytokine type I receptor in the unbound inactive form and in the activated bound...... of the receptor, while the D5 domain is the central leptin-binding domain, implicated in the first steps of activation. Both domains are characterized by a fibronectin type III fold and both contain a conserved WSXWS motif (X represents an unconserved amino acid residue), a distinct feature of the cytokine...... receptors. This motif is thought to play a major role in correct folding and activation of the receptor. The complex between leptin and the D5CA domain was analyzed using nuclear magnetic resonance spectroscopy and the amino acid residues implicated in the binding were determined. To investigate which parts...

  10. Lessons from crystal structures of kainate receptors

    DEFF Research Database (Denmark)

    Møllerud, Stine; Frydenvang, Karla Andrea; Pickering, Darryl S;


    synaptic transmission and modulate network excitability by regulating neurotransmitter release. Dysfunction of kainate receptors has been implicated in several neurological disorders such as epilepsy, schizophrenia and depression. Here we provide a review on the current understanding of kainate receptor...... structure and how they bind agonists, antagonists and ions. The first structure of the ligand-binding domain of the GluK1 subunit was reported in 2005, seven years after publication of the crystal structure of a soluble construct of the ligand-binding domain of the AMPA-type subunit GluA2. Today, a full...

  11. Calcium Sensing Receptor:Signaling Pathways and Physiological Functions%钙敏感受体介导的信号传导通路及生理功能

    Institute of Scientific and Technical Information of China (English)

    赵秀英; 杭苏琴; 朱伟云


    Calcium-sensing receptor ( CaSR) is a member of the G protein-coupled receptors ( GPCRs) C fam-ily, and wildly expressed in nervous system, cardiovascular system, gastrointestinal tract, kidney and bone tis-sue. After combined with its agonists and allosteric modulators, the downstream signaling pathways are activa-ted, which play very important roles in the regulation of calcium homeostasis, cell proliferation and differentia-tion, immune function and endocrine hormone releasing. However, recent researches mostly focus on cattle, human and mice. The studies on livestock and other animals are limited, and undergoing mechanisms of physi-ological functions of CaSR mediation are not clear enough. Therefore, this paper summarized the discovery, the structure, mediated signaling events and physiological effects of the CaSR, in order to lay a theoretical foundation for further study on livestock and other animals.%钙敏感受体( CaSR)是G蛋白偶联受体( GPCRs) C家族的成员,其在神经系统、心血管系统、胃肠道、肾脏及骨组织中有广泛的分布,当与激动剂及变构调节剂结合后,激活下游相关信号通路,对于机体钙稳态的维持、细胞的增殖分化、免疫及多种内分泌激素的释放具有重要调节作用。目前的研究报告主要来自于牛、人、鼠等,对家畜和其他动物的研究还较少,对机体各项生命活动的调控作用也不明确。为此,本文从CaSR的发现、结构、介导的信号活动及生理作用等方面作一综述,以期为研究 CaSR 调节动物生理活动的机制及发挥的生理功能奠定理论基础。

  12. 3D structure of muscle dihydropyridine receptor

    Directory of Open Access Journals (Sweden)

    Montserrat Samsó


    Full Text Available Excitation contraction coupling, the rapid and massive Ca2+ release under control of an action potential that triggers muscle contraction, takes places at specialized regions of the cell called triad junctions. There, a highly ordered supramolecular complex between the dihydropyridine receptor (DHPR and the ryanodine receptor (RyR1 mediates the quasi‐instantaneous conversion from T‐tubule depolarization into Ca2+ release from the sarcoplasmic reticulum (SR. The DHPR has several key modules required for EC coupling: the voltage sensors and II‐III loop in the alpha1s subunit, and the beta subunit. To gain insight into their molecular organization, this review examines the most updated 3D structure of the DHPR as obtained by transmission electron microscopy and image reconstruction. Although structure determination of a heteromeric membrane protein such as the DHPR is challenging, novel technical advances in protein expression and 3D labeling facilitated this task. The 3D structure of the DHPR complex consists of a main body with five irregular corners around its perimeter encompassing the transmembrane alpha 1s subunit besides the intracellular beta subunit, an extended extracellular alpha 2 subunit, and a bulky intracellular II‐III loop. The structural definition attained at 19 Å resolution enabled docking of the atomic coordinates of structural homologs of the alpha1s and beta subunits. These structural features, together with their relative location with respect to the RyR1, are discussed in the context of the functional data.

  13. Crystallization and Structure Determination of Superantigens and Immune Receptor Complexes. (United States)

    Rödström, Karin E J; Lindkvist-Petersson, Karin


    Structure determination of superantigens and the complexes they form with immune receptors have over the years provided insight in their modes of action. This technique requires growing large and highly ordered crystals of the superantigen or receptor-superantigen complex, followed by exposure to X-ray radiation and data collection. Here, we describe methods for crystallizing superantigens and superantigen-receptor complexes using the vapor diffusion technique, how the crystals may be optimized, and lastly data collection and structure determination.

  14. Probing intermolecular protein-protein interactions in the calcium-sensing receptor homodimer using bioluminescence resonance energy transfer (BRET)

    DEFF Research Database (Denmark)

    Jensen, Anders A.; Hansen, Jakob L; Sheikh, Søren P


    -induced intermolecular movements in the CaR homodimer using the new bioluminescence resonance energy transfer technique, BRET2, which is based on the transference of energy from Renilla luciferase (Rluc) to the green fluorescent protein mutant GFP2. We tagged CaR with Rluc and GFP2 at different intracellular locations...

  15. Strontium is a biased agonist of the calcium-sensing receptor in rat medullary thyroid carcinoma 6-23 cells

    DEFF Research Database (Denmark)

    Thomsen, Alex Rojas Bie; Worm, Jesper; Jacobsen, Stine Engesgaard


    of CaSR is poorly understood, the objective of the present study was to investigate biased signaling of CaSR by using rat medullary thyroid carcinoma 6-23 cells as a model of thyroid parafollicular C-cells. By doing concentration-response experiments we focused on the ability of two well known Ca...

  16. Adrenergic Receptors From Molecular Structure to in vivo function. (United States)

    Hein, L; Kobilka, B K


    Adrenergic receptors form the interface between the sympathetic nervous system and the cardiovascular system as well as many endocrine and parenchymal tissues. Although several hundred G-protein-coupled receptors have been identified, adrenergic receptors, along with the visual pigment rhodopsin, have been among the most extensively studied members of this family of receptors. This review focuses on recent advances in understanding the molecular structure, function, and regulation of adrenergic receptors using in vitro systems and integrates recent transgenic animal models that were generated to study the adrenergic system in vivo. (Trends Cardiovasc Med 1997;7:137-145). © 1997, Elsevier Science Inc.

  17. Primary Structure of Nicotinic Acetylcholine Receptor (United States)


    quantities of starting material (for reviews of receptor, see Popot and Changeux, 1984; Stroud and Finer-Moore, 1985). This work led to the...Cloning of the Acetylcholine Receptor. Cold Spring Harbor Symp. on Quant. Biol. XLVIH: 71-78. 15. Popot , J-L. and Changeux, J-P. (1984) The

  18. Structural Studies of Nicotinic Acetylcholine Receptors

    DEFF Research Database (Denmark)

    Shahsavar, Azadeh; Gajhede, Michael; Kastrup, Jette S;


    Nicotinic acetylcholine receptors (nAChRs) are members of the pentameric ligand-gated ion channel superfamily that play important roles in control of neurotransmitter release in the central and peripheral nervous system. These receptors are important therapeutic targets for development of drugs...

  19. Class I Cytokine Receptors: Structure and function in the Membrane

    DEFF Research Database (Denmark)

    Bugge, Katrine Østergaard

    Class I cytokine receptors are involved in important biological functions of both physiological and pathological nature in mammals. However, the molecular details of the cross-membrane signal transduction through these receptors remain obscure. One of the major reasons for this is the lack...... of structural knowledge on their membrane-embedded transmembrane domains (TMDs), which connect the extracellular ligand binding domains to the intracellular signaling platforms. The overall aim of this thesis work was to improve our understanding of the class I cytokine receptor signaling across the membrane...... ample material of high quality for structural studies with NMR spectroscopy of several class I cytokine receptor TMDs. Furthermore, the structure of a class I cytokine receptor TMD in DHPC micelles was solved with solution-state NMR spectroscopy. Additionally, since structural studies of intact proteins...

  20. Molecular cloning, expression, and sequence analysis of GPRC6A, a novel family C G-protein-coupled receptor

    DEFF Research Database (Denmark)

    Wellendorph, Petrine; Bräuner-Osborne, Hans


    with a significant homology to the human calcium-sensing receptor (CaR, 34% aa sequence identity), the taste receptor 1 (T1R1, 28%), and the metabotropic glutamate receptor 1 (mGluR1, 24%), places GPRC6A in family C of the GPCRs. Interestingly, GPRC6A bears the highest resemblance with an odorant goldfish 5...

  1. Structure-Function Studies on the Prolactin Receptor

    DEFF Research Database (Denmark)

    Haxholm, Gitte Wolfsberg

    Class 1 Cytokine receptors are involved in important biological functions mediated through complex networks of intracellular signaling. However, the molecular details of how signaling is regulated are poorly understood. One of the primary reasons for this limited knowledge is the lack of structural...... information on the intracellular domains (ICDs) of these receptors. The overall aim of this study was to obtain an improved understanding of cytokine receptor signaling through structure-function studies on the prolactin receptor (PRLR). The primary focus of this thesis was to structurally characterize...... the PRLR-ICD and the ICD of the related growth hormone receptor (GHR). We showed that both ICDs were intrinsically disordered throughout their entire lengths and that they associated with lipids characteristic of the inner plasma membrane leaflet through conserved motifs, implicating the membrane...

  2. Structural basis for ligand recognition of incretin receptors

    DEFF Research Database (Denmark)

    Underwood, Christina Rye; Parthier, Christoph; Reedtz-Runge, Steffen


    been solved recently by X-ray crystallography. The crystal structures reveal a similar fold of the ECD and a similar mechanism of ligand binding, where the ligand adopts an α-helical conformation. Residues in the C-terminal part of the ligand interact directly with the ECD and hydrophobic interactions...... appear to be the main driving force for ligand binding to the ECD of incretin receptors. Obviously, the-still missing-structures of full-length incretin receptors are required to construct a complete picture of receptor function at the molecular level. However, the progress made recently in structural...

  3. Structure of the [delta]-opioid receptor bound to naltrindole

    Energy Technology Data Exchange (ETDEWEB)

    Granier, Sébastien; Manglik, Aashish; Kruse, Andrew C.; Kobilka, Tong Sun; Thian, Foon Sun; Weis, William I.; Kobilka, Brian K. (Stanford-MED)


    The opioid receptor family comprises three members, the {mu}-, {delta}- and {kappa}-opioid receptors, which respond to classical opioid alkaloids such as morphine and heroin as well as to endogenous peptide ligands like endorphins. They belong to the G-protein-coupled receptor (GPCR) superfamily, and are excellent therapeutic targets for pain control. The {delta}-opioid receptor ({delta}-OR) has a role in analgesia, as well as in other neurological functions that remain poorly understood. The structures of the {mu}-OR and {kappa}-OR have recently been solved. Here we report the crystal structure of the mouse {delta}-OR, bound to the subtype-selective antagonist naltrindole. Together with the structures of the {mu}-OR and {kappa}-OR, the {delta}-OR structure provides insights into conserved elements of opioid ligand recognition while also revealing structural features associated with ligand-subtype selectivity. The binding pocket of opioid receptors can be divided into two distinct regions. Whereas the lower part of this pocket is highly conserved among opioid receptors, the upper part contains divergent residues that confer subtype selectivity. This provides a structural explanation and validation for the 'message-address' model of opioid receptor pharmacology, in which distinct 'message' (efficacy) and 'address' (selectivity) determinants are contained within a single ligand. Comparison of the address region of the {delta}-OR with other GPCRs reveals that this structural organization may be a more general phenomenon, extending to other GPCR families as well.

  4. Study of bioengineered zebra fish olfactory receptor 131-2: receptor purification and secondary structure analysis. (United States)

    Leck, Kwong-Joo; Zhang, Shuguang; Hauser, Charlotte A E


    How fishes are able to detect trace molecules in large bodies of water is not understood. It is plausible that they use olfactory receptors to detect water-soluble compounds. How the zebra fish Danio Rerio, an organism with only 98 functional olfactory receptors, is able to selectively detect and recognize numerous compounds in water remains a puzzling phenomenon. We are interested in studying the biochemical and molecular mechanisms of olfaction in fish. Here, we report on the study of a bioengineered zebra fish olfactory receptor OR131-2, affinity-purified from a HEK293S tetracycline-inducible system. This receptor was expressed and translocated to the cell plasma membrane as revealed by confocal microscopy. Circular dichroism spectroscopy showed that the purified zebra fish receptor folded into an α-helical structure, as observed for other G-protein coupled receptors (GPCRs). Our study shows that it is possible to produce viable quantities of the zebra fish olfactory receptor. This will not only enable detailed structural and functional analyses, but also aid in the design of biosensor devices in order to detect water-soluble metabolites or its intermediates, which are associated with human health.

  5. Study of bioengineered zebra fish olfactory receptor 131-2: receptor purification and secondary structure analysis.

    Directory of Open Access Journals (Sweden)

    Kwong-Joo Leck

    Full Text Available How fishes are able to detect trace molecules in large bodies of water is not understood. It is plausible that they use olfactory receptors to detect water-soluble compounds. How the zebra fish Danio Rerio, an organism with only 98 functional olfactory receptors, is able to selectively detect and recognize numerous compounds in water remains a puzzling phenomenon. We are interested in studying the biochemical and molecular mechanisms of olfaction in fish. Here, we report on the study of a bioengineered zebra fish olfactory receptor OR131-2, affinity-purified from a HEK293S tetracycline-inducible system. This receptor was expressed and translocated to the cell plasma membrane as revealed by confocal microscopy. Circular dichroism spectroscopy showed that the purified zebra fish receptor folded into an α-helical structure, as observed for other G-protein coupled receptors (GPCRs. Our study shows that it is possible to produce viable quantities of the zebra fish olfactory receptor. This will not only enable detailed structural and functional analyses, but also aid in the design of biosensor devices in order to detect water-soluble metabolites or its intermediates, which are associated with human health.

  6. LYRA, a webserver for lymphocyte receptor structural modeling

    DEFF Research Database (Denmark)

    Klausen, Michael Schantz; Anderson, Mads Valdemar; Jespersen, Martin Closter


    a complete and automated method for building of B- and T-cell receptor structural models starting from their amino acid sequence alone. The webserver is freely available and easy to use for non-specialists. Upon submission, LYRA automatically generates alignments using ad hoc profiles, predicts...... the structural class of each hypervariable loop, selects the best templates in an automatic fashion, and provides within minutes a complete 3D model that can be downloaded or inspected online. Experienced users can manually select or exclude template structures according to case specific information. LYRA......The accurate structural modeling of B- and T-cell receptors is fundamental to gain a detailed insight in the mechanisms underlying immunity and in developing new drugs and therapies. The LYRA (LYmphocyte Receptor Automated modeling) web server ( implements...

  7. A structural biology perspective on NMDA receptor pharmacology and function. (United States)

    Regan, Michael C; Romero-Hernandez, Annabel; Furukawa, Hiro


    N-methyld-aspartate receptors (NMDARs) belong to the large family of ionotropic glutamate receptors (iGluRs), which are critically involved in basic brain functions as well as multiple neurological diseases and disorders. The NMDARs are large heterotetrameric membrane protein complexes. The extensive extracellular domains recognize neurotransmitter ligands and allosteric compounds and translate the binding information to regulate activity of the transmembrane ion channel. Here, we review recent advances in the structural biology of NMDARs with a focus on pharmacology and function. Structural analysis of the isolated extracellular domains in combination with the intact heterotetrameric NMDAR structure provides important insights into how this sophisticated ligand-gated ion channel may function.

  8. Structural and pharmacological characterization of phenylalanine-based AMPA receptor antagonists at kainate receptors

    DEFF Research Database (Denmark)

    Venskutonyte, Raminta; Frydenvang, Karla; Valadés, Elena Antón;


    . A new series of phenylalanine derivatives that target iGluRs was reported to bind AMPA receptors. Herein we report our studies of these compounds at the kainate receptors GluK1-3. Several compounds bind with micromolar affinity at GluK1 and GluK3, but do not bind GluK2. The crystal structure of the most...

  9. Structure and dynamics of the M3 muscarinic acetylcholine receptor

    Energy Technology Data Exchange (ETDEWEB)

    Kruse, Andrew C.; Hu, Jianxin; Pan, Albert C.; Arlow, Daniel H.; Rosenbaum, Daniel M.; Rosemond, Erica; Green, Hillary F.; Liu, Tong; Chae, Pil Seok; Dror, Ron O.; Shaw, David E.; Weis, William I.; Wess, Jürgen; Kobilka, Brian K. (Stanford); (NIH); (D.E. Shaw); (Hanyang); (UTSMC)


    Acetylcholine, the first neurotransmitter to be identified, exerts many of its physiological actions via activation of a family of G-protein-coupled receptors (GPCRs) known as muscarinic acetylcholine receptors (mAChRs). Although the five mAChR subtypes (M1-M5) share a high degree of sequence homology, they show pronounced differences in G-protein coupling preference and the physiological responses they mediate. Unfortunately, despite decades of effort, no therapeutic agents endowed with clear mAChR subtype selectivity have been developed to exploit these differences. We describe here the structure of the G{sub q/11}-coupled M3 mAChR ('M3 receptor', from rat) bound to the bronchodilator drug tiotropium and identify the binding mode for this clinically important drug. This structure, together with that of the G{sub i/o}-coupled M2 receptor, offers possibilities for the design of mAChR subtype-selective ligands. Importantly, the M3 receptor structure allows a structural comparison between two members of a mammalian GPCR subfamily displaying different G-protein coupling selectivities. Furthermore, molecular dynamics simulations suggest that tiotropium binds transiently to an allosteric site en route to the binding pocket of both receptors. These simulations offer a structural view of an allosteric binding mode for an orthosteric GPCR ligand and provide additional opportunities for the design of ligands with different affinities or binding kinetics for different mAChR subtypes. Our findings not only offer insights into the structure and function of one of the most important GPCR families, but may also facilitate the design of improved therapeutics targeting these critical receptors.

  10. Structure of a human rhinovirus complexed with its receptor molecule.



    Cryoelectron microscopy has been used to determine the structure of a virus when complexed with its glycoprotein cellular receptor. Human rhinovirus 16 complexed with the two amino-terminal, immunoglobulin-like domains of the intercellular adhesion molecule 1 shows that the intercellular adhesion molecule 1 binds into the 12-A deep "canyon" on the viral surface. This result confirms the prediction that the viral-receptor attachment site lies in a cavity inaccessible to the host's antibodies. ...

  11. In silico discovery of novel Retinoic Acid Receptor agonist structures

    Directory of Open Access Journals (Sweden)

    Samuels Herbert H


    Full Text Available Abstract Background Several Retinoic Acid Receptors (RAR agonists have therapeutic activity against a variety of cancer types; however, unacceptable toxicity profiles have hindered the development of drugs. RAR agonists presenting novel structural and chemical features could therefore open new avenues for the discovery of leads against breast, lung and prostate cancer or leukemia. Results We have analysed the induced fit of the active site residues upon binding of a known ligand. The derived binding site models were used to dock over 150,000 molecules in silico (or virtually to the structure of the receptor with the Internal Coordinates Mechanics (ICM program. Thirty ligand candidates were tested in vitro. Conclusions Two novel agonists resulting from the predicted receptor model were active at 50 nM. One of them displays novel structural features which may translate into the development of new ligands for cancer therapy.

  12. Structure and dynamics of a constitutively active neurotensin receptor

    Energy Technology Data Exchange (ETDEWEB)

    Krumm, Brian E. [National Inst. of Health (NIH), Rockville, MD (United States). National Inst. of Neurological Disorders and Stroke, Dept. of Health and Human Services; Lee, Sangbae [Beckman Research Inst. of the City of Hope, Duarte, CA (United States). Dept. of Molecular Immunology; Bhattacharya, Supriyo [Beckman Research Inst. of the City of Hope, Duarte, CA (United States). Dept. of Molecular Immunology; Botos, Istvan [National Inst. of Health (NIH), Bethesda, MD (United States). National Inst. of Diabetes and; White, Courtney F. [National Inst. of Health (NIH), Rockville, MD (United States). National Inst. of Neurological Disorders and Stroke, Dept. of Health and Human Services; Du, Haijuan [National Inst. of Health (NIH), Rockville, MD (United States). National Inst. of Neurological Disorders and Stroke, Dept. of Health and Human Services; Vaidehi, Nagarajan [Beckman Research Inst. of the City of Hope, Duarte, CA (United States). Dept. of Molecular Immunology; Grisshammer, Reinhard [National Inst. of Health (NIH), Rockville, MD (United States). National Inst. of Neurological Disorders and Stroke, Dept. of Health and Human Services


    Many G protein-coupled receptors show constitutive activity, resulting in the production of a second messenger in the absence of an agonist; and naturally occurring constitutively active mutations in receptors have been implicated in diseases. To gain insight into mechanistic aspects of constitutive activity, we report here the 3.3 Å crystal structure of a constitutively active, agonist-bound neurotensin receptor (NTSR1) and molecular dynamics simulations of agonist-occupied and ligand-free receptor. Comparison with the structure of a NTSR1 variant that has little constitutive activity reveals uncoupling of the ligand-binding domain from conserved connector residues, that effect conformational changes during GPCR activation. Furthermore, molecular dynamics simulations show strong contacts between connector residue side chains and increased flexibility at the intracellular receptor face as features that coincide with robust signalling in cells. The loss of correlation between the binding pocket and conserved connector residues, combined with altered receptor dynamics, possibly explains the reduced neurotensin efficacy in the constitutively active NTSR1 and a facilitated initial engagement with G protein in the absence of agonist.

  13. Structure and function of the human megalin receptor

    DEFF Research Database (Denmark)

    Dagil, Robert

    Megalin is an endocytic lipoprotein receptor expressed widely throughout the body, ranging from the proximal tubule in the kidneys to the cochlea in the inner ear. Megalin is known to bind over 50 different ligands and is involved in protein clearance of the renal ultrafiltrate via endocytosis...... was studied using NMR spectroscopy. The structure of the tenth CR domain from the human megalin receptor was solved using NMR spectroscopy and a HADDOCK model of the complex between this domain and gentamicin was determined. The structural complex showed that a Trp residue and three Asp residues from megalin...

  14. The perception of gibberellins: clues from receptor structure. (United States)

    Ueguchi-Tanaka, Miyako; Matsuoka, Makoto


    The discovery of GID1, a soluble receptor for gibberellins (GAs), has revealed new insights into how GA is perceived. X-ray analysis has demonstrated similarities in the tertiary structure of GID1 to hormone sensitive lipase (HSL), and the GA-binding pocket of GID1 corresponds to the active site of HSL. X-ray analysis has also revealed the structural basis of the GA-GID1 interaction, and evolutionary aspects of GID1 have been discovered by comparison to GID1 from non-flowering plants. Recent studies have also demonstrated the complexity of GA signaling in Arabidopsis, which is mediated by three GID1 and five DELLA proteins. Finally, mechanistic and structural similarities for hormone signaling are compared for GA, auxin and abscisic acid, three hormones where the receptor protein structure was recently described.

  15. Nuclear hormone receptor co-repressors: Structure and function



    Co-repressor proteins, such as SMRT and NCoR, mediate the repressive activity of unliganded nuclear receptors and other transcription factors. They appear to act as intrinsically disordered “hub proteins” that integrate the activities of a range of transcription factors with a number of histone modifying enzymes. Although these co-repressor proteins are challenging targets for structural studies due to their largely unstructured character, a number of structures have recently been determined ...

  16. Crystal structure of mouse coronavirus receptor-binding domain complexed with its murine receptor

    Energy Technology Data Exchange (ETDEWEB)

    Peng, Guiqing; Sun, Dawei; Rajashankar, Kanagalaghatta R.; Qian, Zhaohui; Holmes, Kathryn V.; Li, Fang (Cornell); (UMM-MED); (Colorado)


    Coronaviruses have evolved diverse mechanisms to recognize different receptors for their cross-species transmission and host-range expansion. Mouse hepatitis coronavirus (MHV) uses the N-terminal domain (NTD) of its spike protein as its receptor-binding domain. Here we present the crystal structure of MHV NTD complexed with its receptor murine carcinoembryonic antigen-related cell adhesion molecule 1a (mCEACAM1a). Unexpectedly, MHV NTD contains a core structure that has the same {beta}-sandwich fold as human galectins (S-lectins) and additional structural motifs that bind to the N-terminal Ig-like domain of mCEACAM1a. Despite its galectin fold, MHV NTD does not bind sugars, but instead binds mCEACAM1a through exclusive protein-protein interactions. Critical contacts at the interface have been confirmed by mutagenesis, providing a structural basis for viral and host specificities of coronavirus/CEACAM1 interactions. Sugar-binding assays reveal that galectin-like NTDs of some coronaviruses such as human coronavirus OC43 and bovine coronavirus bind sugars. Structural analysis and mutagenesis localize the sugar-binding site in coronavirus NTDs to be above the {beta}-sandwich core. We propose that coronavirus NTDs originated from a host galectin and retained sugar-binding functions in some contemporary coronaviruses, but evolved new structural features in MHV for mCEACAM1a binding.

  17. Study of structure function correlation of chemokine receptor CXCR4

    Institute of Scientific and Technical Information of China (English)

    ZHENG Hong; Stephen C PEIPER; ZHU Xi-hua


    Objective: To explore the correlation between structure domains and functions of chemokine receptor CXCR4. Methods: After the establishment of wild type chemokine receptor CXCR4 and CXCR2 expressing cell lines, 5 CXCR4/CXCR2 chimeras, 2 CXCR4 mutants were stably expressed on CHO cell line.Binding activities of all variants with the ligand, recombinant human SDF-1β, signal transduction ability after stimulation and their function as coreceptor for HIV-1 were studied with ligand-binding assay, Cytosensor/microphysiometry and cell-cell reporter gene fusion assay. Results: Among all 7 changed CXCR4 receptors, 3 chimeras (2444a, 4442, 4122), and 1 mutant (CXCR4-Tr) bond with SDF-1β in varying degrees, of which only 2444a totally and CXCR4-Tr partially maintain signaling. All changed receptors except for 4222 could act as coreceptors for HIV-1(LAI) in varying degrees. Conclusion: Several structure domains of CXCR4 are involved in the binding with SDF-1β, among which, N-terminal extracellular domain has high affinity of binding with SDF-1β, and the 3rd extracellular loop contributes to the binding, too. Although the C-terminal intracellular domain has no association with the maintenance of the overall structure of the receptor and ligand binding capability, the signaling is decreased when this domain is truncated. For CXCR4 signaling, not only is the conserved motif DRY box needed, but also the characterized conformation of the whole molecule must be formed when activation is required. There are some overlaps between SDF-1β binding domains and coreceptor function domains in molecular structure of CXCR4.

  18. Receptors for myelin inhibitors: Structures and therapeutic opportunities. (United States)

    Cao, Zixuan; Gao, Ying; Deng, Kangwen; Williams, Gareth; Doherty, Patrick; Walsh, Frank S


    Many studies have indicated that the inability of adult mammalian central nervous system (CNS) to regenerate after injury is partly due to the existence of growth-inhibitory molecules associated with CNS myelin. Studies over the years have led to the identification of multiple myelin-associated inhibitors, among which Nogo, myelin-associated glycoprotein (MAG) and oligodendrocyte-myelin glycoprotein (Omgp) represent potentially major contributors to CNS axon regeneration failure. Here we review in vitro and in vivo investigations into these inhibitory ligands and their functional mechanisms, focusing particularly on the neuronal receptors that mediate the inhibitory signals from these myelin molecules. A better understanding of the receptors for myelin-associated inhibitors could provide opportunities to decipher the mechanism of restriction in CNS regeneration, and lead to the development of potential therapeutic targets in neurodegenerative diseases and neurological injury. We will discuss the structures of the receptors and therapeutic opportunities that might arise based on this information.

  19. Structure-based rationale for interleukin 5 receptor antagonism. (United States)

    Ishino, Tetsuya; Harrington, Adrian E; Gopi, Hosahudya; Chaiken, Irwin


    Human interleukin 5 (IL5) is the major hematopoietin that stimulates the proliferation, migration and activation of eosinophils and is implicated in the pathogenesis of inflammatory and other myeloproliferative diseases. IL5 functions through the signaling of a common receptor subunit beta (beta c), in a receptor activation process that requires initial recruitment of an IL5 specific receptor subunit alpha (IL5Ralpha), for cytokine presentation to beta c. Important advances have been made to understand molecular mechanisms of cytokine recognition and receptor antagonism. Mutational studies indicate that a pair of charge complementary regions play an essential role in specific interaction between IL5Ralpha and IL5. Moreover, peptide studies with the IL5 system have identified a cyclic peptide inhibitor, AF17121, which binds specifically to IL5Ralpha by mimicking the cytokine. A key receptor-recognition pharmacophore has been identified in this peptide inhibitor, and sites of inhibitor recognition can be proposed in the homology-deduced structural model of IL5Ralpha. These results provide an experimental platform to derive enhanced-potency peptidomimetic inhibitors. Such inhibitors have potential use as tools to evaluate the role of eosinophilia in disease and as potential leads to antagonists to treat hyper-eosinophilic diseases such as eosinophilic esophagitis, asthma and chronic myeloproliferative leukemias.

  20. μ Opioid receptor: novel antagonists and structural modeling (United States)

    Kaserer, Teresa; Lantero, Aquilino; Schmidhammer, Helmut; Spetea, Mariana; Schuster, Daniela


    The μ opioid receptor (MOR) is a prominent member of the G protein-coupled receptor family and the molecular target of morphine and other opioid drugs. Despite the long tradition of MOR-targeting drugs, still little is known about the ligand-receptor interactions and structure-function relationships underlying the distinct biological effects upon receptor activation or inhibition. With the resolved crystal structure of the β-funaltrexamine-MOR complex, we aimed at the discovery of novel agonists and antagonists using virtual screening tools, i.e. docking, pharmacophore- and shape-based modeling. We suggest important molecular interactions, which active molecules share and distinguish agonists and antagonists. These results allowed for the generation of theoretically validated in silico workflows that were employed for prospective virtual screening. Out of 18 virtual hits evaluated in in vitro pharmacological assays, three displayed antagonist activity and the most active compound significantly inhibited morphine-induced antinociception. The new identified chemotypes hold promise for further development into neurochemical tools for studying the MOR or as potential therapeutic lead candidates.

  1. Central nicotinic receptors: structure, function, ligands, and therapeutic potential. (United States)

    Romanelli, M Novella; Gratteri, Paola; Guandalini, Luca; Martini, Elisabetta; Bonaccini, Claudia; Gualtieri, Fulvio


    The growing interest in nicotinic receptors, because of their wide expression in neuronal and non-neuronal tissues and their involvement in several important CNS pathologies, has stimulated the synthesis of a high number of ligands able to modulate their function. These membrane proteins appear to be highly heterogeneous, and still only incomplete information is available on their structure, subunit composition, and stoichiometry. This is due to the lack of selective ligands to study the role of nAChR under physiological or pathological conditions; so far, only compounds showing selectivity between alpha4beta2 and alpha7 receptors have been obtained. The nicotinic receptor ligands have been designed starting from lead compounds from natural sources such as nicotine, cytisine, or epibatidine, and, more recently, through the high-throughput screening of chemical libraries. This review focuses on the structure of the new agonists, antagonists, and allosteric ligands of nicotinic receptors, it highlights the current knowledge on the binding site models as a molecular modeling approach to design new compounds, and it discusses the nAChR modulators which have entered clinical trials.

  2. Crystal Structure of the Human Laminin Receptor Precursor

    Energy Technology Data Exchange (ETDEWEB)

    Jamieson,K.; Wu, J.; Hubbard, S.; Meruelo, D.


    The human laminin receptor (LamR) interacts with many ligands, including laminin, prions, Sindbis virus, and the polyphenol (-)-epigallocatechin-3-gallate (EGCG), and has been implicated in a number of diseases. LamR is overexpressed on tumor cells, and targeting LamR elicits anti-cancer effects. Here, we report the crystal structure of human LamR, which provides insights into its function and should facilitate the design of novel therapeutics targeting LamR.

  3. Structural dynamics and thermostabilization of neurotensin receptor 1. (United States)

    Lee, Sangbae; Bhattacharya, Supriyo; Tate, Christopher G; Grisshammer, Reinhard; Vaidehi, Nagarajan


    The neurotensin receptor NTSR1 binds the peptide agonist neurotensin (NTS) and signals preferentially via the Gq protein. Recently, Grisshammer and co-workers reported the crystal structure of a thermostable mutant NTSR1-GW5 with NTS bound. Understanding how the mutations thermostabilize the structure would allow efficient design of thermostable mutant GPCRs for protein purification, and subsequent biophysical studies. Using microsecond scale molecular dynamics simulations (4 μs) of the thermostable mutant NTSR1-GW5 and wild type NTSR1, we have elucidated the structural and energetic factors that affect the thermostability and dynamics of NTSR1. The thermostable mutant NTSR1-GW5 is found to be less flexible and less dynamic than the wild type NTSR1. The point mutations confer thermostability by improving the interhelical hydrogen bonds, hydrophobic packing, and receptor interactions with the lipid bilayer, especially in the intracellular regions. During MD, NTSR1-GW5 becomes more hydrated compared to wild type NTSR1, with tight hydrogen bonded water clusters within the transmembrane core of the receptor, thus providing evidence that water plays an important role in improving helical packing in the thermostable mutant. Our studies provide valuable insights into the stability and functioning of NTSR1 that will be useful in future design of thermostable mutants of other peptide GPCRs.

  4. Androgen receptor: structure, role in prostate cancer and drug discovery. (United States)

    Tan, M H Eileen; Li, Jun; Xu, H Eric; Melcher, Karsten; Yong, Eu-leong


    Androgens and androgen receptors (AR) play a pivotal role in expression of the male phenotype. Several diseases, such as androgen insensitivity syndrome (AIS) and prostate cancer, are associated with alterations in AR functions. Indeed, androgen blockade by drugs that prevent the production of androgens and/or block the action of the AR inhibits prostate cancer growth. However, resistance to these drugs often occurs after 2-3 years as the patients develop castration-resistant prostate cancer (CRPC). In CRPC, a functional AR remains a key regulator. Early studies focused on the functional domains of the AR and its crucial role in the pathology. The elucidation of the structures of the AR DNA binding domain (DBD) and ligand binding domain (LBD) provides a new framework for understanding the functions of this receptor and leads to the development of rational drug design for the treatment of prostate cancer. An overview of androgen receptor structure and activity, its actions in prostate cancer, and how structural information and high-throughput screening have been or can be used for drug discovery are provided herein.

  5. A combined computational and structural model of the full-length human prolactin receptor (United States)

    Bugge, Katrine; Papaleo, Elena; Haxholm, Gitte W.; Hopper, Jonathan T. S.; Robinson, Carol V.; Olsen, Johan G.; Lindorff-Larsen, Kresten; Kragelund, Birthe B.


    The prolactin receptor is an archetype member of the class I cytokine receptor family, comprising receptors with fundamental functions in biology as well as key drug targets. Structurally, each of these receptors represent an intriguing diversity, providing an exceptionally challenging target for structural biology. Here, we access the molecular architecture of the monomeric human prolactin receptor by combining experimental and computational efforts. We solve the NMR structure of its transmembrane domain in micelles and collect structural data on overlapping fragments of the receptor with small-angle X-ray scattering, native mass spectrometry and NMR spectroscopy. Along with previously published data, these are integrated by molecular modelling to generate a full receptor structure. The result provides the first full view of a class I cytokine receptor, exemplifying the architecture of more than 40 different receptor chains, and reveals that the extracellular domain is merely the tip of a molecular iceberg.

  6. Structural Dynamics of Insulin Receptor and Transmembrane Signaling. (United States)

    Tatulian, Suren A


    The insulin receptor (IR) is a (αβ)2-type transmembrane tyrosine kinase that plays a central role in cell metabolism. Each αβ heterodimer consists of an extracellular ligand-binding α-subunit and a membrane-spanning β-subunit that comprises the cytoplasmic tyrosine kinase (TK) domain and the phosphorylation sites. The α- and β-subunits are linked via a single disulfide bridge, and the (αβ)2 tetramer is formed by disulfide bonds between the α-chains. Insulin binding induces conformational changes in IR that reach the intracellular β-subunit followed by a protein phosphorylation and activation cascade. Defects in this signaling process, including IR dysfunction caused by mutations, result in type 2 diabetes. Rational drug design aimed at treatment of diabetes relies on knowledge of the detailed structure of IR and the dynamic structural transformations during transmembrane signaling. Recent X-ray crystallographic studies have provided important clues about the mode of binding of insulin to IR, the resulting structural changes and their transmission to the TK domain, but a complete understanding of the structural basis underlying insulin signaling has not been achieved. This review presents a critical analysis of the current status of the structure-function relationship of IR, with a comparative assessment of the other IR family receptors, and discusses potential advancements that may provide insight into the molecular mechanism of insulin signaling.

  7. Crystal Structure of an LSD-Bound Human Serotonin Receptor

    Energy Technology Data Exchange (ETDEWEB)

    Wacker, Daniel; Wang, Sheng; McCorvy, John D.; Betz, Robin M.; Venkatakrishnan, A.J.; Levit, Anat; Lansu, Katherine; Schools, Zachary L.; Che, Tao; Nichols, David E.; Shoichet, Brian K.; Dror, Ron O.; Roth, Bryan L. (UNCSM); (UNC); (Stanford); (Stanford-MED); (UCSF)


    The prototypical hallucinogen LSD acts via serotonin receptors, and here we describe the crystal structure of LSD in complex with the human serotonin receptor 5-HT2B. The complex reveals conformational rearrangements to accommodate LSD, providing a structural explanation for the conformational selectivity of LSD’s key diethylamide moiety. LSD dissociates exceptionally slow from both 5-HT2BR and 5-HT2AR—a major target for its psychoactivity. Molecular dynamics (MD) simulations suggest that LSD’s slow binding kinetics may be due to a “lid” formed by extracellular loop 2 (EL2) at the entrance to the binding pocket. A mutation predicted to increase the mobility of this lid greatly accelerates LSD’s binding kinetics and selectively dampens LSD-mediated β-arrestin2 recruitment. This study thus reveals an unexpected binding mode of LSD; illuminates key features of its kinetics, stereochemistry, and signaling; and provides a molecular explanation for LSD’s actions at human serotonin receptors.

  8. Crystal Structure of the Human Cannabinoid Receptor CB1. (United States)

    Hua, Tian; Vemuri, Kiran; Pu, Mengchen; Qu, Lu; Han, Gye Won; Wu, Yiran; Zhao, Suwen; Shui, Wenqing; Li, Shanshan; Korde, Anisha; Laprairie, Robert B; Stahl, Edward L; Ho, Jo-Hao; Zvonok, Nikolai; Zhou, Han; Kufareva, Irina; Wu, Beili; Zhao, Qiang; Hanson, Michael A; Bohn, Laura M; Makriyannis, Alexandros; Stevens, Raymond C; Liu, Zhi-Jie


    Cannabinoid receptor 1 (CB1) is the principal target of Δ(9)-tetrahydrocannabinol (THC), a psychoactive chemical from Cannabis sativa with a wide range of therapeutic applications and a long history of recreational use. CB1 is activated by endocannabinoids and is a promising therapeutic target for pain management, inflammation, obesity, and substance abuse disorders. Here, we present the 2.8 Å crystal structure of human CB1 in complex with AM6538, a stabilizing antagonist, synthesized and characterized for this structural study. The structure of the CB1-AM6538 complex reveals key features of the receptor and critical interactions for antagonist binding. In combination with functional studies and molecular modeling, the structure provides insight into the binding mode of naturally occurring CB1 ligands, such as THC, and synthetic cannabinoids. This enhances our understanding of the molecular basis for the physiological functions of CB1 and provides new opportunities for the design of next-generation CB1-targeting pharmaceuticals.

  9. A combined computational and structural model of the full-length human prolactin receptor

    DEFF Research Database (Denmark)

    Bugge, Katrine Østergaard; Papaleo, Elena; Haxholm, Gitte Wolfsberg;


    The prolactin receptor is an archetype member of the class I cytokine receptor family, comprising receptors with fundamental functions in biology as well as key drug targets. Structurally, each of these receptors represent an intriguing diversity, providing an exceptionally challenging target...... for structural biology. Here, we access the molecular architecture of the monomeric human prolactin receptor by combining experimental and computational efforts. We solve the NMR structure of its transmembrane domain in micelles and collect structural data on overlapping fragments of the receptor with small......-angle X-ray scattering, native mass spectrometry and NMR spectroscopy. Along with previously published data, these are integrated by molecular modelling to generate a full receptor structure. The result provides the first full view of a class I cytokine receptor, exemplifying the architecture of more than...

  10. Neurotensin receptor-1 inducible palmitoylation is required for efficient receptor-mediated mitogenic-signaling within structured membrane microdomains



    Neurotensin receptor-1 (NTSR-1) is a G-protein coupled receptor (GPCR) that has been recently identified as a mediator of cancer progression. NTSR-1 and its endogenous ligand, neurotensin (NTS), are co-expressed in several breast cancer cell lines and breast cancer tumor samples. Based on our previously published study demonstrating that intact structured membrane microdomains (SMDs) are required for NTSR-1 mitogenic signaling, we hypothesized that regulated receptor palmitoylation is respons...

  11. Crystal structure of pentapeptide-independent chemotaxis receptor methyltransferase (CheR) reveals idiosyncratic structural determinants for receptor recognition. (United States)

    Batra, Monu; Sharma, Rajesh; Malik, Anjali; Dhindwal, Sonali; Kumar, Pravindra; Tomar, Shailly


    Chemotactic methyltransferase, CheR catalyse methylation of specific glutamate residues in the cytoplasmic domain of methyl-accepting chemotactic protein receptors (MCPRs). The methylation of MCPRs is essential for the chemical sensing and chemotactic bacterial mobility towards favorable chemicals or away from unfavorable ones. In this study, crystal structure of B. subtilis CheR (BsCheR) in complex with S-adenosyl-l-homocysteine (SAH) has been determined to 1.8Å resolution. This is the first report of crystal structure belonging to the pentapeptide-independent CheR (PICheR) class. Till date, only one crystal structure of CheR from S. typhimurium (StCheR) belonging to pentapeptide-dependent CheR (PDCheR) class is available. Structural analysis of BsCheR reveals a helix-X-helix motif (HXH) with Asp53 as the linker residue in the N-terminal domain. The key structural features of the PDCheR β-subdomain involved in the formation of a tight complex with the pentapeptide binding motif in MCPRs were found to be absent in the structure of BsCheR. Additionally, isothermal titration calorimetry (ITC) experiments were performed to investigate S-adenosyl-(l)-methionine (SAM) binding affinity and KD was determined to be 0.32mM. The structure of BsCheR reveals that mostly residues of the large C-terminal domain contribute to SAH binding, with contributions of few residues from the linker region and the N-terminal domain. Structural investigations and sequence analysis carried out in this study provide critical insights into the distinct receptor recognition mechanism of the PDCheR and PICheR methyltransferase classes.

  12. The Histamine H3 Receptor: Structure, Pharmacology, and Function. (United States)

    Nieto-Alamilla, Gustavo; Márquez-Gómez, Ricardo; García-Gálvez, Ana-Maricela; Morales-Figueroa, Guadalupe-Elide; Arias-Montaño, José-Antonio


    Among the four G protein-coupled receptors (H1-H4) identified as mediators of the biologic effects of histamine, the H3 receptor (H3R) is distinguished for its almost exclusive expression in the nervous system and the large variety of isoforms generated by alternative splicing of the corresponding mRNA. Additionally, it exhibits dual functionality as autoreceptor and heteroreceptor, and this enables H3Rs to modulate the histaminergic and other neurotransmitter systems. The cloning of the H3R cDNA in 1999 by Lovenberg et al. allowed for detailed studies of its molecular aspects. In this work, we review the characteristics of the H3R, namely, its structure, constitutive activity, isoforms, signal transduction pathways, regional differences in expression and localization, selective agonists, antagonists and inverse agonists, dimerization with other neurotransmitter receptors, and the main presynaptic and postsynaptic effects resulting from its activation. The H3R has attracted interest as a potential drug target for the treatment of several important neurologic and psychiatric disorders, such as Alzheimer and Parkinson diseases, Gilles de la Tourette syndrome, and addiction.

  13. Structure of the homodimeric androgen receptor ligand-binding domain (United States)

    Nadal, Marta; Prekovic, Stefan; Gallastegui, Nerea; Helsen, Christine; Abella, Montserrat; Zielinska, Karolina; Gay, Marina; Vilaseca, Marta; Taulès, Marta; Houtsmuller, Adriaan B.; van Royen, Martin E.; Claessens, Frank; Fuentes-Prior, Pablo; Estébanez-Perpiñá, Eva


    The androgen receptor (AR) plays a crucial role in normal physiology, development and metabolism as well as in the aetiology and treatment of diverse pathologies such as androgen insensitivity syndromes (AIS), male infertility and prostate cancer (PCa). Here we show that dimerization of AR ligand-binding domain (LBD) is induced by receptor agonists but not by antagonists. The 2.15-Å crystal structure of homodimeric, agonist- and coactivator peptide-bound AR-LBD unveils a 1,000-Å2 large dimerization surface, which harbours over 40 previously unexplained AIS- and PCa-associated point mutations. An AIS mutation in the self-association interface (P767A) disrupts dimer formation in vivo, and has a detrimental effect on the transactivating properties of full-length AR, despite retained hormone-binding capacity. The conservation of essential residues suggests that the unveiled dimerization mechanism might be shared by other nuclear receptors. Our work defines AR-LBD homodimerization as an essential step in the proper functioning of this important transcription factor. PMID:28165461

  14. Solution structure of the coxsackievirus and adenovirus receptor domain 2


    Jiang, Shaokai; Caffrey, Michael


    The coxsackievirus and adenovirus receptor (CAR) mediates entry of coxsackievirus and adenovirus. CAR possesses an extracellular region that is comprised of 2 immunoglobulin domains termed CAR–D1 and CAR–D2. In the present work, the solution structure of CAR–D2, consisting of residues 142–235 of human CAR, has been determined by NMR spectroscopy. CAR–D2 is shown to be a β-sandwich motif comprised of two β-sheets, which are stabilized by two disulfide bonds. The first β-sheet is comprised of β...

  15. Structure of the LDL receptor extracellular domain at endosomalpH

    Energy Technology Data Exchange (ETDEWEB)

    Rudenko, Gabby; Henry, Lisa; Henderson, Keith; Ichtchenko,Konstantin; Brown, Michael S.; Goldstein, Joseph L.; Deisenhofer, Johann


    The structure of the low-density lipoprotein receptor extracellular portion has been determined. The document proposes a mechanism for the release of lipoprotein in the endosome. Without this release, the mechanism of receptor recycling cannot function.

  16. Genomic organization of a receptor from sea anemones, structurally and evolutionary related to glycoprotein hormone receptors from mamals

    DEFF Research Database (Denmark)

    Vibede, N; Hauser, Frank; Williamson, M


    Abstract Cnidarians (e.g., sea anemones and corals) are the lowest animal group having a nervous system. Previously, we cloned a receptor from sea anemones that showed a strong structural similarity to the glycoprotein hormone (TSH, FSH, LH/CG) receptors from mammals. Here, we determine the genomic...... organization of this sea anemone receptor. The receptor gene contains eight introns that are all localized within a region coding for the large extracellular N terminus. These introns occur at the same positions and have the same intron phasing as eight introns in the genes coding for the mammalian...

  17. Structural basis for simvastatin competitive antagonism of complement receptor 3

    DEFF Research Database (Denmark)

    Jensen, Maria Risager; Bajic, Goran; Zhang, Xianwei;


    The complement system is an important part of the innate immune response to infection, but may also cause severe complications during inflammation. Small molecule antagonists to complement receptor (CR)3 have been widely sought, but a structural basis for their mode of action is not available. We...... report here on the structure of the human CR3 ligand-binding I domain in complex with simvastatin. Simvastatin targets the metal ion-dependent adhesion site of the open, ligand-binding conformation of the CR3 I domain by direct contact with the chelated Mg2+ ion. Simvastatin antagonizes I domain binding...... to the complement fragments iC3b and C3d, but not to intercellular adhesion molecule-1. By virtue of the I domain's wide distribution in binding kinetics to ligands, it was possible to identify ligand binding kinetics as discriminator for simvastatin antagonism. In static cellular experiments, 15-25 μM simvastatin...

  18. Crystal Structures of the Nuclear Receptor, Liver Receptor Homolog 1, Bound to Synthetic Agonists. (United States)

    Mays, Suzanne G; Okafor, C Denise; Whitby, Richard J; Goswami, Devrishi; Stec, Józef; Flynn, Autumn R; Dugan, Michael C; Jui, Nathan T; Griffin, Patrick R; Ortlund, Eric A


    Liver receptor homolog 1 (NR5A2, LRH-1) is an orphan nuclear hormone receptor that regulates diverse biological processes, including metabolism, proliferation, and the resolution of endoplasmic reticulum stress. Although preclinical and cellular studies demonstrate that LRH-1 has great potential as a therapeutic target for metabolic diseases and cancer, development of LRH-1 modulators has been difficult. Recently, systematic modifications to one of the few known chemical scaffolds capable of activating LRH-1 failed to improve efficacy substantially. Moreover, mechanisms through which LRH-1 is activated by synthetic ligands are entirely unknown. Here, we use x-ray crystallography and other structural methods to explore conformational changes and receptor-ligand interactions associated with LRH-1 activation by a set of related agonists. Unlike phospholipid LRH-1 ligands, these agonists bind deep in the pocket and do not interact with residues near the mouth nor do they expand the pocket like phospholipids. Unexpectedly, two closely related agonists with similar efficacies (GSK8470 and RJW100) exhibit completely different binding modes. The dramatic repositioning is influenced by a differential ability to establish stable face-to-face π-π-stacking with the LRH-1 residue His-390, as well as by a novel polar interaction mediated by the RJW100 hydroxyl group. The differing binding modes result in distinct mechanisms of action for the two agonists. Finally, we identify a network of conserved water molecules near the ligand-binding site that are important for activation by both agonists. This work reveals a previously unappreciated complexity associated with LRH-1 agonist development and offers insights into rational design strategies.

  19. Phosphotyrosine phosphatase R3 receptors: Origin, evolution and structural diversification (United States)

    Chicote, Javier U.; DeSalle, Rob; García-España, Antonio


    Subtype R3 phosphotyrosine phosphatase receptors (R3 RPTPs) are single-spanning membrane proteins characterized by a unique modular composition of extracellular fibronectin repeats and a single cytoplasmatic protein tyrosine phosphatase (PTP) domain. Vertebrate R3 RPTPs consist of five members: PTPRB, PTPRJ, PTPRH and PTPRO, which dephosphorylate tyrosine residues, and PTPRQ, which dephosphorylates phophoinositides. R3 RPTPs are considered novel therapeutic targets in several pathologies such as ear diseases, nephrotic syndromes and cancer. R3 RPTP vertebrate receptors, as well as their known invertebrate counterparts from animal models: PTP52F, PTP10D and PTP4e from the fruitfly Drosophila melanogaster and F44G4.8/DEP-1 from the nematode Caenorhabditis elegans, participate in the regulation of cellular activities including cell growth and differentiation. Despite sharing structural and functional properties, the evolutionary relationships between vertebrate and invertebrate R3 RPTPs are not fully understood. Here we gathered R3 RPTPs from organisms covering a broad evolutionary distance, annotated their structure and analyzed their phylogenetic relationships. We show that R3 RPTPs (i) have probably originated in the common ancestor of animals (metazoans), (ii) are variants of a single ancestral gene in protostomes (arthropods, annelids and nematodes); (iii) a likely duplication of this ancestral gene in invertebrate deuterostomes (echinodermes, hemichordates and tunicates) generated the precursors of PTPRQ and PTPRB genes, and (iv) R3 RPTP groups are monophyletic in vertebrates and have specific conserved structural characteristics. These findings could have implications for the interpretation of past studies and provide a framework for future studies and functional analysis of this important family of proteins. PMID:28257417

  20. New insights into the structural bases of activation of Cys-loop receptors. (United States)

    Bouzat, Cecilia


    Neurotransmitter receptors of the Cys-loop superfamily mediate rapid synaptic transmission throughout the nervous system, and include receptors activated by ACh, GABA, glycine and serotonin. They are involved in physiological processes, including learning and memory, and in neurological disorders, and they are targets for clinically relevant drugs. Cys-loop receptors assemble either from five copies of one type of subunit, giving rise to homomeric receptors, or from several types of subunits, giving rise to heteromeric receptors. Homomeric receptors are invaluable models for probing fundamental relationships between structure and function. Receptors contain a large extracellular domain that carries the binding sites and a transmembrane region that forms the ion pore. How the structural changes elicited by agonist binding are propagated through a distance of 50Å to the ion channel gate is central to understanding receptor function. Depending on the receptor subtype, occupancy of either two, as in the prototype muscle nicotinic receptor, or three binding sites, as in homomeric receptors, is required for full activation. The conformational changes initiated at the binding sites are propagated to the gate through the interface between the extracellular and transmembrane domains. This region forms a network that relays structural changes from the binding site towards the pore, and also contributes to open channel lifetime and rate of desensitization. Thus, this coupling region controls the beginning and duration of a synaptic response. Here we review recent advances in the molecular mechanism by which Cys-loop receptors are activated with particular emphasis on homomeric receptors.

  1. A combined computational and structural model of the full-length human prolactin receptor

    DEFF Research Database (Denmark)

    Bugge, Katrine Østergaard; Papaleo, Elena; Haxholm, Gitte Wolfsberg


    for structural biology. Here, we access the molecular architecture of the monomeric human prolactin receptor by combining experimental and computational efforts. We solve the NMR structure of its transmembrane domain in micelles and collect structural data on overlapping fragments of the receptor with small...

  2. Common structural basis for constitutive activity of the ghrelin receptor family

    DEFF Research Database (Denmark)

    Holst, Birgitte; Holliday, Nicholas D; Bach, Anders


    -independent signaling activity. The structurally homologous motilin receptor served as a constitutively silent control; upon agonist stimulation, however, it signaled with a similar efficacy to the three related receptors. The constitutive activity of the ghrelin receptor and of neurotensin receptor 2 through the G...... demonstrated that the epitope-tagged ghrelin receptor was constitutively internalized but could be trapped at the cell surface by an inverse agonist, whereas GPR39 remained at the cell surface. Mutational analysis showed that the constitutive activity of both the ghrelin receptor and GPR39 could systematically...

  3. Structural basis of transcobalamin recognition by human CD320 receptor (United States)

    Alam, Amer; Woo, Jae-Sung; Schmitz, Jennifer; Prinz, Bernadette; Root, Katharina; Chen, Fan; Bloch, Joël S.; Zenobi, Renato; Locher, Kaspar P.


    Cellular uptake of vitamin B12 (cobalamin) requires capture of transcobalamin (TC) from the plasma by CD320, a ubiquitous cell surface receptor of the LDLR family. Here we present the crystal structure of human holo-TC in complex with the extracellular domain of CD320, visualizing the structural basis of the TC-CD320 interaction. The observed interaction chemistry can rationalize the high affinity of CD320 for TC and lack of haptocorrin binding. The in vitro affinity and complex stability of TC-CD320 were quantitated using a solid-phase binding assay and thermostability analysis. Stable complexes with TC were also observed for the disease-causing CD320ΔE88 mutant and for the isolated LDLR-A2 domain. We also determined the structure of the TC-CD320ΔE88 complex, which revealed only minor changes compared with the wild-type complex. Finally, we demonstrate significantly reduced in vitro affinity of TC for CD320 at low pH, recapitulating the proposed ligand release during the endocytic pathway.

  4. Pharmacodynamic responses to combined treatment regimens with the calcium sensing receptor antagonist JTT-305/MK-5442 and alendronate in osteopenic ovariectomized rats. (United States)

    Fisher, John E; Scott, Kevin; Wei, Nan; Zhao, Jing Z; Cusick, Tara; Tijerina, Monica; Karanam, Bindhu; Duong, Le; Glantschnig, Helmut


    Parathyroid hormone (PTH) is the anabolic standard of care for patients with severe osteoporosis. The CaSR allosteric antagonist JTT-305/MK-5442, a PTH secretagogue, could offer an oral osteoanabolic treatment alternative for postmenopausal women with osteoporosis. Here we disclose the pharmacokinetic profile of JTT-305/MK-5442 and its activity on bone remodeling in ovariectomized (OVX) osteopenic rats. Daily treatments (0.3 to 2.4 mg/kg/d) for 12 weeks resulted in plateaued BMD increases (3.8 to 5.3%) at axial and appendicular skeletal sites. However, treatment effects were not statistically significant, in agreement with effects seen in animals treated with low dose PTH (1-84) (5 μg/kg/d). In a consecutive study we tested JTT-305/MK-5442 effects on bone formation in OVX-rats challenged with combined alendronate (ALN) treatment paradigms. At 7 month, JTT-305/MK-5442 treatment significantly increased BMD in lumbar vertebrae (LV), while no change in BMD was observed in femora or tibiae. ALN add-on co-treatment produced incremental increases in LV, distal femur (DF) and proximal tibia (PT) BMD over the respective ALN control. Histological analyses confirmed modest increases in mineralized surface (MS/BS) and bone formation rate (30.5±1.9%) on trabecular surfaces by JTT-305/MK-5442. As expected, ALN administration profoundly reduced bone formation, however, JTT-305/MK-5442 significantly stimulated MS/BS and BFR in ALN treated groups. In summary, JTT-305/MK-5442 acts as a PTH secretagogue in the osteopenic OVX-rat, eliciting consistent, though modest effects on remediation of BMD due to estrogen depletion. Induction of bone formation by JTT-305/MK-5442 at trabecular bone surfaces appears to be resilient to ALN-mediated suppression of bone formation. This study provides for the first time, a mechanistic evaluation of combination treatment of a PTH secretagogue with ALN.

  5. Structure-function study of the fourth transmembrane segment of the GABAρ1 receptor (United States)

    Estrada-Mondragón, Argel; Reyes-Ruiz, Jorge Mauricio; Martínez-Torres, Ataúlfo; Miledi, Ricardo


    The Cys-loop family of receptors mediates synaptic neurotransmission in the central nervous system of vertebrates. These receptors share several structural characteristics and assemble in the plasma membrane as multimers with fivefold symmetry. Of these, the ionotropic GABA receptors are key players in the pathogenesis of diseases like epilepsy, anxiety, and schizophrenia. Different experimental approaches have shed some light on the mechanisms behind the function of these receptors; but little is known about their structure at high resolution. Sequence homology with the nicotinic acetylcholine receptor predicts that ionotropic GABA receptors possess four transmembrane segments (TM1–4) and that TM2 forms the wall of the ion channel. However, the role of the other three segments is unclear. The GABAρ1 receptor plays a fundamental role in the regulation of neurotransmission along the visual pathway, is highly sensitive to GABA, and exhibits little desensitization. In our recent investigations of the role of TM4 in receptor function, a key residue in this domain (W475) was found to be involved in activation of the receptor. Here we have generated a structural model of the GABAρ1 receptor in silico and assessed its validity by electrophysiologically testing nine amino acid substitutions of W475 and deletions of the neighboring residues (Y474 and S476). The results identify a critical linkage between the ligand-binding domain and the TM4 domain and provide a framework for more detailed structure-function analyses of ionotropic GABA receptors. PMID:20876117

  6. Structure of MERS-CoV spike receptor-binding domain complexed with human receptor DPP4

    Institute of Scientific and Technical Information of China (English)

    Nianshuang Wang; Xuanling Shi; Liwei Jiang; Senyan Zhang; Dongli Wang; Pei Tong; Dongxing Guo


    The spike glycoprotein (S) of recently identified Middle East respiratory syndrome coronavirus (MERS-CoV) targets the cellular receptor,dipeptidyl peptidase 4 (DPP4).Sequence comparison and modeling analysis have revealed a putative receptor-binding domain (RBD) on the viral spike,which mediates this interaction.We report the 3.0 (A)resolution crystal structure of MERS-CoV RBD bound to the extracellular domain of human DPP4.Our results show that MERS-CoV RBD consists of a core and a receptor-binding subdomain.The receptor-binding subdomain interacts with DPP4 p-propeller but not its intrinsic hydrolase domain.MERS-CoV RBD and related SARS-CoV RBD share a high degree of structural similarity in their core subdomains,but are notably divergent in the receptorbinding subdomain.Mutagenesis studies have identified several key residues in the receptor-binding subdomain that are critical for viral binding to DPP4 and entry into the target cell.The atomic details at the interface between MERS-CoV RBD and DPP4 provide structural understanding of the virus and receptor interaction,which can guide development of therapeutics and vaccines against MERS-CoV infection.

  7. Structural and functional diversity of native brain neuronal nicotinic receptors. (United States)

    Gotti, Cecilia; Clementi, Francesco; Fornari, Alice; Gaimarri, Annalisa; Guiducci, Stefania; Manfredi, Irene; Moretti, Milena; Pedrazzi, Patrizia; Pucci, Luca; Zoli, Michele


    Neuronal nicotinic acetylcholine receptors (nAChRs) are a family of ligand-gated ion channels present in the central and peripheral nervous systems, that are permeable to mono- and divalent cations. They share a common basic structure but their pharmacological and functional properties arise from the wide range of different subunit combinations making up distinctive subtypes. nAChRs are involved in many physiological functions in the central and peripheral nervous systems, and are the targets of the widely used drug of abuse nicotine. In addition to tobacco dependence, changes in their number and/or function are associated with neuropsychiatric disorders, ranging from epilepsy to dementia. Although some of the neural circuits involved in the acute and chronic effects of nicotine have been identified, much less is known about which native nAChR subtypes are involved in specific physiological functions and pathophysiological conditions. We briefly review some recent findings concerning the structure and function of native nAChRs, focusing on the subtypes identified in the mesostriatal and habenulo-interpeduncular pathways, two systems involved in nicotine reinforcement and withdrawal. We also discuss recent findings concerning the effect of chronic nicotine on the expression of native subtypes.

  8. Structure-based receptor MIMICS targeted against bacterial superantigen toxins (United States)

    Gupta, Goutam; Hong-Geller, Elizabeth; Shiflett, Patrick R.; Lehnert, Nancy M.


    The invention provides therapeutic compositions useful in the treatment of bacterial superantigen mediated conditions, such as Toxic Shock Syndrome. The compositions comprise genetically engineered bifunctional polypeptides containing a specific T-cell receptor binding domain and a specific MHC class II receptor binding domain, each targeting non-overlapping epitopes on a superantigen molecule against which they are designed. The anti-superantigen "receptor mimetics" or "chimeras" are rationally designed to recreate the modality of superantigen binding directly to both the TCR and the MHC-II receptor, and are capable of acting as decoys for superantigen binding, effectively out-competing the host T-cell and MHC-II receptors, the natural host receptors.

  9. Structural basis and functions of abscisic acid receptors PYLs (United States)

    Zhang, Xing L.; Jiang, Lun; Xin, Qi; Liu, Yang; Tan, Jian X.; Chen, Zhong Z.


    Abscisic acid (ABA) plays a key role in many developmental processes and responses to adaptive stresses in plants. Recently, a new family of nucleocytoplasmic PYR/PYL/RCAR (PYLs) has been identified as bona fide ABA receptors. PYLs together with protein phosphatases type-2C (PP2Cs), Snf1 (Sucrose-non-fermentation 1)-related kinases subfamily 2 (SnRK2s) and downstream substrates constitute the core ABA signaling network. Generally, PP2Cs inactivate SnRK2s kinases by physical interaction and direct dephosphorylation. Upon ABA binding, PYLs change their conformations and then contact and inhibit PP2Cs, thus activating SnRK2s. Here, we reviewed the recent progress in research regarding the structures of the core signaling pathways of ABA, including the (+)-ABA, (−)-ABA and ABA analogs pyrabactin as well as 6AS perception by PYLs, SnRK2s mimicking PYLs in binding PP2Cs. PYLs inhibited PP2Cs in both the presence and absence of ABA and activated SnRK2s. The present review elucidates multiple ABA signal perception and transduction by PYLs, which might shed light on how to design small chemical compounds for improving plant performance in the future. PMID:25745428

  10. Structural basis for activation of G-protein-coupled receptors

    DEFF Research Database (Denmark)

    Gether, Ulrik; Asmar, Fazila; Meinild, Anne Kristine


    -type and mutant beta2-adrenergic receptors purified from Sf-9 insect cells. Our studies have also raised important questions regarding kinetics of receptors activation. These questions should be addressed in the future by application of techniques that will allow for simultaneous measurement of conformational...

  11. Structure of the human M2 muscarinic acetylcholine receptor bound to an antagonist

    Energy Technology Data Exchange (ETDEWEB)

    Haga, Kazuko; Kruse, Andrew C.; Asada, Hidetsugu; Yurugi-Kobayashi, Takami; Shiroishi, Mitsunori; Zhang, Cheng; Weis, William I.; Okada, Tetsuji; Kobilka, Brian K.; Haga, Tatsuya; Kobayashi, Takuya (Stanford-MED); (Kyoto); (Gakushuin); (Kyushu)


    The parasympathetic branch of the autonomic nervous system regulates the activity of multiple organ systems. Muscarinic receptors are G-protein-coupled receptors that mediate the response to acetylcholine released from parasympathetic nerves. Their role in the unconscious regulation of organ and central nervous system function makes them potential therapeutic targets for a broad spectrum of diseases. The M2 muscarinic acetylcholine receptor (M2 receptor) is essential for the physiological control of cardiovascular function through activation of G-protein-coupled inwardly rectifying potassium channels, and is of particular interest because of its extensive pharmacological characterization with both orthosteric and allosteric ligands. Here we report the structure of the antagonist-bound human M2 receptor, the first human acetylcholine receptor to be characterized structurally, to our knowledge. The antagonist 3-quinuclidinyl-benzilate binds in the middle of a long aqueous channel extending approximately two-thirds through the membrane. The orthosteric binding pocket is formed by amino acids that are identical in all five muscarinic receptor subtypes, and shares structural homology with other functionally unrelated acetylcholine binding proteins from different species. A layer of tyrosine residues forms an aromatic cap restricting dissociation of the bound ligand. A binding site for allosteric ligands has been mapped to residues at the entrance to the binding pocket near this aromatic cap. The structure of the M2 receptor provides insights into the challenges of developing subtype-selective ligands for muscarinic receptors and their propensity for allosteric regulation.

  12. Structure, function and physiological consequences of virally encoded chemokine seven transmembrane receptors

    DEFF Research Database (Denmark)

    Rosenkilde, M M; Smit, M J; Waldhoer, M


    A number of human and animal herpes viruses encode G-protein coupled receptors with seven transmembrane (7TM) segments-most of which are clearly related to human chemokine receptors. It appears, that these receptors are used by the virus for immune evasion, cellular transformation, tissue targeting......, and possibly for cell entry. In addition, many virally-encoded chemokine 7TM receptors have been suggested to be causally involved in pathogenic phenotypes like Kaposi sarcoma, atherosclerosis, HIV-infection and tumour development. The role of these receptors during the viral life cycle and in viral...... pathogenesis is still poorly understood. Here we focus on the current knowledge of structure, function and trafficking patterns of virally encoded chemokine receptors and further address the putative roles of these receptors in virus survival and host -cell and/or -immune system modulation. Finally, we...

  13. Structure-function relationships for the interleukin 2 receptor system

    Directory of Open Access Journals (Sweden)

    Richard J. Robb


    Full Text Available Receptors for interleukin 2 (IL-2 esit in at least three forms which differ in their subunit compositio, their affinity for ligand and their ability to mediate a cellular reponse. Type I receptors occur following cellular acitivation and consist of the 55,000 m. w. glycoprotein Tac. These receptors bind IL-2 with a low affinity, do not internalize ligand and have not been definitively associated with any response. Type II receptors, on the other hand, conssit of one or more glycoproteins of 70,000 m. w. which have been termed "beta ([beta] chains." They bind IL-2 with an intermediate affinity and rapidly internalize the ligand. [Beta] proteins mediate many cellular IL-2-dependent reponses, including the short-term activation of natural killer cells and the induction of Tac protein expression. Type III receptors consist of a ternary complex of the Tac protein, the [beta] chain(s and IL-2. They are characterized by a paricularly high affinity for ligand association. Type III receptors also internalize ligand and mediate IL-2-dependent responses at low factor concentrations. The identification of two independent IL-2-binding molecules, Tac and [beta], thus provides the elusive molecular explanation for the differences in IL-2 receptor affinity and suggests the potential for selective therapeutic manipulation of IL-2 reponses.

  14. [Opioid receptors of the CNS: function, structure and distribution]. (United States)

    Slamberová, R


    Even though the alkaloids of opium, such as morphine and codeine, were isolated at the beginning of 19th century, the opioid receptors were not determined until 1970's. The discovery of endogenous opioid peptides, such as endorphins, enkephalins and dynorphins, has helped to differentiate between the specific opioid receptor subtypes, mu, delta and kappa, that are used up to now. Opioid receptors are distributed in the central nervous system unevenly. Each receptor subtype has its own specific and nonspecific agonists and antagonists. Opioides, as exogenous opioid receptor agonists, are drugs that are often used in medicine for their analgesic effects, but they are also some of the most heavily abused drugs in the world. Opioides may also induce long-term changes in the numbers and binding activities of opioid receptors. Some of our studies in fact demonstrate that prenatal morphine exposure can alter opioid receptors of adult rats. This may begin to provide insight into the sources of some of the morphological and behavioral changes in the progeny of mothers that received or abused opioides during pregnancy.

  15. Muscarinic acetylcholine receptor subtypes: localization and structure/function

    DEFF Research Database (Denmark)

    Brann, M R; Ellis, J; Jørgensen, H


    Based on the sequence of the five cloned muscarinic receptor subtypes (m1-m5), subtype selective antibody and cDNA probes have been prepared. Use of these probes has demonstrated that each of the five subtypes has a markedly distinct distribution within the brain and among peripheral tissues....... The distributions of these subtypes and their potential physiological roles are discussed. By use of molecular genetic manipulation of cloned muscarinic receptor cDNAs, the regions of muscarinic receptors that specify G-protein coupling and ligand binding have been defined in several recent studies. Overall...

  16. The structure of the third intracellular loop of the muscarinic acetylcholine receptor M2 subtype. (United States)

    Ichiyama, Susumu; Oka, Yoshiaki; Haga, Kazuko; Kojima, Shuichi; Tateishi, Yukihiro; Shirakawa, Masahiro; Haga, Tatsuya


    We have examined whether the long third intracellular loop (i3) of the muscarinic acetylcholine receptor M2 subtype has a rigid structure. Circular dichroism (CD) and nuclear magnetic resonance spectra of M2i3 expressed in and purified from Escherichia coli indicated that M2i3 consists mostly of random coil. In addition, the differential CD spectrum between the M2 and M2deltai3 receptors, the latter of which lacks most of i3 except N- and C-terminal ends, gave no indication of secondary structure. These results suggest that the central part of i3 of the M2 receptor has a flexible structure.

  17. The Structure of the Mouse Serotonin 5-HT3 Receptor in Lipid Vesicles. (United States)

    Kudryashev, Mikhail; Castaño-Díez, Daniel; Deluz, Cédric; Hassaine, Gherici; Grasso, Luigino; Graf-Meyer, Alexandra; Vogel, Horst; Stahlberg, Henning


    The function of membrane proteins is best understood if their structure in the lipid membrane is known. Here, we determined the structure of the mouse serotonin 5-HT3 receptor inserted in lipid bilayers to a resolution of 12 Å without stabilizing antibodies by cryo electron tomography and subtomogram averaging. The reconstruction reveals protein secondary structure elements in the transmembrane region, the extracellular pore, and the transmembrane channel pathway, showing an overall similarity to the available X-ray model of the truncated 5-HT3 receptor determined in the presence of a stabilizing nanobody. Structural analysis of the 5-HT3 receptor embedded in a lipid bilayer allowed the position of the membrane to be determined. Interactions between the densely packed receptors in lipids were visualized, revealing that the interactions were maintained by the short horizontal helices. In combination with methodological improvements, our approach enables the structural analysis of membrane proteins in response to voltage and ligand gating.

  18. Selective Negative Allosteric Modulation Of Metabotropic Glutamate Receptors - A Structural Perspective of Ligands and Mutants

    DEFF Research Database (Denmark)

    Harpsøe, Kasper; Isberg, Vignir; Tehan, Benjamin G


    modulators. In this analysis, we make the first comprehensive structural comparison of all metabotropic glutamate receptors, placing selective negative allosteric modulators and critical mutants into the detailed context of the receptor binding sites. A better understanding of how the different m...

  19. Structure of the murine constitutive androstane receptor complexed to androstenol: a molecular basis for inverse agonism

    Energy Technology Data Exchange (ETDEWEB)

    Shan, L.; Vincent, J.; Brunzelle, J.S.; Dussault, I.; Lin, M.; Ianculescu, I.; Sherman, M.A.; Forman, B.M.; Fernandez, E. (Tennesse)


    The nuclear receptor CAR is a xenobiotic responsive transcription factor that plays a central role in the clearance of drugs and bilirubin while promoting cocaine and acetaminophen toxicity. In addition, CAR has established a 'reverse' paradigm of nuclear receptor action where the receptor is active in the absence of ligand and inactive when bound to inverse agonists. We now report the crystal structure of murine CAR bound to the inverse agonist androstenol. Androstenol binds within the ligand binding pocket, but unlike many nuclear receptor ligands, it makes no contacts with helix H12/AF2. The transition from constitutive to basal activity (androstenol bound) appears to be associated with a ligand-induced kink between helices H10 and H11. This disrupts the previously predicted salt bridge that locks H12 in the transcriptionally active conformation. This mechanism of inverse agonism is distinct from traditional nuclear receptor antagonists thereby offering a new approach to receptor modulation.

  20. Cardiac nuclear receptors: architects of mitochondrial structure and function. (United States)

    Vega, Rick B; Kelly, Daniel P


    The adult heart is uniquely designed and equipped to provide a continuous supply of energy in the form of ATP to support persistent contractile function. This high-capacity energy transduction system is the result of a remarkable surge in mitochondrial biogenesis and maturation during the fetal-to-adult transition in cardiac development. Substantial evidence indicates that nuclear receptor signaling is integral to dynamic changes in the cardiac mitochondrial phenotype in response to developmental cues, in response to diverse postnatal physiologic conditions, and in disease states such as heart failure. A subset of cardiac-enriched nuclear receptors serve to match mitochondrial fuel preferences and capacity for ATP production with changing energy demands of the heart. In this Review, we describe the role of specific nuclear receptors and their coregulators in the dynamic control of mitochondrial biogenesis and energy metabolism in the normal and diseased heart.

  1. Structural Insights into the Interactions between Platelet Receptors and Fibrillar Collagen*


    Herr, Andrew B.; Farndale, Richard W.


    Collagen peptides have been used to identify binding sites for several important collagen receptors, including integrin α2β1, glycoprotein VI, and von Willebrand factor. In parallel, the structures of these collagen receptors have been reported, and their interactions with collagen peptides have been studied. Recently, the three-dimensional structure of the intact type I collagen fiber from rat tail tendon has been resolved by fiber diffraction. It is now possible to map the binding sites of ...

  2. Crystal structure of the human beta2 adrenergic G-protein-coupled receptor

    DEFF Research Database (Denmark)

    Rasmussen, Søren Gøgsig Faarup; Choi, Hee-Jung; Rosenbaum, Daniel M;


    Structural analysis of G-protein-coupled receptors (GPCRs) for hormones and neurotransmitters has been hindered by their low natural abundance, inherent structural flexibility, and instability in detergent solutions. Here we report a structure of the human beta2 adrenoceptor (beta2AR), which...

  3. Illuminating the structure and function of Cys-loop receptors

    DEFF Research Database (Denmark)

    Pless, Stephan Alexander; Lynch, Joseph W


    Cys-loop receptors are an important class of ligand-gated ion channels. They mediate fast synaptic neurotransmission, are implicated in various 'channelopathies' and are important pharmacological targets. Recent progress in X-ray crystallography and electron microscopy has provided a considerable...

  4. Structure-based drug design for G protein-coupled receptors. (United States)

    Congreve, Miles; Dias, João M; Marshall, Fiona H


    Our understanding of the structural biology of G protein-coupled receptors has undergone a transformation over the past 5 years. New protein-ligand complexes are described almost monthly in high profile journals. Appreciation of how small molecules and natural ligands bind to their receptors has the potential to impact enormously how medicinal chemists approach this major class of receptor targets. An outline of the key topics in this field and some recent examples of structure- and fragment-based drug design are described. A table is presented with example views of each G protein-coupled receptor for which there is a published X-ray structure, including interactions with small molecule antagonists, partial and full agonists. The possible implications of these new data for drug design are discussed.

  5. Concomitant Action of Structural Elements and Receptor Phosphorylation Determines Arrestin-3 Interaction with the Free Fatty Acid Receptor FFA4* (United States)

    Butcher, Adrian J.; Hudson, Brian D.; Shimpukade, Bharat; Alvarez-Curto, Elisa; Prihandoko, Rudi; Ulven, Trond; Milligan, Graeme; Tobin, Andrew B.


    In addition to being nutrients, free fatty acids act as signaling molecules by activating a family of G protein-coupled receptors. Among these is FFA4, previously called GPR120, which responds to medium and long chain fatty acids, including health-promoting ω-3 fatty acids, which have been implicated in the regulation of metabolic and inflammatory responses. Here we show, using mass spectrometry, mutagenesis, and phosphospecific antibodies, that agonist-regulated phosphorylation of the human FFA4 receptor occurred primarily at five residues (Thr347, Thr349, Ser350, Ser357, and Ser360) in the C-terminal tail. Mutation of these residues reduced both the efficacy and potency of ligand-mediated arrestin-3 recruitment as well as affecting recruitment kinetics. Combined mutagenesis of all five of these residues was insufficient to fully abrogate interaction with arrestin-3, but further mutagenesis of negatively charged residues revealed additional structural components for the interaction with arrestin-3 within the C-terminal tail of the receptor. These elements consist of the acidic residues Glu341, Asp348, and Asp355 located close to the phosphorylation sites. Receptor phosphorylation thus operates in concert with structural elements within the C-terminal tail of FFA4 to allow for the recruitment of arrestin-3. Importantly, these mechanisms of arrestin-3 recruitment operate independently from Gq/11 coupling, thereby offering the possibility that ligands showing stimulus bias could be developed that exploit these differential coupling mechanisms. Furthermore, this provides a strategy for the design of biased receptors to probe physiologically relevant signaling. PMID:24817122

  6. X-ray structures define human P2X3 receptor gating cycle and antagonist action (United States)

    Mansoor, Steven E.; Lü, Wei; Oosterheert, Wout; Shekhar, Mrinal; Tajkhorshid, Emad; Gouaux, Eric


    P2X receptors are trimeric, non-selective cation channels activated by ATP that have important roles in the cardiovascular, neuronal and immune systems. Despite their central function in human physiology and although they are potential targets of therapeutic agents, there are no structures of human P2X receptors. The mechanisms of receptor desensitization and ion permeation, principles of antagonism, and complete structures of the pore-forming transmembrane domains of these receptors remain unclear. Here we report X-ray crystal structures of the human P2X3 receptor in apo/resting, agonist-bound/open-pore, agonist-bound/closed-pore/desensitized and antagonist-bound/closed states. The open state structure harbours an intracellular motif we term the ‘cytoplasmic cap’, which stabilizes the open state of the ion channel pore and creates lateral, phospholipid-lined cytoplasmic fenestrations for water and ion egress. The competitive antagonists TNP-ATP and A-317491 stabilize the apo/resting state and reveal the interactions responsible for competitive inhibition. These structures illuminate the conformational rearrangements that underlie P2X receptor gating and provide a foundation for the development of new pharmacological agents.

  7. Structural Characterization of the Hemagglutinin Receptor Specificity from the 2009 H1N1 Influenza Pandemic

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Rui; McBride, Ryan; Nycholat, Corwin M.; Paulson, James C.; Wilson, Ian A. (Scripps)


    Influenza virus hemagglutinin (HA) is the viral envelope protein that mediates viral attachment to host cells and elicits membrane fusion. The HA receptor-binding specificity is a key determinant for the host range and transmissibility of influenza viruses. In human pandemics of the 20th century, the HA normally has acquired specificity for human-like receptors before widespread infection. Crystal structures of the H1 HA from the 2009 human pandemic (A/California/04/2009 [CA04]) in complex with human and avian receptor analogs reveal conserved recognition of the terminal sialic acid of the glycan ligands. However, favorable interactions beyond the sialic acid are found only for {alpha}2-6-linked glycans and are mediated by Asp190 and Asp225, which hydrogen bond with Gal-2 and GlcNAc-3. For {alpha}2-3-linked glycan receptors, no specific interactions beyond the terminal sialic acid are observed. Our structural and glycan microarray analyses, in the context of other high-resolution HA structures with {alpha}2-6- and {alpha}2-3-linked glycans, now elucidate the structural basis of receptor-binding specificity for H1 HAs in human and avian viruses and provide a structural explanation for the preference for {alpha}2-6 siaylated glycan receptors for the 2009 pandemic swine flu virus.

  8. Structural Disorder in the Complex of Human Pregnane X Receptor and the Macrolide Antibiotic Rifampicin

    Energy Technology Data Exchange (ETDEWEB)

    Chrencik, Jill E.; Orans, Jillian; Moore, Linda B.; Xue, Yu; Peng, Li; Collins, Jon L.; Wisely, G. Bruce; Lambert, Millard H.; Kliewer, Steven A.; Redinbo, Matthew R. (U. of Texas-SMED); (UNC)


    The human nuclear xenobiotic receptor, pregnane X receptor (PXR), detects a variety of structurally distinct endogenous and xenobiotic compounds and controls expression of genes central to drug and cholesterol metabolism. The macrolide antibiotic rifampicin, a front-line treatment for tuberculosis, is an established PXR agonist and, at 823 Da, is one of the largest known ligands for the receptor. We present the 2.8 {angstrom} crystal structure of the ligand-binding domain of human PXR in complex with rifampicin. We also use structural and mutagenesis data to examine the origins of the directed promiscuity exhibited by the PXRs across species. Three structurally flexible loops adjacent to the ligand-binding pocket of PXR are disordered in this crystal structure, including the 200-210 region that is part of a sequence insert novel to the promiscuous PXRs relative to other members of the nuclear receptor superfamily. The 4-methyl-1-piperazinyl ring of rifampicin, which would lie adjacent to the disordered protein regions, is also disordered and not observed in the structure. Taken together, our results indicate that one wall of the PXR ligand-binding cavity can remain flexible even when the receptor is in complex with an activating ligand. These observations highlight the key role that structural flexibility plays in PXR's promiscuous response to xenobiotics.

  9. Molecular dynamics simulation of the structure and dynamics of 5-HT3 serotonin receptor (United States)

    Antonov, M. Yu.; Popinako, A. V.; Prokopiev, G. A.


    In this work, we investigated structure, dynamics and ion transportation in transmembrane domain of the 5-HT3 serotonin receptor. High-resolution (0.35 nm) structure of the 5-HT3 receptor in complex with stabilizing nanobodies was determined by protein crystallography in 2014 (Protein data bank (PDB) code 4PIR). Transmembrane domain of the structure was prepared in complex with explicit membrane environment (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC)) and solvent (TIP3P water model). Molecular dynamics protocols for simulation and stabilization of the transmembrane domain of the 5-HT3 receptor model were developed and 60 ns simulation of the structure was conducted in order to explore structural parameters of the system. We estimated the mean force profile for Na+ ions using umbrella sampling method.

  10. Structural and Molecular Evidence Suggesting Coronavirus-driven Evolution of Mouse Receptor. (United States)

    Peng, Guiqing; Yang, Yang; Pasquarella, Joseph R; Xu, Liqing; Qian, Zhaohui; Holmes, Kathryn V; Li, Fang


    Hosts and pathogens are locked in an evolutionary arms race. To infect mice, mouse hepatitis coronavirus (MHV) has evolved to recognize mouse CEACAM1a (mCEACAM1a) as its receptor. To elude MHV infections, mice may have evolved a variant allele from the Ceacam1a gene, called Ceacam1b, producing mCEACAM1b, which is a much poorer MHV receptor than mCEACAM1a. Previous studies showed that sequence differences between mCEACAM1a and mCEACAM1b in a critical MHV-binding CC' loop partially account for the low receptor activity of mCEACAM1b, but detailed structural and molecular mechanisms for the differential MHV receptor activities of mCEACAM1a and mCEACAM1b remained elusive. Here we have determined the crystal structure of mCEACAM1b and identified the structural differences and additional residue differences between mCEACAM1a and mCEACAM1b that affect MHV binding and entry. These differences include conformational alterations of the CC' loop as well as residue variations in other MHV-binding regions, including β-strands C' and C'' and loop C'C''. Using pseudovirus entry and protein-protein binding assays, we show that substituting the structural and residue features from mCEACAM1b into mCEACAM1a reduced the viral receptor activity of mCEACAM1a, whereas substituting the reverse changes from mCEACAM1a into mCEACAM1b increased the viral receptor activity of mCEACAM1b. These results elucidate the detailed molecular mechanism for how mice may have kept pace in the evolutionary arms race with MHV by undergoing structural and residue changes in the MHV receptor, providing insight into this possible example of pathogen-driven evolution of a host receptor protein.

  11. Structural basis for bitter taste receptor activation and its potential role in targeting diabetes

    Directory of Open Access Journals (Sweden)

    Ravinder Abrol


    Full Text Available Background: Taste receptors are G protein-coupled receptors that, besides being present in the taste buds, have also been shown to be present in the gastrointestinal (GI system, respiratory system, and brain, though their function at these locations is not well understood. Objective: To understand the nutrient mediated release of gut peptides like GLP-1 from enteroendocrine L-cells of the GI system, we focused on a bitter taste receptor TAS2R38 (based on animal models to investigate the structural basis of its potential role in the release of gut peptides. Methods: The atomic-level structure of bitter taste receptor TAS2R38 was predicted using GEnSeMBLE, a first-principle based GPCR structure prediction method. These structures were obtained for the dominant taster haplotype (PAV as well as for the nontaster haplotype (AVI of the receptor. The known ligands phenylthiocarbamide (PTC and 6-n-propylthiouracil (PTU were docked to these structures to provide a structural basis for the taster and nontaster haplotypes. Results: Docking of known ligands PTU and PTC to taster and nontaster haplotypes of the bitter taste receptor showed a backbone hydrogen bond to residue 262 in taster but not in nontaster haplotype, suggesting a potential mode of action of these molecules in the activation of the bitter taste receptor. Conclusion: These results, combined with the ability of PTC to release gut peptides from in vitro models of the enteroendocrine L-cells, suggest a potential structural basis for TAS2R38 activation that can lead to the release of those peptides. This release has a therapeutic benefit for type 2 diabetes and implies a role for bitter tasting (but safe natural compounds targeting TAS2R38 as potential drug candidates for curing type 2 diabetes.

  12. Cloning and characterization of a human orphan family C G-protein coupled receptor GPRC5D

    DEFF Research Database (Denmark)

    Bräuner-Osborne, H; Jensen, A A; Sheppard, P O


    predicted to encode an additional subtype. The full length coding regions of mouse mGprc5d and human GPRC5D were cloned and shown to contain predicted open reading frames of 300 and 345 amino acids, respectively. GPRC5D has seven putative transmembrane segments and is expressed in the cell membrane...... intestine, whereas other organs only express a subset of the genes. In an attempt to delineate the signal transduction pathway(s) of the orphan receptors, a series of chimeric receptors containing the amino terminal domain of the calcium sensing receptor or metabotropic glutamate receptor subtype 1...

  13. Structure of the CCR5 Chemokine Receptor-HIV Entry Inhibitor Maraviroc Complex

    Energy Technology Data Exchange (ETDEWEB)

    Tan, Qiuxiang; Zhu, Ya; Li, Jian; Chen, Zhuxi; Han, Gye Won; Kufareva, Irina; Li, Tingting; Ma, Limin; Fenalti, Gustavo; Li, Jing; Zhang, Wenru; Xie, Xin; Yang, Huaiyu; Jiang, Hualiang; Cherezov, Vadim; Liu, Hong; Stevens, Raymond C.; Zhao, Qiang; Wu, Beili [Scripps; (Chinese Aca. Sci.); (UCSD)


    The CCR5 chemokine receptor acts as a co-receptor for HIV-1 viral entry. Here we report the 2.7 angstrom–resolution crystal structure of human CCR5 bound to the marketed HIV drug maraviroc. The structure reveals a ligand-binding site that is distinct from the proposed major recognition sites for chemokines and the viral glycoprotein gp120, providing insights into the mechanism of allosteric inhibition of chemokine signaling and viral entry. A comparison between CCR5 and CXCR4 crystal structures, along with models of co-receptor–gp120-V3 complexes, suggests that different charge distributions and steric hindrances caused by residue substitutions may be major determinants of HIV-1 co-receptor selectivity. These high-resolution insights into CCR5 can enable structure-based drug discovery for the treatment of HIV-1 infection.

  14. Structural features of the Nogo receptor signaling complexes at the neuron/myelin interface. (United States)

    Saha, Nayanendu; Kolev, Momchil; Nikolov, Dimitar B


    Upon spinal cord injury, the central nervous system axons are unable to regenerate, partially due to the repulsive action of myelin inhibitors, such as the myelin-associated glycoprotein (MAG), Nogo-A and the oligodendrocyte myelin glycoprotein (OMgp). These inhibitors bind and signal through a single receptor/co-receptor complex that comprises of NgR1/LINGO-1 and either p75 or TROY, triggering intracellular downstream signaling that impedes the re-growth of axons. Structure-function analysis of myelin inhibitors and their neuronal receptors, particularly the NgRs, have provided novel information regarding the molecular details of the inhibitor/receptor/co-receptor interactions. Structural and biochemical studies have revealed the architecture of many of these proteins and identified the molecular regions important for assembly of the inhibitory signaling complexes. It was also recently shown that gangliosides, such as GT1b, mediate receptor/co-receptor binding. In this review, we highlight these studies and summarize our current understanding of the multi-protein cell-surface complexes mediating inhibitory signaling events at the neuron/myelin interface.

  15. Structures and receptor binding of hemagglutinins from human-infecting H7N9 influenza viruses. (United States)

    Shi, Yi; Zhang, Wei; Wang, Fei; Qi, Jianxun; Wu, Ying; Song, Hao; Gao, Feng; Bi, Yuhai; Zhang, Yanfang; Fan, Zheng; Qin, Chengfeng; Sun, Honglei; Liu, Jinhua; Haywood, Joel; Liu, Wenjun; Gong, Weimin; Wang, Dayan; Shu, Yuelong; Wang, Yu; Yan, Jinghua; Gao, George F


    An avian-origin human-infecting influenza (H7N9) virus was recently identified in China. We have evaluated the viral hemagglutinin (HA) receptor-binding properties of two human H7N9 isolates, A/Shanghai/1/2013 (SH-H7N9) (containing the avian-signature residue Gln(226)) and A/Anhui/1/2013 (AH-H7N9) (containing the mammalian-signature residue Leu(226)). We found that SH-H7N9 HA preferentially binds the avian receptor analog, whereas AH-H7N9 HA binds both avian and human receptor analogs. Furthermore, an AH-H7N9 mutant HA (Leu(226) → Gln) was found to exhibit dual receptor-binding property, indicating that other amino acid substitutions contribute to the receptor-binding switch. The structures of SH-H7N9 HA, AH-H7N9 HA, and its mutant in complex with either avian or human receptor analogs show how AH-H7N9 can bind human receptors while still retaining the avian receptor-binding property.

  16. Structure of adenovirus bound to cellular receptor car (United States)

    Freimuth, Paul I.


    Disclosed is a mutant CAR-DI-binding adenovirus which has a genome comprising one or more mutations in sequences which encode the fiber protein knob domain wherein the mutation causes the encoded viral particle to have a significantly weakened binding affinity for CAR-DI relative to wild-type adenovirus. Such mutations may be in sequences which encode either the AB loop, or the HI loop of the fiber protein knob domain. Specific residues and mutations are described. Also disclosed is a method for generating a mutant adenovirus which is characterized by a receptor binding affinity or specificity which differs substantially from wild type.

  17. Effect of Receptor Structure and Length on the Wrapping of a Nanoparticle by a Lipid Membrane

    Directory of Open Access Journals (Sweden)

    Haizhen Zhang


    Full Text Available Nanoparticles have been considered as a type of powerful tool to deliver drugs and genes into cells for disease diagnosis and therapies. It has been generally accepted that the internalization of nanoparticles into cells is mostly realized by receptor-mediated endocytosis. However, for the influence of structural factors of receptors on endocytosis, this is still largely unknown. In this paper, computer simulations are applied to investigate the effects of structure (i.e., the number of constituent chains of the receptor and the length of the receptor on the wrapping behavior of nanoparticles by the lipid membrane, which is a key step of receptor-medicated endocytosis. It is found that these structural factors of receptors have strong effects on the nanoparticle’s final interaction configuration with the membrane in the simulations, such as adhering on the membrane surface or being partly or fully wrapped by the membrane. Furthermore, in some cases, the rupture of the lipid membrane occurs. These results are helpful for the understanding of endocytosis and the preparation of advanced nanoscale drug-delivery vectors.

  18. Structural Basis for Antibody Discrimination between Two Hormones That Recognize the Parathyroid Hormone Receptor

    Energy Technology Data Exchange (ETDEWEB)

    McKinstry, William J.; Polekhina, Galina; Diefenbach-Jagger, Hannelore; Ho, Patricia W.M.; Sato, Koh; Onuma, Etsuro; Gillespie, Matthew T.; Martin, T. John; Parker, Michael W.; (SVIMR-A); (Chugai); (Melbourne)


    Parathyroid hormone-related protein (PTHrP) plays a vital role in the embryonic development of the skeleton and other tissues. When it is produced in excess by cancers it can cause hypercalcemia, and its local production by breast cancer cells has been implicated in the pathogenesis of bone metastasis formation in that disease. Antibodies have been developed that neutralize the action of PTHrP through its receptor, parathyroid hormone receptor 1, without influencing parathyroid hormone action through the same receptor. Such neutralizing antibodies against PTHrP are therapeutically effective in animal models of the humoral hypercalcemia of malignancy and of bone metastasis formation. We have determined the crystal structure of the complex between PTHrP (residues 1-108) and a neutralizing monoclonal anti-PTHrP antibody that reveals the only point of contact is an {alpha}-helical structure extending from residues 14-29. Another striking feature is that the same residues that interact with the antibody also interact with parathyroid hormone receptor 1, showing that the antibody and the receptor binding site on the hormone closely overlap. The structure explains how the antibody discriminates between the two hormones and provides information that could be used in the development of novel agonists and antagonists of their common receptor.

  19. The genomic structure of the human UFO receptor. (United States)

    Schulz, A S; Schleithoff, L; Faust, M; Bartram, C R; Janssen, J W


    Using a DNA transfection-tumorigenicity assay we have recently identified the UFO oncogene. It encodes a tyrosine kinase receptor characterized by the juxtaposition of two immunoglobulin-like and two fibronectin type III repeats in its extracellular domain. Here we describe the genomic organization of the human UFO locus. The UFO receptor is encoded by 20 exons that are distributed over a region of 44 kb. Different isoforms of UFO mRNA are generated by alternative splicing of exon 10 and differential usage of two imperfect polyadenylation sites resulting in the presence or absence of 1.5-kb 3' untranslated sequences. Primer extension and S1 nuclease analyses revealed multiple transcriptional initiation sites including a major site 169 bp upstream of the translation start site. The promoter region is GC rich, lacks TATA and CAAT boxes, but contains potential recognition sites for a variety of trans-acting factors, including Sp1, AP-2 and the cyclic AMP response element-binding protein. Proto-UFO and its oncogenic counterpart exhibit identical cDNA and promoter regions sequences. Possible modes of UFO activation are discussed.

  20. Structure-Function Similarities between a Plant Receptor-like Kinase and the Human Interleukin-1 Receptor-associated Kinase-4*



    Phylogenetic analysis has previously shown that plant receptor-like kinases (RLKs) are monophyletic with respect to the kinase domain and share an evolutionary origin with the animal interleukin-1 receptor-associated kinase/Pelle-soluble kinases. The lysin motif domain-containing receptor-like kinase-3 (LYK3) of the legume Medicago truncatula shows 33% amino acid sequence identity with human IRAK-4 over the kinase domain. Using the structure of this animal kinase as a template, homology model...

  1. Structural evidence for evolution of shark Ig new antigen receptor variable domain antibodies from a cell-surface receptor. (United States)

    Streltsov, V A; Varghese, J N; Carmichael, J A; Irving, R A; Hudson, P J; Nuttall, S D


    The Ig new antigen receptors (IgNARs) are single-domain antibodies found in the serum of sharks. Here, we report 2.2- and 2.8-A structures of the type 2 IgNAR variable domains 12Y-1 and 12Y-2. Structural features include, first, an Ig superfamily topology transitional between cell adhesion molecules, antibodies, and T cell receptors; and, second, a vestigial complementarity-determining region 2 at the "bottom" of the molecule, apparently discontinuous from the antigen-binding paratope and similar to that observed in cell adhesion molecules. Thus, we suggest that IgNARs originated as cell-surface adhesion molecules coopted to the immune repertoire and represent an evolutionary lineage independent of variable heavy chain/variable light chain type antibodies. Additionally, both 12Y-1 and 12Y-2 form unique crystallographic dimers, predominantly mediated by main-chain framework interactions, which represent a possible model for primordial cell-based interactions. Unusually, the 12Y-2 complementarity-determining region 3 also adopts an extended beta-hairpin structure, suggesting a distinct selective advantage in accessing cryptic antigenic epitopes.

  2. Structural Basis of Intracellular TGF-β Signaling: Receptors and Smads. (United States)

    Chaikuad, Apirat; Bullock, Alex N


    Stimulation of the transforming growth factor β (TGF-β) family receptors activates an intracellular phosphorylation-dependent signaling cascade that culminates in Smad transcriptional activation and turnover. Structural studies have identified a number of allosteric mechanisms that control the localization, conformation, and oligomeric state of the receptors and Smads. Such mechanisms dictate the ordered binding of substrate and adaptor proteins that determine the directionality of the signaling process. Activation of the pathway has been illustrated by the various structures of the receptor-activated Smads (R-Smads) with SARA, Smad4, and YAP, respectively, whereas mechanisms of down-regulation have been elucidated by the structural complexes of FKBP12, Ski, and Smurf1. Interesting parallels have emerged between the R-Smads and the Forkhead-associated (FHA) and interferon regulatory factor (IRF)-associated domains, as well as the Hippo pathway. However, important questions remain as to the mechanism of Smad-independent signaling.

  3. Structure of the measles virus hemagglutinin bound to its cellular receptor SLAM. (United States)

    Hashiguchi, Takao; Ose, Toyoyuki; Kubota, Marie; Maita, Nobuo; Kamishikiryo, Jun; Maenaka, Katsumi; Yanagi, Yusuke


    Measles virus, a major cause of childhood morbidity and mortality worldwide, predominantly infects immune cells using signaling lymphocyte activation molecule (SLAM) as a cellular receptor. Here we present crystal structures of measles virus hemagglutinin (MV-H), the receptor-binding glycoprotein, in complex with SLAM. The MV-H head domain binds to a β-sheet of the membrane-distal ectodomain of SLAM using the side of its β-propeller fold. This is distinct from attachment proteins of other paramyxoviruses that bind receptors using the top of their β-propeller. The structure provides templates for antiviral drug design, an explanation for the effectiveness of the measles virus vaccine, and a model of the homophilic SLAM-SLAM interaction involved in immune modulations. Notably, the crystal structures obtained show two forms of the MV-H-SLAM tetrameric assembly (dimer of dimers), which may have implications for the mechanism of fusion triggering.

  4. DMPD: Structure, function and regulation of the Toll/IL-1 receptor adaptor proteins. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 17667936 Structure, function and regulation of the Toll/IL-1 receptor adaptor prote... (.svg) (.html) (.csml) Show Structure, function and regulation of the Toll/IL-1 receptor adaptor proteins. ...PubmedID 17667936 Title Structure, function and regulation of the Toll/IL-1 recep

  5. Structure of the nociceptin/orphanin FQ receptor in complex with a peptide mimetic

    Energy Technology Data Exchange (ETDEWEB)

    Thompson, Aaron A.; Liu, Wei; Chun, Eugene; Katritch, Vsevolod; Wu, Huixian; Vardy, Eyal; Huang, Xi-Ping; Trapella, Claudio; Guerrini, Remo; Calo, Girolamo; Roth, Bryan L.; Cherezov, Vadim; Stevens, Raymond C. (Ferrara); (Scripps); (UNC)


    Members of the opioid receptor family of G-protein-coupled receptors (GPCRs) are found throughout the peripheral and central nervous system, where they have key roles in nociception and analgesia. Unlike the 'classical' opioid receptors, {delta}, {kappa} and {mu} ({delta}-OR, {kappa}-OR and {mu}-OR), which were delineated by pharmacological criteria in the 1970s and 1980s, the nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP, also known as ORL-1) was discovered relatively recently by molecular cloning and characterization of an orphan GPCR. Although it shares high sequence similarity with classical opioid GPCR subtypes ({approx}60%), NOP has a markedly distinct pharmacology, featuring activation by the endogenous peptide N/OFQ, and unique selectivity for exogenous ligands. Here we report the crystal structure of human NOP, solved in complex with the peptide mimetic antagonist compound-24 (C-24) (ref. 4), revealing atomic details of ligand-receptor recognition and selectivity. Compound-24 mimics the first four amino-terminal residues of the NOP-selective peptide antagonist UFP-101, a close derivative of N/OFQ, and provides important clues to the binding of these peptides. The X-ray structure also shows substantial conformational differences in the pocket regions between NOP and the classical opioid receptors {kappa} (ref. 5) and {mu} (ref. 6), and these are probably due to a small number of residues that vary between these receptors. The NOP-compound-24 structure explains the divergent selectivity profile of NOP and provides a new structural template for the design of NOP ligands.

  6. Hepatic structural enhancement and insulin resistance amelioration due to AT1 receptor blockade. (United States)

    Souza-Mello, Vanessa


    Over the last decade, the role of renin-angiotensin system (RAS) on the development of obesity and its comorbidities has been extensively addressed. Both circulating and local RAS components are up-regulated in obesity and involved in non-alcoholic fatty liver disease onset. Pharmacological manipulations of RAS are viable strategies to tackle metabolic impairments caused by the excessive body fat mass. Renin inhibitors rescue insulin resistance, but do not have marked effects on hepatic steatosis. However, angiotensin-converting enzyme inhibitors and angiotensin receptor blockers (ARB) yield beneficial hepatic remodeling. ARBs elicit body mass loss and normalize insulin levels, tackling insulin resistance. Also, this drug class increases adiponectin levels, besides countering interleukin-6, tumoral necrosis factor-alpha, and transforming growth factor-beta 1. The latter is essential to prevent from liver fibrosis. When conjugated with peroxisome proliferator-activated receptor (PPAR)-alpha activation, ARB fully rescues fatty liver. These effects might be orchestrated by an indirect up-regulation of MAS receptor due to angiotensin II receptor type 1 (AT1R) blockade. These associations of ARB with PPAR activation and ACE2-angiotensin (ANG) (1-7)-MAS receptor axis deserve a better understanding. This editorial provides a brief overview of the current knowledge regarding AT1R blockade effects on sensitivity to insulin and hepatic structural alterations as well as the intersections of AT1R blockade with peroxisome proliferator-activated receptor activation and ACE2-ANG (1-7) - MAS receptor axis.

  7. Pharmacological and structural characterization of conformationally restricted (S)-glutamate analogues at ionotropic glutamate receptors

    DEFF Research Database (Denmark)

    Juknaite, Lina; Venskutonyte, Raminta; Assaf, Zeinab


    A2 and the kainate receptor GluK3. These structures show that CBG-IV interacts with the binding pocket in the same way as (S)-glutamate. The binding affinities reveal that CBG-IV has high affinity at the AMPA and kainate receptor subtypes. Appreciable binding affinity of CBG-IV was not observed......Conformationally restricted glutamate analogues have been pharmacologically characterized at AMPA and kainate receptors and the crystal structures have been solved of the ligand (2S,1'R,2'S)-2-(2'-carboxycyclobutyl)glycine (CBG-IV) in complex with the ligand binding domains of the AMPA receptor Glu...... at NMDA receptors, where the introduction of the carbocyclic ring is expected to lead to a steric clash with binding site residues. CBG-IV was demonstrated to be an agonist at both GluA2 and the kainate receptor GluK1. CBG-IV showed high affinity binding to GluK1 compared to GluA2, GluK2 and GluK3, which...

  8. GPCR engineering yields high-resolution structural insights into beta2-adrenergic receptor function

    DEFF Research Database (Denmark)

    Rosenbaum, Daniel M; Cherezov, Vadim; Hanson, Michael A


    crystallization, we engineered a beta2AR fusion protein in which T4 lysozyme (T4L) replaces most of the third intracellular loop of the GPCR ("beta2AR-T4L") and showed that this protein retains near-native pharmacologic properties. Analysis of adrenergic receptor ligand-binding mutants within the context...... of the reported high-resolution structure of beta2AR-T4L provides insights into inverse-agonist binding and the structural changes required to accommodate catecholamine agonists. Amino acids known to regulate receptor function are linked through packing interactions and a network of hydrogen bonds, suggesting...

  9. Structures of pattern recognition receptors reveal molecular mechanisms of autoinhibition, ligand recognition and oligomerization. (United States)

    Chuenchor, Watchalee; Jin, Tengchuan; Ravilious, Geoffrey; Xiao, T Sam


    Pattern recognition receptors (PRRs) are essential sentinels for pathogens or tissue damage and integral components of the innate immune system. Recent structural studies have provided unprecedented insights into the molecular mechanisms of ligand recognition and signal transduction by several PRR families at distinct subcellular compartments. Here we highlight some of the recent discoveries and summarize the common themes that are emerging from these exciting studies. Better mechanistic understanding of the structure and function of the PRRs will improve future prospects of therapeutic targeting of these important innate immune receptors.

  10. The second PGD(2) receptor CRTH2: structure, properties, and functions in leukocytes. (United States)

    Nagata, Kinya; Hirai, Hiroyuki


    Prostaglandin (PG) D(2) plays a broad range of physiological and pathophysiological functions. Until just a few years ago, it was thought that most of the biological actions of PGD(2) are mediated via the classical PGD(2) receptor DP. Recently, we identified a second PGD(2) receptor, chemoattractant receptor-homologous molecule expressed on T helper (Th)2 cells (CRTH2), with different functions relative to DP. Here, we review the recent findings on the structure, tissue distribution, ligand selectivity, signalling pathways, and functions in leukocytes of this receptor. The data suggest that the PGD(2)/CRTH2 system play important roles in allergic inflammation through its stimulatory effects on Th2 cells, eosinophils, and basophils.

  11. Structure-function Aspects of Extracellular Leucine-rich Repeat-containing Cell Surface Receptors in Plants

    Institute of Scientific and Technical Information of China (English)

    Zhao Zhang; Bart PHJ Thomma


    Plants exploit several types of cell surface receptors for perception of extracellular signals, of which the extracellular leucine-rich repeat (eLRR)-containing receptors form the major class. Although the function of most plant eLRR receptors remains unclear, an increasing number of these receptors are shown to play roles in innate immunity and a wide variety of developmental processes. Recent efforts using domain swaps, gene shuffling analyses, site-directed mutagenesis, interaction studies, and crystallographic analyses resulted in the current knowledge on ligand binding and the mechanism of activation of plant eLRR receptors. This review provides an overview of eLRR receptor research, specifically summarizing the recent understanding of interactions among plant eLRR receptors, their co-receptors and corresponding ligands. The functions of distinct eLRR receptor domains, and their role in structure, ligand perception and multimeric complex formation are discussed.

  12. From G Protein-coupled Receptor Structure Resolution to Rational Drug Design. (United States)

    Jazayeri, Ali; Dias, Joao M; Marshall, Fiona H


    A number of recent technical solutions have led to significant advances in G protein-coupled receptor (GPCR) structural biology. Apart from a detailed mechanistic view of receptor activation, the new structures have revealed novel ligand binding sites. Together, these insights provide avenues for rational drug design to modulate the activities of these important drug targets. The application of structural data to GPCR drug discovery ushers in an exciting era with the potential to improve existing drugs and discover new ones. In this review, we focus on technical solutions that have accelerated GPCR crystallography as well as some of the salient findings from structures that are relevant to drug discovery. Finally, we outline some of the approaches used in GPCR structure based drug design.

  13. Structure and mechanism for recognition of peptidehormones by Class B G-protein-coupled receptors

    Institute of Scientific and Technical Information of China (English)

    Kuntal PAL; Karsten MELCHER; H Eric XU


    Class B G-protein-coupled receptors (GPCRs) are receptors for peptide hormones that include glucagon,parathyroid hormone,and calcitonin.These receptors are involved in a wide spectrum of physiological activities,from metabolic regulation and stress control to development and maintenance of the skeletal system.As such,they are important drug targets for the treatment of diabetes,osteo-porosis,and stress related disorders.Class B GPCRs are organized into two modular domains:an extracellular domain (ECD) and ahelical bundle that contains seven transmembrane helices (TM domain).The ECD is responsible for the high affinity and specificity of hormone binding,and the TM domain is required for receptor activation and signal coupling to downstream G-proteins.Although the structure of the full-length receptor remains unknown,the ECD structures have been well characterized for a number of Class BGPCRs,revealing a common fold for ligand recognition.This review summarizes the general structural principles that guide hormone binding by Class B ECDs and their implications in the design of peptide hormone analogs for therapeutic purposes.

  14. Structure of CC chemokine receptor 2 with orthosteric and allosteric antagonists

    Energy Technology Data Exchange (ETDEWEB)

    Zheng, Yi; Qin, Ling; Ortiz Zacarías, Natalia V.; de Vries, Henk; Han, Gye Won; Gustavsson, Martin; Dabros, Marta; Zhao, Chunxia; Cherney, Robert J.; Carter, Percy; Stamos, Dean; Abagyan, Ruben; Cherezov, Vadim; Stevens, Raymond C.; IJzerman, Adriaan P.; Heitman, Laura H.; Tebben, Andrew; Kufareva, Irina; Handel , Tracy M. (Vertex Pharm); (Leiden-MC); (USC); (BMS); (UCSD)


    CC chemokine receptor 2 (CCR2) is one of 19 members of the chemokine receptor subfamily of human class A G-protein-coupled receptors. CCR2 is expressed on monocytes, immature dendritic cells, and T-cell subpopulations, and mediates their migration towards endogenous CC chemokine ligands such as CCL2 (ref. 1). CCR2 and its ligands are implicated in numerous inflammatory and neurodegenerative diseases2 including atherosclerosis, multiple sclerosis, asthma, neuropathic pain, and diabetic nephropathy, as well as cancer3. These disease associations have motivated numerous preclinical studies and clinical trials4 (see in search of therapies that target the CCR2–chemokine axis. To aid drug discovery efforts5, here we solve a structure of CCR2 in a ternary complex with an orthosteric (BMS-681 (ref. 6)) and allosteric (CCR2-RA-[R]7) antagonist. BMS-681 inhibits chemokine binding by occupying the orthosteric pocket of the receptor in a previously unseen binding mode. CCR2-RA-[R] binds in a novel, highly druggable pocket that is the most intracellular allosteric site observed in class A G-protein-coupled receptors so far; this site spatially overlaps the G-protein-binding site in homologous receptors. CCR2-RA-[R] inhibits CCR2 non-competitively by blocking activation-associated conformational changes and formation of the G-protein-binding interface. The conformational signature of the conserved microswitch residues observed in double-antagonist-bound CCR2 resembles the most inactive G-protein-coupled receptor structures solved so far. Like other protein–protein interactions, receptor–chemokine complexes are considered challenging therapeutic targets for small molecules, and the present structure suggests diverse pocket epitopes that can be exploited to overcome obstacles in drug design.

  15. Structural variation and uniformity among tetraloop-receptor interactions and other loop-helix interactions in RNA crystal structures.

    Directory of Open Access Journals (Sweden)

    Li Wu

    Full Text Available Tetraloop-receptor interactions are prevalent structural units in RNAs, and include the GAAA/11-nt and GNRA-minor groove interactions. In this study, we have compiled a set of 78 nonredundant loop-helix interactions from X-ray crystal structures, and examined them for the extent of their sequence and structural variation. Of the 78 interactions in the set, only four were classical GAAA/11-nt motifs, while over half (48 were GNRA-minor groove interactions. The GNRA-minor groove interactions were not a homogeneous set, but were divided into five subclasses. The most predominant subclass is characterized by two triple base pair interactions in the minor groove, flanked by two ribose zipper contacts. This geometry may be considered the "standard" GNRA-minor groove interaction, while the other four subclasses are alternative ways to form interfaces between a minor groove and tetraloop. The remaining 26 structures in the set of 78 have loops interacting with mostly idiosyncratic receptors. Among the entire set, a number of sequence-structure correlations can be identified, which may be used as initial hypotheses in predicting three-dimensional structures from primary sequences. Conversely, other sequence patterns are not predictive; for example, GAAA loop sequences and GG/CC receptors bind to each other with three distinct geometries. Finally, we observe an example of structural evolution in group II introns, in which loop-receptor motifs are substituted for each other while maintaining the larger three-dimensional geometry. Overall, the study gives a more complete view of RNA loop-helix interactions that exist in nature.

  16. Structural combination of established 5-HT(2A) receptor ligands: new aspects of the binding mode

    DEFF Research Database (Denmark)

    Kramer, Vasko; Herth, Matthias M; Santini, Martin A;


    MH.MZ, MDL 100907, and altanserin are structurally similar 4-benzoyl-piperidine derivatives and are well accommodated to receptor interaction models. We combined structural elements of different high-affinity and selective 5-HT(2A) antagonists, as MH.MZ, altanserin, and SR 46349B, to improve......) with a moderate affinity toward the 5-HT(2A) receptor (K(i) = 57 nm). The remarkably reduced affinity of other compounds (4a), (4b), and (4c) (K(i) = 411, 360 and 356 nm respectively) indicates that MH.MZ can only bind to the 5-HT(2A) receptor with the p-fluorophenylethyl residue in a sterically restricted...

  17. A new crystal structure fragment-based pharmacophore method for G protein-coupled receptors

    DEFF Research Database (Denmark)

    Fidom, Kimberley; Isberg, Vignir; Hauser, Alexander Sebastian;


    We have developed a new method for the building of pharmacophores for G protein-coupled receptors, a major drug target family. The method is a combination of the ligand- and target-based pharmacophore methods and founded on the extraction of structural fragments, interacting ligand moiety...... for new targets. A validating retrospective virtual screening of histamine H1 and H3 receptor pharmacophores yielded area-under-the-curves of 0.88 and 0.82, respectively. The fragment-based method has the unique advantage that it can be applied to targets for which no (homologous) crystal structures...... or ligands are known. 47% of the class A G protein-coupled receptors can be targeted with at least four-element pharmacophores. The fragment libraries can also be used to grow known ligands or for rotamer refinement of homology models. Researchers can download the complete fragment library or a subset...

  18. Structural insights into the nucleotide base specificity of P2X receptors (United States)

    Kasuya, Go; Fujiwara, Yuichiro; Tsukamoto, Hisao; Morinaga, Satoshi; Ryu, Satoshi; Touhara, Kazushige; Ishitani, Ryuichiro; Furutani, Yuji; Hattori, Motoyuki; Nureki, Osamu


    P2X receptors are trimeric ATP-gated cation channels involved in diverse physiological processes, ranging from muscle contraction to nociception. Despite the recent structure determination of the ATP-bound P2X receptors, the molecular mechanism of the nucleotide base specificity has remained elusive. Here, we present the crystal structure of zebrafish P2X4 in complex with a weak affinity agonist, CTP, together with structure-based electrophysiological and spectroscopic analyses. The CTP-bound structure revealed a hydrogen bond, between the cytosine base and the side chain of the basic residue in the agonist binding site, which mediates the weak but significant affinity for CTP. The cytosine base is further recognized by two main chain atoms, as in the ATP-bound structure, but their bond lengths seem to be extended in the CTP-bound structure, also possibly contributing to the weaker affinity for CTP over ATP. This work provides the structural insights for the nucleotide base specificity of P2X receptors. PMID:28332633

  19. A comparative structural bioinformatics analysis of the insulin receptor family ectodomain based on phylogenetic information.

    Directory of Open Access Journals (Sweden)

    Miguel E Rentería

    Full Text Available The insulin receptor (IR, the insulin-like growth factor 1 receptor (IGF1R and the insulin receptor-related receptor (IRR are covalently-linked homodimers made up of several structural domains. The molecular mechanism of ligand binding to the ectodomain of these receptors and the resulting activation of their tyrosine kinase domain is still not well understood. We have carried out an amino acid residue conservation analysis in order to reconstruct the phylogeny of the IR Family. We have confirmed the location of ligand binding site 1 of the IGF1R and IR. Importantly, we have also predicted the likely location of the insulin binding site 2 on the surface of the fibronectin type III domains of the IR. An evolutionary conserved surface on the second leucine-rich domain that may interact with the ligand could not be detected. We suggest a possible mechanical trigger of the activation of the IR that involves a slight 'twist' rotation of the last two fibronectin type III domains in order to face the likely location of insulin. Finally, a strong selective pressure was found amongst the IRR orthologous sequences, suggesting that this orphan receptor has a yet unknown physiological role which may be conserved from amphibians to mammals.

  20. Structure-based discovery of selective serotonin 5-HT(1B) receptor ligands. (United States)

    Rodríguez, David; Brea, José; Loza, María Isabel; Carlsson, Jens


    The development of safe and effective drugs relies on the discovery of selective ligands. Serotonin (5-hydroxytryptamine [5-HT]) G protein-coupled receptors are therapeutic targets for CNS disorders but are also associated with adverse drug effects. The determination of crystal structures for the 5-HT1B and 5-HT2B receptors provided an opportunity to identify subtype selective ligands using structure-based methods. From docking screens of 1.3 million compounds, 22 molecules were predicted to be selective for the 5-HT1B receptor over the 5-HT2B subtype, a requirement for safe serotonergic drugs. Nine compounds were experimentally verified as 5-HT1B-selective ligands, with up to 300-fold higher affinities for this subtype. Three of the ligands were agonists of the G protein pathway. Analysis of state-of-the-art homology models of the two 5-HT receptors revealed that the crystal structures were critical for predicting selective ligands. Our results demonstrate that structure-based screening can guide the discovery of ligands with specific selectivity profiles.

  1. X-ray structures define human P2X3 receptor gating cycle and antagonist action

    NARCIS (Netherlands)

    Mansoor, Steven E.; Lü, Wei; Oosterheert, W.; Shekhar, Mrinal; Tajkhorshid, Emad; Gouaux, Eric


    P2X receptors are trimeric, non-selective cation channels activated by ATP that have important roles in the cardiovascular, neuronal and immune systems. Despite their central function in human physiology and although they are potential targets of therapeutic agents, there are no structures of human

  2. Pyrazolo Derivatives as Potent Adenosine Receptor Antagonists: An Overview on the Structure-Activity Relationships

    Directory of Open Access Journals (Sweden)

    Siew Lee Cheong


    Full Text Available In the past few decades, medicinal chemistry research towards potent and selective antagonists of human adenosine receptors (namely, A1, A2A, A2B, and A3 has been evolving rapidly. These antagonists are deemed therapeutically beneficial in several pathological conditions including neurological and renal disorders, cancer, inflammation, and glaucoma. Up to this point, many classes of compounds have been successfully synthesized and identified as potent human adenosine receptor antagonists. In this paper, an overview of the structure-activity relationship (SAR profiles of promising nonxanthine pyrazolo derivatives is reported and discussed. We have emphasized the SAR for some representative structures such as pyrazolo-[4,3-e]-1,2,4-triazolo-[1,5-c]pyrimidines; pyrazolo-[3,4-c] or -[4,3-c]quinolines; pyrazolo-[4,3-d]pyrimidinones; pyrazolo-[3,4-d]pyrimidines and pyrazolo-[1,5-a]pyridines. This overview not only clarifies the structural requirements deemed essential for affinity towards individual adenosine receptor subtypes, but it also sheds light on the rational design and optimization of existing structural templates to allow us to conceive new, more potent adenosine receptor antagonists.

  3. X-ray structure of the mouse serotonin 5-HT3 receptor

    NARCIS (Netherlands)

    Hassaine, Gherici; Deluz, Cedric; Grasso, Luigino; Wyss, Romain; Tol, Menno B.; Hovius, Ruud; Graff, Alexandra; Stahlberg, Henning; Tomizaki, Takashi; Desmyter, Aline; Moreau, Christophe; Li, Xiao-Dan; Poitevin, Frederic; Vogel, Horst; Nury, Hugues


    Neurotransmitter-gated ion channels of the Cys-loop receptor family mediate fast neurotransmission throughout the nervous system. The molecular processes of neurotransmitter binding, subsequent opening of the ion channel and ion permeation remain poorly understood. Here we present the X-ray structur

  4. Structure and Mode of Peptide Binding of Pheromone Receptor PrgZ

    NARCIS (Netherlands)

    Berntsson, Ronnie P. -A.; Schuurman-Wolters, Gea K.; Dunny, Gary; Slotboom, Dirk-Jan; Poolman, Bert


    Wepresent the crystal structure of the pheromone receptor protein PrgZ from Enterococcus faecalis in complex with the heptapeptide cCF10 (LVTLVFV), which is used in signaling between conjugative recipient and donor cells. Comparison of PrgZ with homologous oligopeptide-binding proteins (AppA and Opp

  5. Structural insight into antibody-mediated antagonism of the Glucagon-like peptide-1 Receptor. (United States)

    Hennen, Stephanie; Kodra, János T; Soroka, Vladyslav; Krogh, Berit O; Wu, Xiaoai; Kaastrup, Peter; Ørskov, Cathrine; Rønn, Sif G; Schluckebier, Gerd; Barbateskovic, Silvia; Gandhi, Prafull S; Reedtz-Runge, Steffen


    The Glucagon-like peptide-1 receptor (GLP-1R) is a member of the class B G protein-coupled receptor (GPCR) family and a well-established target for the treatment of type 2 diabetes. The N-terminal extracellular domain (ECD) of GLP-1R is important for GLP-1 binding and the crystal structure of the GLP-1/ECD complex was reported previously. The first structure of a class B GPCR transmembrane (TM) domain was solved recently, but the full length receptor structure is still not well understood. Here we describe the molecular details of antibody-mediated antagonism of the GLP-1R using both in vitro pharmacology and x-ray crystallography. We showed that the antibody Fab fragment (Fab 3F52) blocked the GLP-1 binding site of the ECD directly and thereby acts as a competitive antagonist of native GLP-1. Interestingly, Fab 3F52 also blocked a short peptide agonist believed to engage primarily the transmembrane and extracellular loop region of GLP-1R, whereas functionality of an allosteric small-molecule agonist was not inhibited. This study has implications for the structural understanding of the GLP-1R and related class B GPCRs, which is important for the development of new and improved therapeutics targeting these receptors.

  6. Structural Insights into Divalent Cation Modulations of ATP-Gated P2X Receptor Channels

    Directory of Open Access Journals (Sweden)

    Go Kasuya


    Full Text Available P2X receptors are trimeric ATP-gated cation channels involved in physiological processes ranging widely from neurotransmission to pain and taste signal transduction. The modulation of the channel gating, including that by divalent cations, contributes to these diverse physiological functions of P2X receptors. Here, we report the crystal structure of an invertebrate P2X receptor from the Gulf Coast tick Amblyomma maculatum in the presence of ATP and Zn2+ ion, together with electrophysiological and computational analyses. The structure revealed two distinct metal binding sites, M1 and M2, in the extracellular region. The M1 site, located at the trimer interface, is responsible for Zn2+ potentiation by facilitating the structural change of the extracellular domain for pore opening. In contrast, the M2 site, coupled with the ATP binding site, might contribute to regulation by Mg2+. Overall, our work provides structural insights into the divalent cation modulations of P2X receptors.

  7. Structure-based, rational design of T cell receptors

    Directory of Open Access Journals (Sweden)

    Vincent eZoete


    Full Text Available Adoptive cell transfer using engineered T cells is emerging as a promising treatment for metastatic melanoma. Such an approach allows one to introduce TCR modifications that, while maintaining the specificity for the targeted antigen, can enhance the binding and kinetic parameters for the interaction pMHC. Using the well-characterized 2C TCR/SIYR/H-2K(b structure as a model system, we demonstrated that a binding free energy decomposition based on the MM-GBSA approach provides a detailed and reliable description of the TCR/pMHC interactions at the structural and thermodynamic levels. Starting from this result, we developed a new structure-based approach, to rationally design new TCR sequences, and applied it to the BC1 TCR targeting the HLA-A2 restricted NY-ESO-1157-165 cancer-testis epitope. 54% of the designed sequence replacements exhibited improved pMHC-binding as compared to the native TCR, with up to 150 fold increase in affinity, while preserving specificity. Genetically-engineered CD8+ T cells expressing these modified TCRs showed an improved functional activity compared to those expressing BC1 TCR. We measured maximum levels of activities for TCRs within the upper limit of natural affinity. Beyond the affinity threshold at KD < 1 μM we observed an attenuation in cellular function. We have also developed a homology modeling-based approach, TCRep 3D, to obtain accurate structural models of any TCR-pMHC complexes. We have complemented the approach with a simplified rigid method to predict the TCR orientation over pMHC. These methods potentially extend the use of our TCR engineering method to entire TCR repertoires for which no X-ray structure is available. We have also performed a steered molecular dynamics study of the unbinding of the TCR-pMHC complex to get a better understanding of how TCRs interact with pMHCs. This entire rational TCR design pipeline is now being used to produce rationally optimized TCRs for adoptive cell therapies of

  8. A structural feature of the non-peptide ligand interactions with mice mu-opioid receptors. (United States)

    Noori, Hamid R; Mucksch, Christian; Urbassek, Herbert M


    By binding to and activating the G-protein coupled μ-, κ- and δ-opioid receptors in the central nervous system, opiates are known to induce analgesic and sedative effects. In particular, non-peptide opioid ligands are often used in clinical applications to induce these therapeutically beneficial effects, due to their superior pharmacokinetics and bioavailability in comparison to endogenous neuropeptides. However, since opioid alkaloids are highly addictive substances, it is necessary to understand the exact mechanisms of their actions, specifically the ligand-binding properties of the target receptors, in order to safely apply opiates for therapeutic purposes. Using an in silico molecular docking approach (AutoDock Vina) combined with two-step cluster analysis, we have computationally obtained the docking scores and the ligand-binding pockets of twelve representative non-peptide nonendogenous agonists and antagonists at the crystallographically identified μ-opioid receptor. Our study predicts the existence of two main binding sites that are congruently present in all opioid receptor types. Interestingly, in terms of the agonist or antagonist properties of the substances on the receptors, the clustering analysis suggests a relationship with the position of the ligand-binding pockets, particularly its depth within the receptor structure. Furthermore, the binding affinity of the substances is directly correlated to the proximity of the binding pockets to the extracellular space. In conclusion, the results provide further insights into the structural features of the functional pharmacology of opioid receptors, suggesting the importance of the binding position of non-peptide agonists and antagonists- specifically the distance and the level of exposure to the extracellular space- to their dissociation kinetics and subsequent potency.

  9. Crystal structure of the human OX2 orexin receptor bound to the insomnia drug suvorexant (United States)

    Yin, Jie; Mobarec, Juan Carlos; Kolb, Peter; Rosenbaum, Daniel M.


    The orexin (also known as hypocretin) G protein-coupled receptors (GPCRs) respond to orexin neuropeptides in the central nervous system to regulate sleep and other behavioural functions in humans. Defects in orexin signalling are responsible for the human diseases of narcolepsy and cataplexy; inhibition of orexin receptors is an effective therapy for insomnia. The human OX2 receptor (OX2R) belongs to the β branch of the rhodopsin family of GPCRs, and can bind to diverse compounds including the native agonist peptides orexin-A and orexin-B and the potent therapeutic inhibitor suvorexant. Here, using lipid-mediated crystallization and protein engineering with a novel fusion chimaera, we solved the structure of the human OX2R bound to suvorexant at 2.5 Å resolution. The structure reveals how suvorexant adopts a π-stacked horseshoe-like conformation and binds to the receptor deep in the orthosteric pocket, stabilizing a network of extracellular salt bridges and blocking transmembrane helix motions necessary for activation. Computational docking suggests how other classes of synthetic antagonists may interact with the receptor at a similar position in an analogous π-stacked fashion. Elucidation of the molecular architecture of the human OX2R expands our understanding of peptidergic GPCR ligand recognition and will aid further efforts to modulate orexin signalling for therapeutic ends.

  10. Functional and Structural Overview of G-Protein-Coupled Receptors Comprehensively Obtained from Genome Sequences

    Directory of Open Access Journals (Sweden)

    Makiko Suwa


    Full Text Available An understanding of the functional mechanisms of G-protein-coupled receptors (GPCRs is very important for GPCR-related drug design. We have developed an integrated GPCR database (SEVENS that includes 64,090 reliable GPCR genes comprehensively identified from 56 eukaryote genome sequences, and overviewed the sequences and structure spaces of the GPCRs. In vertebrates, the number of receptors for biological amines, peptides, etc. is conserved in most species, whereas the number of chemosensory receptors for odorant, pheromone, etc. significantly differs among species. The latter receptors tend to be single exon type or a few exon type and show a high ratio in the numbers of GPCRs, whereas some families, such as Class B and Class C receptors, have long lengths due to the presence of many exons. Statistical analyses of amino acid residues reveal that most of the conserved residues in Class A GPCRs are found in the cytoplasmic half regions of transmembrane (TM helices, while residues characteristic to each subfamily found on the extracellular half regions. The 69 of Protein Data Bank (PDB entries of complete or fragmentary structures could be mapped on the TM/loop regions of Class A GPCRs covering 14 subfamilies.

  11. in Silico investigation of the structural requirements for the AMPA receptor antagonism by quinoxaline derivatives. (United States)

    Azam, Faizul; Abugrain, Ismaiel Mohamed; Sanalla, Mohamed Hussin; Elnaas, Radwan Fatahalla; Rajab, Ibrahim Abdassalam Ibn


    Glutamate receptors have been implicated in various neurological disorders and their antagonism offers a suitable approach for the treatment of such disorders. The field of drug design and discovery aims to find best medicines to prevent, treat and cure diseases quickly and efficiently. In this regard, computational tools have helped medicinal chemists modify and optimize molecules to potent drug candidates with better pharmacokinetic profiles, and guiding biologists and pharmacologists to explore new disease genes as well as novel drug targets. In the present study, to understand the structural requirements for AMPA receptor antagonism, molecular docking study was performed on 41 structurally diverse antagonists based on quinoxaline nucleus. Lamarckian genetic algorithm methodology was employed for docking simulations using AutoDock 4.2 program. The results obtained signify that the molecular docking approach is reliable and produces a good correlation coefficient (r(2) = 0.6) between experimental and docking predicted AMPA receptor antagonistic activity. The aromatic moiety of quinoxaline core has been proved to be vital for hydrophobic contacts exhibiting - interactions in docked conformations. However, polar moieties such as carboxylic group and 1,2,4-triazole moieties were noted to be sites for hydrophilic interactions in terms of hydrogen bonding with the receptor. These analyses can be exploited to design and develop novel AMPA receptor antagonists for the treatment of different neurological disorders.

  12. Hydrazone based luminescent receptors for fluorescent sensing of Cu{sup 2+}: Structure and spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Mukherjee, Soma, E-mail: [Department of Environmental Science, University of Kalyani, Kalyani, Nadia, 741235 West Bengal (India); Mal, Palash [Department of Environmental Science, University of Kalyani, Kalyani, Nadia, 741235 West Bengal (India); Stoeckli-Evans, Helen [Institute of Physics, University of Neuchâtel, rue Emile-Argand 11, CH-2000 Neuchâtel (Switzerland)


    Two new luminescent hydrazones, HL1 and HL2 were investigated for selective and sensitive fluorescent recognition of Cu{sup 2+} in aqueous medium (CH{sub 3}CN/H{sub 2}O (1:4, v/v) solvent system) with a 1:1 binding stoichiometry. The emission peak of HL (λ{sub em}=405 nm), undergoes significant quenching upon complexation with Cu{sup 2+}. The quantum yields for the receptors and in situ formed Cu{sup 2+} complexes were determined. The absorption ratiometric analysis was carried out in presence of various metal ions to confirm the selectivity of the receptors towards Cu{sup 2+}. They were able to detect Cu{sup 2+} with a ∼0.9 µM detection limit as indicated by fluorimetric measurements. The molecular structures of the receptors were determined by single crystal X-ray diffraction analysis. - Highlights: • Small molecule luminescent hydrazones were developed for recognition of Cu{sup 2+}. • Selectivity and sensitivity were studied spectroscopically in aqueous medium. • Binding stoichiometry, association constant, and quantum yields were calculated. • Receptors have low detection limit for Cu{sup 2+}. • Crystal structures of the receptors were solved by X-ray diffractometry.

  13. Dimeric Arrangement of the Parathyroid Hormone Receptor and a Structural Mechanism for Ligand-induced Dissociation

    Energy Technology Data Exchange (ETDEWEB)

    Pioszak, Augen A.; Harikumar, Kaleeckal G.; Parker, Naomi R.; Miller, Laurence J.; Xu, H. Eric (Van Andel); (Mayo)


    The parathyroid hormone receptor (PTH1R) is a class B G protein-coupled receptor that is activated by parathyroid hormone (PTH) and PTH-related protein (PTHrP). Little is known about the oligomeric state of the receptor and its regulation by hormone. The crystal structure of the ligand-free PTH1R extracellular domain (ECD) reveals an unexpected dimer in which the C-terminal segment of both ECD protomers forms an {alpha}-helix that mimics PTH/PTHrP by occupying the peptide binding groove of the opposing protomer. ECD-mediated oligomerization of intact PTH1R was confirmed in living cells by bioluminescence and fluorescence resonance energy transfer experiments. As predicted by the structure, PTH binding disrupted receptor oligomerization. A receptor rendered monomeric by mutations in the ECD retained wild-type PTH binding and cAMP signaling ability. Our results are consistent with the hypothesis that PTH1R forms constitutive dimers that are dissociated by ligand binding and that monomeric PTH1R is capable of activating G protein.

  14. Structural Dynamics of the Glycine-binding Domain of the N-Methyl-d-Aspartate Receptor* (United States)

    Dolino, Drew M.; Cooper, David; Ramaswamy, Swarna; Jaurich, Henriette; Landes, Christy F.; Jayaraman, Vasanthi


    N-Methyl-d-aspartate receptors mediate the slow component of excitatory neurotransmission in the central nervous system. These receptors are obligate heteromers containing glycine- and glutamate-binding subunits. The ligands bind to a bilobed agonist-binding domain of the receptor. Previous x-ray structures of the glycine-binding domain of NMDA receptors showed no significant changes between the partial and full agonist-bound structures. Here we have used single molecule fluorescence resonance energy transfer (smFRET) to investigate the cleft closure conformational states that the glycine-binding domain of the receptor adopts in the presence of the antagonist 5,7-dichlorokynurenic acid (DCKA), the partial agonists 1-amino-1-cyclobutanecarboxylic acid (ACBC) and l-alanine, and full agonists glycine and d-serine. For these studies, we have incorporated the unnatural amino acid p-acetyl-l-phenylalanine for specific labeling of the protein with hydrazide derivatives of fluorophores. The single molecule fluorescence resonance energy transfer data show that the agonist-binding domain can adopt a wide range of cleft closure states with significant overlap in the states occupied by ligands of varying efficacy. The difference lies in the fraction of the protein in a more closed-cleft form, with full agonists having a larger fraction in the closed-cleft form, suggesting that the ability of ligands to select for these states could dictate the extent of activation. PMID:25404733

  15. Studies decode crystal structure of a neurotrophic factor and the p75 neurotrophin receptor complex

    Institute of Scientific and Technical Information of China (English)


    @@ CAS biophysicists have made new discoveries about the p75 neurotrophin receptor (p75NTR).Under the guidance of Prof.JIANG Tao of the CAS Institute of Biophysics (IBP),Dr.GONG Yong and Dr.CAO Peng revealed crystal structure of a symmetrical complex of the neurotrophin-3 and p75NTR,bringing to light the mode of their interaction and their structural basis.The work was published by the ioumal Nature on 7 August.

  16. Structures of Receptor Complexes of a North American H7N2 Influenza Hemagglutinin with a Loop Deletion in the Receptor Binding Site

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Hua; Chen, Li-Mei; Carney, Paul J.; Donis, Ruben O.; Stevens, James (CDC)


    Human infections with subtype H7 avian influenza viruses have been reported as early as 1979. In 1996, a genetically stable 24-nucleotide deletion emerged in North American H7 influenza virus hemagglutinins, resulting in an eight amino acid deletion in the receptor-binding site. The continuous circulation of these viruses in live bird markets, as well as its documented ability to infect humans, raises the question of how these viruses achieve structural stability and functionality. Here we report a detailed molecular analysis of the receptor binding site of the North American lineage subtype H7N2 virus A/New York/107/2003 (NY107), including complexes with an avian receptor analog (3'-sialyl-N-acetyllactosamine, 3'SLN) and two human receptor analogs (6'-sialyl-N-acetyllactosamine, 6'SLN; sialyllacto-N-tetraose b, LSTb). Structural results suggest a novel mechanism by which residues Arg220 and Arg229 (H3 numbering) are used to compensate for the deletion of the 220-loop and form interactions with the receptor analogs. Glycan microarray results reveal that NY107 maintains an avian-type ({alpha}2-3) receptor binding profile, with only moderate binding to human-type ({alpha}2-6) receptor. Thus despite its dramatically altered receptor binding site, this HA maintains functionality and confirms a need for continued influenza virus surveillance of avian and other animal reservoirs to define their zoonotic potential.

  17. NCBI nr-aa BLAST: CBRC-OCUN-01-1504 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-OCUN-01-1504 ref|NP_058692.1| calcium-sensing receptor [Rattus norvegicus] sp|...P48442|CASR_RAT Extracellular calcium-sensing receptor precursor (CaSR) (Parathyroid Cell calcium-sensing re...ceptor) gb|AAC52149.1| kidney extracellular calcium-sensing receptor gb|AAC52195.1| calcium sensing receptor... gb|EDM11279.1| calcium-sensing receptor, isoform CRA_a [Rattus norvegicus] gb|EDM11281.1| calcium-sensing... receptor, isoform CRA_a [Rattus norvegicus] gb|EDM11282.1| calcium-sensing recepto

  18. Structures of receptor complexes formed by hemagglutinins from the Asian Influenza pandemic of 1957


    Liu, Junfeng; Stevens, David J.; Lesley F Haire; Walker, Philip A.; Coombs, Peter J.; Russell, Rupert J.; Gamblin, Steven J.; John J Skehel


    The viruses that caused the three influenza pandemics of the twentieth century in 1918, 1957, and 1968 had distinct hemagglutinin receptor binding glycoproteins that had evolved the capacity to recognize human cell receptors. We have determined the structure of the H2 hemagglutinin from the second pandemic, the “Asian Influenza” of 1957. We compare it with the 1918 “Spanish Influenza” hemagglutinin, H1, and the 1968 “Hong Kong Influenza” hemagglutinin, H3, and show that despite its close over...

  19. 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


    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).

  20. Structure and function of florigen and the receptor complex. (United States)

    Taoka, Ken-ichiro; Ohki, Izuru; Tsuji, Hiroyuki; Kojima, Chojiro; Shimamoto, Ko


    In the 1930s, the flowering hormone, florigen, was proposed to be synthesized in leaves under inductive day length and transported to the shoot apex, where it induces flowering. More recently, generated genetic and biochemical data suggest that florigen is a protein encoded by the gene, FLOWERING LOCUS T (FT). A rice (Oryza sativa) FT homolog, Hd3a, interacts with the rice FD homolog, OsFD1, via a 14-3-3 protein. Formation of this tri-protein complex is essential for flowering promotion by Hd3a in rice. In addition, the multifunctionality of FT homologs, other than for flowering promotion, is an emerging concept. Here we review the structural and biochemical features of the florigen protein complex and discuss the molecular basis for the multifunctionality of FT proteins.

  1. Structural modeling of G-protein coupled receptors: An overview on automatic web-servers. (United States)

    Busato, Mirko; Giorgetti, Alejandro


    Despite the significant efforts and discoveries during the last few years in G protein-coupled receptor (GPCR) expression and crystallization, the receptors with known structures to date are limited only to a small fraction of human GPCRs. The lack of experimental three-dimensional structures of the receptors represents a strong limitation that hampers a deep understanding of their function. Computational techniques are thus a valid alternative strategy to model three-dimensional structures. Indeed, recent advances in the field, together with extraordinary developments in crystallography, in particular due to its ability to capture GPCRs in different activation states, have led to encouraging results in the generation of accurate models. This, prompted the community of modelers to render their methods publicly available through dedicated databases and web-servers. Here, we present an extensive overview on these services, focusing on their advantages, drawbacks and their role in successful applications. Future challenges in the field of GPCR modeling, such as the predictions of long loop regions and the modeling of receptor activation states are presented as well.

  2. Structures of human folate receptors reveal biological trafficking states and diversity in folate and antifolate recognition. (United States)

    Wibowo, Ardian S; Singh, Mirage; Reeder, Kristen M; Carter, Joshua J; Kovach, Alexander R; Meng, Wuyi; Ratnam, Manohar; Zhang, Faming; Dann, Charles E


    Antifolates, folate analogs that inhibit vitamin B9 (folic acid)-using cellular enzymes, have been used over several decades for the treatment of cancer and inflammatory diseases. Cellular uptake of the antifolates in clinical use occurs primarily via widely expressed facilitative membrane transporters. More recently, human folate receptors (FRs), high affinity receptors that transport folate via endocytosis, have been proposed as targets for the specific delivery of new classes of antifolates or folate conjugates to tumors or sites of inflammation. The development of specific, FR-targeted antifolates would be accelerated if additional biophysical data, particularly structural models of the receptors, were available. Here we describe six distinct crystallographic models that provide insight into biological trafficking of FRs and distinct binding modes of folate and antifolates to these receptors. From comparison of the structures, we delineate discrete structural conformations representative of key stages in the endocytic trafficking of FRs and propose models for pH-dependent conformational changes. Additionally, we describe the molecular details of human FR in complex with three clinically prevalent antifolates, pemetrexed (also Alimta), aminopterin, and methotrexate. On the whole, our data form the basis for rapid design and implementation of unique, FR-targeted, folate-based drugs for the treatment of cancer and inflammatory diseases.

  3. Cloning of rat thymic stromal lymphopoietin receptor (TSLPR) and characterization of genomic structure of murine Tslpr gene

    DEFF Research Database (Denmark)

    Blagoev, Blagoy; Nielsen, Mogens M; Angrist, Misha


    , a cytokine involved in B- and T-cell function. We have cloned the TSLP receptor from rat and find that the WSXWX motif commonly found in extracellular domains of cytokine receptors is conserved as a W(T/S)XV(T/A) motif among TSLP receptors from mouse, rat and human. As in the mouse, TSLP receptor is widely...... is similar to the expression of several other cytokine receptors that have been characterized thus far. We have also characterized the genomic structure of the murine Tslpr gene which shows that in addition to primary sequence homology, it shares a common genomic organization of coding exons with the murine...

  4. Structure and Notch receptor binding of the tandem WWE domain of Deltex. (United States)

    Zweifel, Mark E; Leahy, Daniel J; Barrick, Doug


    Deltex is a cytosolic effector of Notch signaling thought to bind through its N-terminal domain to the Notch receptor. Here we report the structure of the Drosophila Deltex N-terminal domain, which contains two tandem WWE sequence repeats. The WWE repeats, which adopt a novel fold, are related by an approximate two-fold axis of rotation. Although the WWE repeats are structurally distinct, they interact extensively and form a deep cleft at their junction that appears well suited for ligand binding. The two repeats are thermodynamically coupled; this coupling is mediated in part by a conserved segment that is immediately C-terminal to the second WWE domain. We demonstrate that although the Deltex WWE tandem is monomeric in solution, it forms a heterodimer with the ankyrin domain of the Notch receptor. These results provide structural and functional insight into how Deltex modulates Notch signaling, and how WWE modules recognize targets for ubiquitination.

  5. Recent advances in structure of progestins and their binding to progesterone receptors. (United States)

    Cabeza, Marisa; Heuze, Yvonne; Sánchez, Araceli; Garrido, Mariana; Bratoeff, Eugene


    The role of progesterone in women's cancers as well as the knowledge of the progesterone receptor (PR) structure has prompted the design of different therapies. The aim of this review is to describe the basic structure of PR agonists and antagonists as well as the recent treatments for illness associated with the progesterone receptor. The rational design for potent and effective drugs for the treatment of female cancer must consider the structural changes of the androgen and progestogen skeleton which are an indicator of their activity as progestins or antiprogestins. The presence of a hydroxyl group at C-17 in the progesterone skeleton brings about a loss of progestational activity whereas acetylation induces a progestational effect. The incorporation of an ethynyl functional group to the testosterone framework results in a loss of androgenic activity with a concomitant enhancement of the progestational effect. On the other hand, an ester function at C-3 of dehydroepiandrosterone skeleton induces partial antagonism to the PR.

  6. Phytoestrogens and Mycoestrogens Induce Signature Structure Dynamics Changes on Estrogen Receptor α

    Directory of Open Access Journals (Sweden)

    Xueyan Chen


    Full Text Available Endocrine disrupters include a broad spectrum of chemicals such as industrial chemicals, natural estrogens and androgens, synthetic estrogens and androgens. Phytoestrogens are widely present in diet and food supplements; mycoestrogens are frequently found in grains. As human beings and animals are commonly exposed to phytoestrogens and mycoestrogens in diet and environment, it is important to understand the potential beneficial or hazardous effects of estrogenic compounds. Many bioassays have been established to study the binding of estrogenic compounds with estrogen receptor (ER and provided rich data in the literature. However, limited assays can offer structure information with regard to the ligand/ER complex. Our current study surveys the global structure dynamics changes for ERα ligand binding domain (LBD when phytoestrogens and mycoestrogens bind. The assay is based on the structure dynamics information probed by hydrogen deuterium exchange mass spectrometry and offers a unique viewpoint to elucidate the mechanism how phytoestrogens and mycoestrogens interact with estrogen receptor. The cluster analysis based on the hydrogen deuterium exchange (HDX assay data reveals a unique pattern when phytoestrogens and mycoestrogens bind with ERα LBD compared to that of estradiol and synthetic estrogen modulators. Our study highlights that structure dynamics could play an important role in the structure function relationship when endocrine disrupters interact with estrogen receptors.

  7. Structure-based rational design of a Toll-like receptor 4 (TLR4 decoy receptor with high binding affinity for a target protein.

    Directory of Open Access Journals (Sweden)

    Jieun Han

    Full Text Available Repeat proteins are increasingly attracting much attention as alternative scaffolds to immunoglobulin antibodies due to their unique structural features. Nonetheless, engineering interaction interface and understanding molecular basis for affinity maturation of repeat proteins still remain a challenge. Here, we present a structure-based rational design of a repeat protein with high binding affinity for a target protein. As a model repeat protein, a Toll-like receptor4 (TLR4 decoy receptor composed of leucine-rich repeat (LRR modules was used, and its interaction interface was rationally engineered to increase the binding affinity for myeloid differentiation protein 2 (MD2. Based on the complex crystal structure of the decoy receptor with MD2, we first designed single amino acid substitutions in the decoy receptor, and obtained three variants showing a binding affinity (K(D one-order of magnitude higher than the wild-type decoy receptor. The interacting modes and contributions of individual residues were elucidated by analyzing the crystal structures of the single variants. To further increase the binding affinity, single positive mutations were combined, and two double mutants were shown to have about 3000- and 565-fold higher binding affinities than the wild-type decoy receptor. Molecular dynamics simulations and energetic analysis indicate that an additive effect by two mutations occurring at nearby modules was the major contributor to the remarkable increase in the binding affinities.

  8. A Structural Investigation into Oct4 Regulation by Orphan Nuclear Receptors, Germ Cell Nuclear Factor (GCNF) and Liver Receptor Homolog-1 (LRH-1). (United States)

    Weikum, Emily R; Tuntland, Micheal L; Murphy, Michael N; Ortlund, Eric A


    Oct4 is a transcription factor required for maintaining pluripotency and self-renewal in stem cells. Prior to differentiation, Oct4 must be silenced to allow for the development of the three germ layers in the developing embryo. This fine-tuning is controlled by the nuclear receptors, liver receptor homolog-1 and germ cell nuclear factor. Liver receptor homolog-1 is responsible for driving the expression of Oct4 where germ cell nuclear factor represses its expression upon differentiation. Both receptors bind to a DR0 motif located within the Oct4 promoter. Here, we present the first structure of mouse germ cell nuclear factor DNA binding domain in complex with the Oct4 DR0. The overall structure revealed two molecules bound in a head-to-tail fashion on opposite sides of the DNA. Additionally, we solved the structure of the human liver receptor homolog-1 DNA binding domain bound to the same element. We explore the structural elements that govern Oct4 recognition by these two nuclear receptors.

  9. Structure-Driven Pharmacology of Transient Receptor Potential Channel Vanilloid 1. (United States)

    Díaz-Franulic, Ignacio; Caceres-Molina, Javier; Sepulveda, Romina V; Gonzalez-Nilo, Fernando; Latorre, Ramon


    The transient receptor potential vanilloid 1 (TRPV1) ion channel is a polymodal receptor that mediates the flux of cations across the membrane in response to several stimuli, including heat, voltage, and ligands. The best known agonist of TRPV1 channels is capsaicin, the pungent component of "hot" chili peppers. In addition, peptides found in the venom of poisonous animals, along with the lipids phosphatidylinositol 4,5-biphosphate, lysophosphatidic acid, and cholesterol, bind to TRPV1 with high affinity to modulate channel gating. Here, we discuss the functional evidence regarding ligand-dependent activation of TRPV1 channels in light of structural data recently obtained by cryoelectron microscopy. This review focuses on the mechanistic insights into ligand binding and allosteric gating of TRPV1 channels and the relevance of accurate polymodal receptor biophysical characterization for drug design in novel pain therapies.

  10. Structural and functional characterization of alternative transmembrane domain conformations in VEGF receptor 2 activation. (United States)

    Manni, Sandro; Mineev, Konstantin S; Usmanova, Dinara; Lyukmanova, Ekaterina N; Shulepko, Mikhail A; Kirpichnikov, Mikhail P; Winter, Jonas; Matkovic, Milos; Deupi, Xavier; Arseniev, Alexander S; Ballmer-Hofer, Kurt


    Transmembrane signaling by receptor tyrosine kinases (RTKs) entails ligand-mediated dimerization and structural rearrangement of the extracellular domains. RTK activation also depends on the specific orientation of the transmembrane domain (TMD) helices, as suggested by pathogenic, constitutively active RTK mutants. Such mutant TMDs carry polar amino acids promoting stable transmembrane helix dimerization, which is essential for kinase activation. We investigated the effect of polar amino acids introduced into the TMD of vascular endothelial growth factor receptor 2, regulating blood vessel homeostasis. Two mutants showed constitutive kinase activity, suggesting that precise TMD orientation is mandatory for kinase activation. Nuclear magnetic resonance spectroscopy revealed that TMD helices in activated constructs were rotated by 180° relative to the interface of the wild-type conformation, confirming that ligand-mediated receptor activation indeed results from transmembrane helix rearrangement. A molecular dynamics simulation confirmed the transmembrane helix arrangement of wild-type and mutant TMDs revealed by nuclear magnetic resonance spectroscopy.

  11. Electrospun Gelatin/β-TCP Composite Nanofibers Enhance Osteogenic Differentiation of BMSCs and In Vivo Bone Formation by Activating Ca2+-Sensing Receptor Signaling

    Directory of Open Access Journals (Sweden)

    Xuehui Zhang


    Full Text Available Calcium phosphate- (CaP- based composite scaffolds have been used extensively for the bone regeneration in bone tissue engineering. Previously, we developed a biomimetic composite nanofibrous membrane of gelatin/β-tricalcium phosphate (TCP and confirmed their biological activity in vitro and bone regeneration in vivo. However, how these composite nanofibers promote the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs is unknown. Here, gelatin/β-TCP composite nanofibers were fabricated by incorporating 20 wt% β-TCP nanoparticles into electrospun gelatin nanofibers. Electron microscopy showed that the composite β-TCP nanofibers had a nonwoven structure with a porous network and a rough surface. Spectral analyses confirmed the presence and chemical stability of the β-TCP and gelatin components. Compared with pure gelatin nanofibers, gelatin/β-TCP composite nanofibers caused increased cell attachment, proliferation, alkaline phosphatase activity, and osteogenic gene expression in rat BMSCs. Interestingly, the expression level of the calcium-sensing receptor (CaSR was significantly higher on the composite nanofibrous scaffolds than on pure gelatin. For rat calvarial critical sized defects, more extensive osteogenesis and neovascularization occurred in the composite scaffolds group compared with the gelatin group. Thus, gelatin/β-TCP composite scaffolds promote osteogenic differentiation of BMSCs in vitro and bone regeneration in vivo by activating Ca2+-sensing receptor signaling.

  12. Electrospun Gelatin/β-TCP Composite Nanofibers Enhance Osteogenic Differentiation of BMSCs and In Vivo Bone Formation by Activating Ca (2+) -Sensing Receptor Signaling. (United States)

    Zhang, Xuehui; Meng, Song; Huang, Ying; Xu, Mingming; He, Ying; Lin, Hong; Han, Jianmin; Chai, Yuan; Wei, Yan; Deng, Xuliang


    Calcium phosphate- (CaP-) based composite scaffolds have been used extensively for the bone regeneration in bone tissue engineering. Previously, we developed a biomimetic composite nanofibrous membrane of gelatin/β-tricalcium phosphate (TCP) and confirmed their biological activity in vitro and bone regeneration in vivo. However, how these composite nanofibers promote the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) is unknown. Here, gelatin/β-TCP composite nanofibers were fabricated by incorporating 20 wt% β-TCP nanoparticles into electrospun gelatin nanofibers. Electron microscopy showed that the composite β-TCP nanofibers had a nonwoven structure with a porous network and a rough surface. Spectral analyses confirmed the presence and chemical stability of the β-TCP and gelatin components. Compared with pure gelatin nanofibers, gelatin/β-TCP composite nanofibers caused increased cell attachment, proliferation, alkaline phosphatase activity, and osteogenic gene expression in rat BMSCs. Interestingly, the expression level of the calcium-sensing receptor (CaSR) was significantly higher on the composite nanofibrous scaffolds than on pure gelatin. For rat calvarial critical sized defects, more extensive osteogenesis and neovascularization occurred in the composite scaffolds group compared with the gelatin group. Thus, gelatin/β-TCP composite scaffolds promote osteogenic differentiation of BMSCs in vitro and bone regeneration in vivo by activating Ca(2+)-sensing receptor signaling.

  13. Structural model for gamma-aminobutyric acid receptor noncompetitive antagonist binding: widely diverse structures fit the same site. (United States)

    Chen, Ligong; Durkin, Kathleen A; Casida, John E


    Several major insecticides, including alpha-endosulfan, lindane, and fipronil, and the botanical picrotoxinin are noncompetitive antagonists (NCAs) for the GABA receptor. We showed earlier that human beta(3) homopentameric GABA(A) receptor recognizes all of the important GABAergic insecticides and reproduces the high insecticide sensitivity and structure-activity relationships of the native insect receptor. Despite large structural diversity, the NCAs are proposed to fit a single binding site in the chloride channel lumen lined by five transmembrane 2 segments. This hypothesis is examined with the beta(3) homopentamer by mutagenesis, pore structure studies, NCA binding, and molecular modeling. The 15 amino acids in the cytoplasmic half of the pore were mutated to cysteine, serine, or other residue for 22 mutants overall. Localization of A-1'C, A2'C, T6'C, and L9'C (index numbers for the transmembrane 2 region) in the channel lumen was established by disulfide cross-linking. Binding of two NCA radioligands [(3)H]1-(4-ethynylphenyl)-4-n-propyl-2,6,7-trioxabicyclo[2.2.2]octane and [(3)H] 3,3-bis-trifluoromethyl-bicyclo[2,2,1]heptane-2,2-dicarbonitrile was dramatically reduced with 8 of the 15 mutated positions, focusing attention on A2', T6', and L9' as proposed binding sites, consistent with earlier mutagenesis studies. The cytoplasmic half of the beta3 homopentamer pore was modeled as an alpha-helix. The six NCAs listed above plus t-butylbicyclophosphorothionate fit the 2' to 9' pore region forming hydrogen bonds with the T6' hydroxyl and hydrophobic interactions with A2', T6', and L9' alkyl substituents, thereby blocking the channel. Thus, widely diverse NCA structures fit the same GABA receptor beta subunit site with important implications for insecticide cross-resistance and selective toxicity between insects and mammals.

  14. Structural comparison of phospholipase-A2-binding regions in phospholipase-A2 receptors from various mammals. (United States)

    Higashino, K; Ishizaki, J; Kishino, J; Ohara, O; Arita, H


    We determined the nucleotide sequence of a mouse cDNA encoding the receptor for pancreatic group I phospholipase A2 (PLA2-I). Interspecies structural comparison of the mouse receptor with bovine PLA2-I receptor, whose structure had been clarified, revealed that the fourth carbohydrate-recognition domain (CRD)-like domain (CRD-like 4) was the most conserved among the domains in the PLA2-I receptor, suggesting the functional importance of CRD-like 4. A transient expression experiment with a truncated form of the receptor consisting of three CRD-like domains, from the third to the fifth, demonstrated that the PLA2-I-binding site of the receptor is constituted from these three CRD-like domains, supporting the functional indispensability of CRD-like 4 in the receptor. Since the PLA2-I-binding region was thus assigned to be CRD-like domains 3-5, we further analyzed the structures of the PLA2-I-binding regions in the PLA2-I receptors from the rat, rabbit and human. Furthermore, the obtained PLA2-I receptor cDNA fragments from these animals made it possible to examine the tissue expression patterns of this receptor in various mammals. The results, together with the results of the genomic structural analysis of this gene, indicated that a PLA2 receptor recently characterized by Lambeau et al. [Lambeau, G., Ancian, P., Barhanin, J. & Lazdunski, M. (1994) J. Biol. Chem. 269, 1575-1578] is a rabbit counterpart of the PLA2-I receptor although these two PLA2 receptors have distinctive PLA2-binding specificities.

  15. A novel dualistic profile of an allosteric AMPA receptor modulator identified through studies on recombinant receptors, mouse hippocampal synapses and crystal structures

    DEFF Research Database (Denmark)

    Bundgaard Christiansen, Gitte; Harbak, Barbara; E. Hede, Susanne;


    -mediated neurotransmission. The aim of this study was to investigate functional and structural aspects of a novel analog of the AMPA receptor PAM cyclothiazide (CTZ) on recombinant and native glutamate receptors. We expressed rat GluA4flip and flop in Xenopus oocytes and characterized NS1376 and CTZ under two...... information through X-ray structures, docking and molecular dynamics, which revealed that NS1376 interacts at the dimer interface of the ligand-binding domain in a manner overall similar to CTZ. NS1376 reveals that minor structural changes in CTZ can result in an altered modulatory profile, both enhancing...

  16. receptores

    Directory of Open Access Journals (Sweden)

    Salete Regina Daronco Benetti


    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”.

  17. Anticonvulsant activity of artificial sweeteners: a structural link between sweet-taste receptor T1R3 and brain glutamate receptors. (United States)

    Talevi, Alan; Enrique, Andrea V; Bruno-Blanch, Luis E


    A virtual screening campaign based on application of a topological discriminant function capable of identifying novel anticonvulsant agents indicated several widely-used artificial sweeteners as potential anticonvulsant candidates. Acesulfame potassium, cyclamate and saccharin were tested in the Maximal Electroshock Seizure model (mice, ip), showing moderate anticonvulsant activity. We hypothesized a probable structural link between the receptor responsible of sweet taste and anticonvulsant molecular targets. Bioinformatic tools confirmed a highly significant sequence-similarity between taste-related protein T1R3 and several metabotropic glutamate receptors from different species, including glutamate receptors upregulated in epileptogenesis and certain types of epilepsy.

  18. New insights for drug design from the X-ray crystallographic structures of G-protein-coupled receptors. (United States)

    Jacobson, Kenneth A; Costanzi, Stefano


    Methodological advances in X-ray crystallography have made possible the recent solution of X-ray structures of pharmaceutically important G protein-coupled receptors (GPCRs), including receptors for biogenic amines, peptides, a nucleoside, and a sphingolipid. These high-resolution structures have greatly increased our understanding of ligand recognition and receptor activation. Conformational changes associated with activation common to several receptors entail outward movements of the intracellular side of transmembrane helix 6 (TM6) and movements of TM5 toward TM6. Movements associated with specific agonists or receptors have also been described [e.g., extracellular loop (EL) 3 in the A(2A) adenosine receptor]. The binding sites of different receptors partly overlap but differ significantly in ligand orientation, depth, and breadth of contact areas in TM regions and the involvement of the ELs. A current challenge is how to use this structural information for the rational design of novel potent and selective ligands. For example, new chemotypes were discovered as antagonists of various GPCRs by subjecting chemical libraries to in silico docking in the X-ray structures. The vast majority of GPCR structures and their ligand complexes are still unsolved, and no structures are known outside of family A GPCRs. Molecular modeling, informed by supporting information from site-directed mutagenesis and structure-activity relationships, has been validated as a useful tool to extend structural insights to related GPCRs and to analyze docking of other ligands in already crystallized GPCRs.

  19. Beyond small-molecule SAR: using the dopamine D3 receptor crystal structure to guide drug design. (United States)

    Keck, Thomas M; Burzynski, Caitlin; Shi, Lei; Newman, Amy Hauck


    The dopamine D3 receptor is a target of pharmacotherapeutic interest in a variety of neurological disorders including schizophrenia, restless leg syndrome, and drug addiction. The high protein sequence homology between the D3 and D2 receptors has posed a challenge to developing D3 receptor-selective ligands whose behavioral actions can be attributed to D3 receptor engagement, in vivo. However, through primarily small-molecule structure-activity relationship (SAR) studies, a variety of chemical scaffolds have been discovered over the past two decades that have resulted in several D3 receptor-selective ligands with high affinity and in vivo activity. Nevertheless, viable clinical candidates remain limited. The recent determination of the high-resolution crystal structure of the D3 receptor has invigorated structure-based drug design, providing refinements to the molecular dynamic models and testable predictions about receptor-ligand interactions. This chapter will highlight recent preclinical and clinical studies demonstrating potential utility of D3 receptor-selective ligands in the treatment of addiction. In addition, new structure-based rational drug design strategies for D3 receptor-selective ligands that complement traditional small-molecule SAR to improve the selectivity and directed efficacy profiles are examined.

  20. Structure-activity relationship study of spider polyamine toxins as inhibitors of ionotropic glutamate receptors. (United States)

    Xiong, Xiao-Feng; Poulsen, Mette H; Hussein, Rama A; Nørager, Niels G; Strømgaard, Kristian


    The spider polyamine toxins Joro spider toxin-3 (JSTX-3) and Nephila polyamine toxins-1 and -8 (NPTX-1 and NPTX-8) are isolated from the venom of the orb-weaver spider Nephila clavata (Joro spider). They share a high degree of structural resemblance, their aromatic head groups being the only difference, and were recently found to be very potent open-channel blockers of ionotropic glutamate (iGlu) receptors. In this study we designed and synthesized a collection of 24 analogues of these toxins using a recently developed solid-phase synthetic methodology. Systematic variation in two regions of the toxins and subsequent evaluation of biological activity at AMPA and NMDA subtypes of iGlu receptors provided succinct information on structure-activity relationships. In particular, one set of analogues were found to display exquisite selectivity and potency for AMPA receptors relative to the natural products. Thus, this systematic SAR study has provided new pharmacological tools for studies of iGlu receptors.

  1. Artonin E and Structural Analogs from Artocarpus Species Abrogates Estrogen Receptor Signaling in Breast Cancer

    Directory of Open Access Journals (Sweden)

    Imaobong Etti


    Full Text Available The increasing rate of mortality ensued from breast cancer has encouraged research into safer and efficient therapy. The human Estrogen receptor α has been implicated in the majority of reported breast cancer cases. Molecular docking employing Glide, Schrodinger suite 2015, was used to study the binding affinities of small molecules from the Artocarpus species after their drug-like properties were ascertained. The structure of the ligand-binding domain of human Estrogen receptor α was retrieved from Protein Data Bank while the structures of compounds were collected from PubChem database. The binding interactions of the studied compounds were reported as well as their glide scores. The best glide scored ligand, was Artonin E with a score of −12.72 Kcal when compared to other studied phytomolecules and it evoked growth inhibition of an estrogen receptor positive breast cancer cells in submicromolar concentration (3.8–6.9 µM in comparison to a reference standard Tamoxifen (18.9–24.1 µM within the tested time point (24–72 h. The studied ligands, which had good interactions with the target receptor, were also drug-like when compared with 95% of orally available drugs with the exception of Artoelastin, whose predicted physicochemical properties rendered it less drug-like. The in silico physicochemical properties, docking interactions and growth inhibition of the best glide scorer are indications of the anti-breast cancer relevance of the studied molecules.

  2. [Critical role of peptidic toxins in the functional and structural analysis of nicotinic acetylcholine receptors]. (United States)

    Fruchart-Gaillard, Carole; Ménez, André; Servent, Denis


    Animal toxins which interact on various receptors and channels have been often used in the studies of the functional roles of these targets. Nicotinic toxins have been purified from snake and cone venoms and are characterized by high affinity and various selectivity of interactions on the different nicotinic receptors subtypes. Since 30 years they have been used as molecular probes to identify, localize and purify these receptors. Furthermore, they have played a crucial role in the better understanding of their functional properties and have been useful in their structural studies. These peptidic toxins could be chemically synthetized or recombinantly expressed and nonnatural residues could be introduced in their sequences in order to delineate their functional interaction sites. The structural modelisation of toxin-nAChR interaction allows us to understand the antagonistic property of these toxins and open the way to the design of engineered ligands with predetermined specificity, useful as pharmacological tools or therapeutic agents in the numerous diseases involving this receptor family.

  3. Structural and energetic effects of A2A adenosine receptor mutations on agonist and antagonist binding.

    Directory of Open Access Journals (Sweden)

    Henrik Keränen

    Full Text Available To predict structural and energetic effects of point mutations on ligand binding is of considerable interest in biochemistry and pharmacology. This is not only useful in connection with site-directed mutagenesis experiments, but could also allow interpretation and prediction of individual responses to drug treatment. For G-protein coupled receptors systematic mutagenesis has provided the major part of functional data as structural information until recently has been very limited. For the pharmacologically important A(2A adenosine receptor, extensive site-directed mutagenesis data on agonist and antagonist binding is available and crystal structures of both types of complexes have been determined. Here, we employ a computational strategy, based on molecular dynamics free energy simulations, to rationalize and interpret available alanine-scanning experiments for both agonist and antagonist binding to this receptor. These computer simulations show excellent agreement with the experimental data and, most importantly, reveal the molecular details behind the observed effects which are often not immediately evident from the crystal structures. The work further provides a distinct validation of the computational strategy used to assess effects of point-mutations on ligand binding. It also highlights the importance of considering not only protein-ligand interactions but also those mediated by solvent water molecules, in ligand design projects.

  4. Nuclear receptor engineering based on novel structure activity relationships revealed by farnesyl pyrophosphate. (United States)

    Goyanka, Ritu; Das, Sharmistha; Samuels, Herbert H; Cardozo, Timothy


    Nuclear receptors (NRs) comprise the second largest protein family targeted by currently available drugs, acting via specific ligand interactions within the ligand binding domain (LBD). Recently, farnesyl pyrophosphate (FPP) was shown to be a unique promiscuous NR ligand, activating a subset of NR family members and inhibiting wound healing in skin. The current study aimed at visualizing the unique basis of FPP interaction with multiple receptors in order to identify general structure-activity relationships that operate across the NR family. Docking of FPP to the 3D structures of the LBDs of a diverse set of NRs consistently revealed an electrostatic FPP pyrophosphate contact with an NR arginine conserved in the NR family, a hydrophobic farnesyl contact with NR helix-12 and a ligand binding pocket volume between 300 and 430 Å(3) as the minimal requirements for FPP activation of any NR. Lack of any of these structural features appears to render a given NR resistant to FPP activation. We used these structure-activity relationships to rationally design and successfully engineer several mutant human estrogen receptors that retain responsiveness to estradiol but no longer respond to FPP.

  5. Insights into the structural biology of G-protein coupled receptors impacts drug design for central nervous system neurodegenerative processes


    Dalet, Farfán-García Eunice; Guadalupe, Trujillo-Ferrara José; María del Carmen, Castillo-Hernández; Humberto, Guerra-Araiza Christian; Antonio, Soriano-Ursúa Marvin


    In the last few years, there have been important new insights into the structural biology of G-protein coupled receptors. It is now known that allosteric binding sites are involved in the affinity and selectivity of ligands for G-protein coupled receptors, and that signaling by these receptors involves both G-protein dependent and independent pathways. The present review outlines the physiological and pharmacological implications of this perspective for the design of new drugs to treat disord...

  6. Linking structure to function: Recent lessons from inositol 1,4,5-trisphosphate receptor mutagenesis. (United States)

    Yule, David I; Betzenhauser, Matthew J; Joseph, Suresh K


    Great insight has been gained into the structure and function of the inositol 1,4,5 trisphosphate receptor (InsP(3)R) by studies employing mutagenesis of the cDNA encoding the receptor. Notably, early studies using this approach defined the key constituents required for InsP(3) binding in the N-terminus and the membrane spanning regions in the C-terminal domain responsible for channel formation, targeting and function. In this article we evaluate recent studies which have used a similar approach to investigate key residues underlying the in vivo modulation by select regulatory factors. In addition, we review studies defining the structural requirements in the channel domain which comprise the conduction pathway and are suggested to be involved in the gating of the channel.

  7. The Structural Basis for the Function of Two Anti-VEGF Receptor 2 Antibodies

    Energy Technology Data Exchange (ETDEWEB)

    M Franklin; E Navarro; Y Wang; S Patel; P Singh; Y Zhang; K Persaud; A Bari; H Griffith; et al.


    The anti-VEGF receptor 2 antibody IMC-1121B is a promising antiangiogenic drug being tested for treatment of breast and gastric cancer. We have determined the structure of the 1121B Fab fragment in complex with domain 3 of VEGFR2, as well as the structure of a different neutralizing anti-VEGFR2 antibody, 6.64, also in complex with VEGFR2 domain 3. The two Fab fragments bind at opposite ends of VEGFR2 domain 3; 1121B directly blocks VEGF binding, whereas 6.64 may prevent receptor dimerization by perturbing the domain 3:domain 4 interface. Mutagenesis reveals that residues essential for VEGF, 1121B, and 6.64 binding are nonoverlapping among the three contact patches.

  8. The Structural Basis for the Function of Two Anti-VEGF Receptor 2 Antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Franklin, Matthew C.; Navarro, Elizabeth C.; Wang, Yujie; Patel, Sheetal; Singh, Pinki; Zhang, Yi; Persaud, Kris; Bari, Amtul; Griffith, Heather; Shen, Leyi; Balderes, Paul; Kussie, Paul (ImClone)


    The anti-VEGF receptor 2 antibody IMC-1121B is a promising antiangiogenic drug being tested for treatment of breast and gastric cancer. We have determined the structure of the 1121B Fab fragment in complex with domain 3 of VEGFR2, as well as the structure of a different neutralizing anti-VEGFR2 antibody, 6.64, also in complex with VEGFR2 domain 3. The two Fab fragments bind at opposite ends of VEGFR2 domain 3; 1121B directly blocks VEGF binding, whereas 6.64 may prevent receptor dimerization by perturbing the domain 3:domain 4 interface. Mutagenesis reveals that residues essential for VEGF, 1121B, and 6.64 binding are nonoverlapping among the three contact patches.

  9. Structural Basis for Negative Cooperativity in Growth Factor Binding to an EGF Receptor

    Energy Technology Data Exchange (ETDEWEB)

    Alvarado, Diego; Klein, Daryl E.; Lemmon, Mark A. (UPENN-MED)


    Transmembrane signaling by the epidermal growth factor receptor (EGFR) involves ligand-induced dimerization and allosteric regulation of the intracellular tyrosine kinase domain. Crystallographic studies have shown how ligand binding induces dimerization of the EGFR extracellular region but cannot explain the high-affinity and low-affinity classes of cell-surface EGF-binding sites inferred from curved Scatchard plots. From a series of crystal structures of the Drosophila EGFR extracellular region, we show here how Scatchard plot curvature arises from negatively cooperative ligand binding. The first ligand-binding event induces formation of an asymmetric dimer with only one bound ligand. The unoccupied site in this dimer is structurally restrained, leading to reduced affinity for binding of the second ligand, and thus negative cooperativity. Our results explain the cell-surface binding characteristics of EGF receptors and suggest how individual EGFR ligands might stabilize distinct dimeric species with different signaling properties.

  10. All-Atom Structural Models of the Transmembrane Domains of Insulin and Type 1 Insulin-Like Growth Factor Receptors. (United States)

    Mohammadiarani, Hossein; Vashisth, Harish


    The receptor tyrosine kinase superfamily comprises many cell-surface receptors including the insulin receptor (IR) and type 1 insulin-like growth factor receptor (IGF1R) that are constitutively homodimeric transmembrane glycoproteins. Therefore, these receptors require ligand-triggered domain rearrangements rather than receptor dimerization for activation. Specifically, binding of peptide ligands to receptor ectodomains transduces signals across the transmembrane domains for trans-autophosphorylation in cytoplasmic kinase domains. The molecular details of these processes are poorly understood in part due to the absence of structures of full-length receptors. Using MD simulations and enhanced conformational sampling algorithms, we present all-atom structural models of peptides containing 51 residues from the transmembrane and juxtamembrane regions of IR and IGF1R. In our models, the transmembrane regions of both receptors adopt helical conformations with kinks at Pro961 (IR) and Pro941 (IGF1R), but the C-terminal residues corresponding to the juxtamembrane region of each receptor adopt unfolded and flexible conformations in IR as opposed to a helix in IGF1R. We also observe that the N-terminal residues in IR form a kinked-helix sitting at the membrane-solvent interface, while homologous residues in IGF1R are unfolded and flexible. These conformational differences result in a larger tilt-angle of the membrane-embedded helix in IGF1R in comparison to IR to compensate for interactions with water molecules at the membrane-solvent interfaces. Our metastable/stable states for the transmembrane domain of IR, observed in a lipid bilayer, are consistent with a known NMR structure of this domain determined in detergent micelles, and similar states in IGF1R are consistent with a previously reported model of the dimerized transmembrane domains of IGF1R. Our all-atom structural models suggest potentially unique structural organization of kinase domains in each receptor.

  11. Structural dynamics and energetics underlying allosteric inactivation of the cannabinoid receptor CB1. (United States)

    Fay, Jonathan F; Farrens, David L


    G protein-coupled receptors (GPCRs) are surprisingly flexible molecules that can do much more than simply turn on G proteins. Some even exhibit biased signaling, wherein the same receptor preferentially activates different G-protein or arrestin signaling pathways depending on the type of ligand bound. Why this behavior occurs is still unclear, but it can happen with both traditional ligands and ligands that bind allosterically outside the orthosteric receptor binding pocket. Here, we looked for structural mechanisms underlying these phenomena in the marijuana receptor CB1. Our work focused on the allosteric ligand Org 27569, which has an unusual effect on CB1-it simultaneously increases agonist binding, decreases G--protein activation, and induces biased signaling. Using classical pharmacological binding studies, we find that Org 27569 binds to a unique allosteric site on CB1 and show that it can act alone (without need for agonist cobinding). Through mutagenesis studies, we find that the ability of Org 27569 to bind is related to how much receptor is in an active conformation that can couple with G protein. Using these data, we estimated the energy differences between the inactive and active states. Finally, site-directed fluorescence labeling studies show the CB1 structure stabilized by Org 27569 is different and unique from that stabilized by antagonist or agonist. Specifically, transmembrane helix 6 (TM6) movements associated with G-protein activation are blocked, but at the same time, helix 8/TM7 movements are enhanced, suggesting a possible mechanism for the ability of Org 27569 to induce biased signaling.

  12. Receptor Polymorphism and Genomic Structure Interact to Shape Bitter Taste Perception. (United States)

    Roudnitzky, Natacha; Behrens, Maik; Engel, Anika; Kohl, Susann; Thalmann, Sophie; Hübner, Sandra; Lossow, Kristina; Wooding, Stephen P; Meyerhof, Wolfgang


    The ability to taste bitterness evolved to safeguard most animals, including humans, against potentially toxic substances, thereby leading to food rejection. Nonetheless, bitter perception is subject to individual variations due to the presence of genetic functional polymorphisms in bitter taste receptor (TAS2R) genes, such as the long-known association between genetic polymorphisms in TAS2R38 and bitter taste perception of phenylthiocarbamide. Yet, due to overlaps in specificities across receptors, such associations with a single TAS2R locus are uncommon. Therefore, to investigate more complex associations, we examined taste responses to six structurally diverse compounds (absinthin, amarogentin, cascarillin, grosheimin, quassin, and quinine) in a sample of the Caucasian population. By sequencing all bitter receptor loci, inferring long-range haplotypes, mapping their effects on phenotype variation, and characterizing functionally causal allelic variants, we deciphered at the molecular level how a subjects' genotype for the whole-family of TAS2R genes shapes variation in bitter taste perception. Within each haplotype block implicated in phenotypic variation, we provided evidence for at least one locus harboring functional polymorphic alleles, e.g. one locus for sensitivity to amarogentin, one of the most bitter natural compounds known, and two loci for sensitivity to grosheimin, one of the bitter compounds of artichoke. Our analyses revealed also, besides simple associations, complex associations of bitterness sensitivity across TAS2R loci. Indeed, even if several putative loci harbored both high- and low-sensitivity alleles, phenotypic variation depended on linkage between these alleles. When sensitive alleles for bitter compounds were maintained in the same linkage phase, genetically driven perceptual differences were obvious, e.g. for grosheimin. On the contrary, when sensitive alleles were in opposite phase, only weak genotype-phenotype associations were seen

  13. Structural determinants for binding to angiotensin converting enzyme 2 (ACE2 and angiotensin receptors

    Directory of Open Access Journals (Sweden)

    Daniel eClayton


    Full Text Available Angiotensin converting enzyme 2 (ACE2 is a zinc carboxypeptidase involved in the renin angiotensin system (RAS and inactivates the potent vasopressive peptide angiotensin II (Ang II by removing the C-terminal phenylalanine residue to yield Ang1-7. This conversion inactivates the vasoconstrictive action of Ang II and yields a peptide that acts as a vasodilatory molecule at the Mas receptor and potentially other receptors. Given the growing complexity of RAS and level of cross-talk between ligands and their corresponding enzymes and receptors, the design of molecules with selectivity for the major RAS binding partners to control cardiovascular tone is an on-going challenge. In previous studies we used single β-amino acid substitutions to modulate the structure of Ang II and its selectivity for ACE2, AT1R and angiotensin type 2 (AT2R receptor. We showed that modification at the C-terminus of Ang II generally resulted in more pronounced changes to secondary structure and ligand binding, and here we further explore this region for the potential to modulate ligand specificity. In this study, 1 a library of forty-seven peptides derived from the C-terminal tetra-peptide sequence (-IHPF of Ang II was synthesised and assessed for ACE2 binding, 2 the terminal group requirements for high affinity ACE2 binding were explored by and N- and C-terminal modification, 3 high affinity ACE2 binding chimeric AngII analogues were then synthesized and assessed, 4 the structure of the full-length Ang II analogues were assessed by circular dichroism, and 5 the Ang II analogues were assessed for AT1R/AT2R selectivity by cell-based assays. Studies on the C-terminus of Ang II demonstrated varied specificity at different residue positions for ACE2 binding and four Ang II chimeric peptides were identified as selective ligands for the AT2 receptor. Overall, these results provide insight into the residue and structural requirements for ACE2 binding and angiotensin receptor

  14. X-ray structures of AMPA receptor-cone snail toxin complexes illuminate activation mechanism. (United States)

    Chen, Lei; Dürr, Katharina L; Gouaux, Eric


    AMPA-sensitive glutamate receptors are crucial to the structural and dynamic properties of the brain, to the development and function of the central nervous system, and to the treatment of neurological conditions from depression to cognitive impairment. However, the molecular principles underlying AMPA receptor activation have remained elusive. We determined multiple x-ray crystal structures of the GluA2 AMPA receptor in complex with a Conus striatus cone snail toxin, a positive allosteric modulator, and orthosteric agonists, at 3.8 to 4.1 angstrom resolution. We show how the toxin acts like a straightjacket on the ligand-binding domain (LBD) "gating ring," restraining the domains via both intra- and interdimer cross-links such that agonist-induced closure of the LBD "clamshells" is transduced into an irislike expansion of the gating ring. By structural analysis of activation-enhancing mutants, we show how the expansion of the LBD gating ring results in pulling forces on the M3 helices that, in turn, are coupled to ion channel gating.

  15. Crystal structure of the[mu]-opioid receptor bound to a morphinan antagonist

    Energy Technology Data Exchange (ETDEWEB)

    Manglik, Aashish; Kruse, Andrew C.; Kobilka, Tong Sun; Thian, Foon Sun; Mathiesen, Jesper M.; Sunahara, Roger K.; Pardo, Leonardo; Weis, William I.; Kobilka, Brian K.; Granier, Sébastien (Michigan-Med); (Stanford-MED); (UAB, Spain)


    Opium is one of the world's oldest drugs, and its derivatives morphine and codeine are among the most used clinical drugs to relieve severe pain. These prototypical opioids produce analgesia as well as many undesirable side effects (sedation, apnoea and dependence) by binding to and activating the G-protein-coupled {mu}-opioid receptor ({mu}-OR) in the central nervous system. Here we describe the 2.8 {angstrom} crystal structure of the mouse {mu}-OR in complex with an irreversible morphinan antagonist. Compared to the buried binding pocket observed in most G-protein-coupled receptors published so far, the morphinan ligand binds deeply within a large solvent-exposed pocket. Of particular interest, the {mu}-OR crystallizes as a two-fold symmetrical dimer through a four-helix bundle motif formed by transmembrane segments 5 and 6. These high-resolution insights into opioid receptor structure will enable the application of structure-based approaches to develop better drugs for the management of pain and addiction.

  16. A Hybrid Approach to Structure and Function Modeling of G Protein-Coupled Receptors. (United States)

    Latek, Dorota; Bajda, Marek; Filipek, Sławomir


    The recent GPCR Dock 2013 assessment of serotonin receptor 5-HT1B and 5-HT2B, and smoothened receptor SMO targets, exposed the strengths and weaknesses of the currently used computational approaches. The test cases of 5-HT1B and 5-HT2B demonstrated that both the receptor structure and the ligand binding mode can be predicted with the atomic-detail accuracy, as long as the target-template sequence similarity is relatively high. On the other hand, the observation of a low target-template sequence similarity, e.g., between SMO from the frizzled GPCR family and members of the rhodopsin family, hampers the GPCR structure prediction and ligand docking. Indeed, in GPCR Dock 2013, accurate prediction of the SMO target was still beyond the capabilities of most research groups. Another bottleneck in the current GPCR research, as demonstrated by the 5-HT2B target, is the reliable prediction of global conformational changes induced by activation of GPCRs. In this work, we report details of our protocol used during GPCR Dock 2013. Our structure prediction and ligand docking protocol was especially successful in the case of 5-HT1B and 5-HT2B-ergotamine complexes for which we provide one of the most accurate predictions. In addition to a description of the GPCR Dock 2013 results, we propose a novel hybrid computational methodology to improve GPCR structure and function prediction. This computational methodology employs two separate rankings for filtering GPCR models. The first ranking is ligand-based while the second is based on the scoring scheme of the recently published BCL method. In this work, we prove that the use of knowledge-based potentials implemented in BCL is an efficient way to cope with major bottlenecks in the GPCR structure prediction. Thereby, we also demonstrate that the knowledge-based potentials for membrane proteins were significantly improved, because of the recent surge in available experimental structures.

  17. Novel indole and azaindole (pyrrolopyridine) cannabinoid (CB) receptor agonists: design, synthesis, structure-activity relationships, physicochemical properties and biological activity

    NARCIS (Netherlands)

    Blaazer, A.R.; Lange, J.H.M.; van der Neut, M.A.W.; Mulder, A.; den Boon, F.S.; Werkman, T.R.; Kruse, C.G.; Wadman, W.J.


    The discovery, synthesis and structure-activity relationship (SAR) of a novel series of cannabinoid 1 (CB1) and cannabinoid 2 (CB2) receptor ligands are reported. Based on the aminoalkylindole class of cannabinoid receptor agonists, a biphenyl moiety was introduced as novel lipophilic indole 3-acyl

  18. Structural determinants of diphenethylamines for interaction with the κ opioid receptor: Synthesis, pharmacology and molecular modeling studies. (United States)

    Guerrieri, Elena; Bermudez, Marcel; Wolber, Gerhard; Berzetei-Gurske, Ilona P; Schmidhammer, Helmut; Spetea, Mariana


    The κ opioid (KOP) receptor crystal structure in an inactive state offers nowadays a valuable platform for inquiry into receptor function. We describe the synthesis, pharmacological evaluation and docking calculations of KOP receptor ligands from the class of diphenethylamines using an active-like structure of the KOP receptor attained by molecular dynamics simulations. The structure-activity relationships derived from computational studies was in accordance with pharmacological activities of targeted diphenethylamines at the KOP receptor established by competition binding and G protein activation in vitro assays. Our analysis identified that agonist binding results in breaking of the Arg156-Thr273 hydrogen bond, which stabilizes the inactive receptor conformation, and a crucial hydrogen bond with His291 is formed. Compounds with a phenolic 4-hydroxy group do not form the hydrogen bond with His291, an important residue for KOP affinity and agonist activity. The size of the N-substituent hosted by the hydrophobic pocket formed by Val108, Ile316 and Tyr320 considerably influences binding and selectivity, with the n-alkyl size limit being five carbon atoms, while bulky substituents turn KOP agonists in antagonists. Thus, combination of experimental and molecular modeling strategies provides an initial framework for understanding the structural features of diphenethylamines that are essential to promote binding affinity and selectivity for the KOP receptor, and may be involved in transduction of the ligand binding event into molecular changes, ultimately leading to receptor activation.

  19. Evidence for homogeneity of thromboxane A2 receptor using structurally different antagonists. (United States)

    Swayne, G T; Maguire, J; Dolan, J; Raval, P; Dane, G; Greener, M; Owen, D A


    Nine structurally dissimilar thromboxane antagonists (SQ 29548, ICI 185282, AH 23848, BM 13505 (Daltroban), BM 13177 (Sulotroban), SK&F 88046, L-636499, L-640035 and a Bayer compound SK&F 47821) were studied for activity as thromboxane A2 receptor antagonists. The assays used were inhibition of responses induced by the thromboxane mimetic, U46619, on human washed platelet aggregation, rabbit platelet aggregation, rabbit aortic strip contraction, anaesthetised guinea-pig bronchoconstriction, and a radio-labelled ligand (125I-PTA-OH) binding assay as a measure of affinity for the human platelet receptor. The results of the present study, with activities spanning at least four orders of magnitude along with statistically significant correlations (at least P less than 0.01), strongly suggests that between assays, antagonists and species a homogenous population of thromboxane A2 receptors exists. This finding is in contrast to those of a close series of 13-azapinane antagonists studied by other workers which have suggested receptor heterogeneity.

  20. Structure-Based Understanding of Binding Affinity and Mode of Estrogen Receptor α Agonists and Antagonists (United States)

    Barron, Mace G.


    The flexible hydrophobic ligand binding pocket (LBP) of estrogen receptor α (ERα) allows the binding of a wide variety of endocrine disruptors. Upon ligand binding, the LBP reshapes around the contours of the ligand and stabilizes the complex by complementary hydrophobic interactions and specific hydrogen bonds with the ligand. Here we present a framework for quantitative analysis of the steric and electronic features of the human ERα-ligand complex using three dimensional (3D) protein-ligand interaction description combined with 3D-QSAR approach. An empirical hydrophobicity density field is applied to account for hydrophobic contacts of ligand within the LBP. The obtained 3D-QSAR model revealed that hydrophobic contacts primarily determine binding affinity and govern binding mode with hydrogen bonds. Several residues of the LBP appear to be quite flexible and adopt a spectrum of conformations in various ERα-ligand complexes, in particular His524. The 3D-QSAR was combined with molecular docking based on three receptor conformations to accommodate receptor flexibility. The model indicates that the dynamic character of the LBP allows accommodation and stable binding of structurally diverse ligands, and proper representation of the protein flexibility is critical for reasonable description of binding of the ligands. Our results provide a quantitative and mechanistic understanding of binding affinity and mode of ERα agonists and antagonists that may be applicable to other nuclear receptors. PMID:28061508

  1. The structure of cytomegalovirus immune modulator UL141 highlights structural Ig-fold versatility for receptor binding

    Energy Technology Data Exchange (ETDEWEB)

    Nemčovičová, Ivana [La Jolla Institute for Allergy and Immunology, 9420 Athena Circle, La Jolla, CA 92037 (United States); Slovak Academy of Sciences, Dúbravská cesta 9, SK 84505 Bratislava (Slovakia); Zajonc, Dirk M., E-mail: [La Jolla Institute for Allergy and Immunology, 9420 Athena Circle, La Jolla, CA 92037 (United States)


    The crystal structure of Human cytomegalovirus immune modulator UL141 was solved at 3.25 Å resolution. Here, a detailed analysis of its intimate dimerization interface and the biophysical properties of its receptor (TRAIL-R2 and CD155) binding interactions are presented. Natural killer (NK) cells are critical components of the innate immune system as they rapidly detect and destroy infected cells. To avoid immune recognition and to allow long-term persistence in the host, Human cytomegalovirus (HCMV) has evolved a number of genes to evade or inhibit immune effector pathways. In particular, UL141 can inhibit cell-surface expression of both the NK cell-activating ligand CD155 as well as the TRAIL death receptors (TRAIL-R1 and TRAIL-R2). The crystal structure of unliganded HCMV UL141 refined to 3.25 Å resolution allowed analysis of its head-to-tail dimerization interface. A ‘dimerization-deficient’ mutant of UL141 (ddUL141) was further designed, which retained the ability to bind to TRAIL-R2 or CD155 while losing the ability to cross-link two receptor monomers. Structural comparison of unliganded UL141 with UL141 bound to TRAIL-R2 further identified a mobile loop that makes intimate contacts with TRAIL-R2 upon receptor engagement. Superposition of the Ig-like domain of UL141 on the CD155 ligand T-cell immunoreceptor with Ig and ITIM domains (TIGIT) revealed that UL141 can potentially engage CD155 similar to TIGIT by using the C′C′′ and GF loops. Further mutations in the TIGIT binding site of CD155 (Q63R and F128R) abrogated UL141 binding, suggesting that the Ig-like domain of UL141 is a viral mimic of TIGIT, as it targets the same binding site on CD155 using similar ‘lock-and-key’ interactions. Sequence alignment of the UL141 gene and its orthologues also showed conservation in this highly hydrophobic (L/A)X{sub 6}G ‘lock’ motif for CD155 binding as well as conservation of the TRAIL-R2 binding patches, suggesting that these host–receptor

  2. Structure of metabotropic glutamate receptor C-terminal domains in contact with interacting proteins

    Directory of Open Access Journals (Sweden)

    Ralf eEnz


    Full Text Available Metabotropic glutamate receptors (mGluRs regulate intracellular signal pathways that control several physiological tasks, including neuronal excitability, learning and memory. This is achieved by the formation of synaptic signal complexes, in which mGluRs assemble with functionally related proteins such as enzymes, scaffolds and cytoskeletal anchor proteins. Thus, mGluR associated proteins actively participate in the regulation of glutamatergic neurotransmission. Importantly, dysfunction of mGluRs and interacting proteins may lead to impaired signal transduction and finally result in neurological disorders, e.g. night blindness, addiction, epilepsy, schizophrenia, autism spectrum disorders and Parkinson´s disease. In contrast to solved crystal structures of extracellular N-terminal domains of some mGluR types, only a few studies analyzed the conformation of intracellular receptor domains. Intracellular C-termini of most mGluR types are subject to alternative splicing and can be further modified by phosphorylation and SUMOylation. In this way, diverse interaction sites for intracellular proteins that bind to and regulate the glutamate receptors are generated. Indeed, most of the known mGluR binding partners interact with the receptors´ C-terminal domains. Within the last years, different laboratories analyzed the structure of these domains and described the geometry of the contact surface between mGluR C-termini and interacting proteins. Here, I will review recent progress in the structure characterization of mGluR C-termini and provide an up-to-date summary of the geometry of these domains in contact with binding partners.

  3. Structure of metabotropic glutamate receptor C-terminal domains in contact with interacting proteins. (United States)

    Enz, Ralf


    Metabotropic glutamate receptors (mGluRs) regulate intracellular signal pathways that control several physiological tasks, including neuronal excitability, learning, and memory. This is achieved by the formation of synaptic signal complexes, in which mGluRs assemble with functionally related proteins such as enzymes, scaffolds, and cytoskeletal anchor proteins. Thus, mGluR associated proteins actively participate in the regulation of glutamatergic neurotransmission. Importantly, dysfunction of mGluRs and interacting proteins may lead to impaired signal transduction and finally result in neurological disorders, e.g., night blindness, addiction, epilepsy, schizophrenia, autism spectrum disorders and Parkinson's disease. In contrast to solved crystal structures of extracellular N-terminal domains of some mGluR types, only a few studies analyzed the conformation of intracellular receptor domains. Intracellular C-termini of most mGluR types are subject to alternative splicing and can be further modified by phosphorylation and SUMOylation. In this way, diverse interaction sites for intracellular proteins that bind to and regulate the glutamate receptors are generated. Indeed, most of the known mGluR binding partners interact with the receptors' C-terminal domains. Within the last years, different laboratories analyzed the structure of these domains and described the geometry of the contact surface between mGluR C-termini and interacting proteins. Here, I will review recent progress in the structure characterization of mGluR C-termini and provide an up-to-date summary of the geometry of these domains in contact with binding partners.

  4. Molecular and Structural Characterization of a Novel Escherichia coli Interleukin Receptor Mimic Protein

    Directory of Open Access Journals (Sweden)

    Danilo G. Moriel


    Full Text Available Urinary tract infection (UTI is a disease of extremely high incidence in both community and nosocomial settings. UTIs cause significant morbidity and mortality, with approximately 150 million cases globally per year. Uropathogenic Escherichia coli (UPEC is the primary cause of UTI and is generally treated empirically. However, the rapidly increasing incidence of UTIs caused by multidrug-resistant UPEC strains has led to limited available treatment options and highlights the urgent need to develop alternative treatment and prevention strategies. In this study, we performed a comprehensive analysis to define the regulation, structure, function, and immunogenicity of recently identified UPEC vaccine candidate C1275 (here referred to as IrmA. We showed that the irmA gene is highly prevalent in UPEC, is cotranscribed with the biofilm-associated antigen 43 gene, and is regulated by the global oxidative stress response OxyR protein. Localization studies identified IrmA in the UPEC culture supernatant. We determined the structure of IrmA and showed that it adopts a unique domain-swapped dimer architecture. The dimeric structure of IrmA displays similarity to those of human cytokine receptors, including the interleukin-2 receptor (IL-2R, interleukin-4 receptor (IL-4R, and interleukin-10 receptor (IL-10R binding domains, and we showed that purified IrmA can bind to their cognate cytokines. Finally, we showed that plasma from convalescent urosepsis patients contains high IrmA antibody titers, demonstrating the strong immunogenicity of IrmA. Taken together, our results indicate that IrmA may play an important role during UPEC infection.

  5. Modeling structure of G protein-coupled receptors in huan genome

    KAUST Repository

    Zhang, Yang


    G protein-coupled receptors (or GPCRs) are integral transmembrane proteins responsible to various cellular signal transductions. Human GPCR proteins are encoded by 5% of human genes but account for the targets of 40% of the FDA approved drugs. Due to difficulties in crystallization, experimental structure determination remains extremely difficult for human GPCRs, which have been a major barrier in modern structure-based drug discovery. We proposed a new hybrid protocol, GPCR-I-TASSER, to construct GPCR structure models by integrating experimental mutagenesis data with ab initio transmembrane-helix assembly simulations, assisted by the predicted transmembrane-helix interaction networks. The method was tested in recent community-wide GPCRDock experiments and constructed models with a root mean square deviation 1.26 Å for Dopamine-3 and 2.08 Å for Chemokine-4 receptors in the transmembrane domain regions, which were significantly closer to the native than the best templates available in the PDB. GPCR-I-TASSER has been applied to model all 1,026 putative GPCRs in the human genome, where 923 are found to have correct folds based on the confidence score analysis and mutagenesis data comparison. The successfully modeled GPCRs contain many pharmaceutically important families that do not have previously solved structures, including Trace amine, Prostanoids, Releasing hormones, Melanocortins, Vasopressin and Neuropeptide Y receptors. All the human GPCR models have been made publicly available through the GPCR-HGmod database at The results demonstrate new progress on genome-wide structure modeling of transmembrane proteins which should bring useful impact on the effort of GPCR-targeted drug discovery.

  6. Structure, signaling mechanism and regulation of the natriuretic peptide receptor guanylate cyclase.

    Energy Technology Data Exchange (ETDEWEB)

    Misono, K. S.; Philo, J. S.; Arakawa, T.; Ogata, C. M.; Qiu, Y.; Ogawa, H.; Young, H. S. (Biosciences Division); (Univ. of Nevada); (Alliance Protein Labs.)


    Atrial natriuretic peptide (ANP) and the homologous B-type natriuretic peptide are cardiac hormones that dilate blood vessels and stimulate natriuresis and diuresis, thereby lowering blood pressure and blood volume. ANP and B-type natriuretic peptide counterbalance the actions of the renin-angiotensin-aldosterone and neurohormonal systems, and play a central role in cardiovascular regulation. These activities are mediated by natriuretic peptide receptor-A (NPRA), a single transmembrane segment, guanylyl cyclase (GC)-linked receptor that occurs as a homodimer. Here, we present an overview of the structure, possible chloride-mediated regulation and signaling mechanism of NPRA and other receptor GCs. Earlier, we determined the crystal structures of the NPRA extracellular domain with and without bound ANP. Their structural comparison has revealed a novel ANP-induced rotation mechanism occurring in the juxtamembrane region that apparently triggers transmembrane signal transduction. More recently, the crystal structures of the dimerized catalytic domain of green algae GC Cyg12 and that of cyanobacterium GC Cya2 have been reported. These structures closely resemble that of the adenylyl cyclase catalytic domain, consisting of a C1 and C2 subdomain heterodimer. Adenylyl cyclase is activated by binding of G{sub s}{alpha} to C2 and the ensuing 7{sup o} rotation of C1 around an axis parallel to the central cleft, thereby inducing the heterodimer to adopt a catalytically active conformation. We speculate that, in NPRA, the ANP-induced rotation of the juxtamembrane domains, transmitted across the transmembrane helices, may induce a similar rotation in each of the dimerized GC catalytic domains, leading to the stimulation of the GC catalytic activity.

  7. Crystal structure of the β2 adrenergic receptor-Gs protein complex

    Energy Technology Data Exchange (ETDEWEB)

    Rasmussen, Søren G.F.; DeVree, Brian T; Zou, Yaozhong; Kruse, Andrew C; Chung, Ka Young; Kobilka, Tong Sun; Thian, Foon Sun; Chae, Pil Seok; Pardon, Els; Calinski, Diane; Mathiesen, Jesper M; Shah, Syed T.A.; Lyons, Joseph A; Caffrey, Martin; Gellman, Samuel H; Steyaert, Jan; Skiniotis, Georgios; Weis, William I; Sunahara, Roger K; Kobilka, Brian K [Brussels; (Trinity); (Michigan); (Stanford-MED); (Michigan-Med); (UW)


    G protein-coupled receptors (GPCRs) are responsible for the majority of cellular responses to hormones and neurotransmitters as well as the senses of sight, olfaction and taste. The paradigm of GPCR signalling is the activation of a heterotrimeric GTP binding protein (G protein) by an agonist-occupied receptor. The β2 adrenergic receptor2AR) activation of Gs, the stimulatory G protein for adenylyl cyclase, has long been a model system for GPCR signalling. Here we present the crystal structure of the active state ternary complex composed of agonist-occupied monomeric β2AR and nucleotide-free Gs heterotrimer. The principal interactions between the β2AR and Gs involve the amino- and carboxy-terminal α-helices of Gs, with conformational changes propagating to the nucleotide-binding pocket. The largest conformational changes in the β2AR include a 14Å outward movement at the cytoplasmic end of transmembrane segment 6 (TM6) and an α-helical extension of the cytoplasmic end of TM5. The most surprising observation is a major displacement of the α-helical domain of Gαs relative to the Ras-like GTPase domain. This crystal structure represents the first high-resolution view of transmembrane signalling by a GPCR.

  8. Molecular And Structural Basis of Cytokine Receptor Pleiotropy in the Interleukin-4/13 System

    Energy Technology Data Exchange (ETDEWEB)

    LaPorte, S.L.; Juo, Z.S.; Vaclavikova, J.; Colf, L.A.; Qi, X.; Heller, N.M.; Keegan, A.D.; Garcia, K.C.


    Interleukin-4 and Interleukin-13 are cytokines critical to the development of T cell-mediated humoral immune responses, which are associated with allergy and asthma, and exert their actions through three different combinations of shared receptors. Here we present the crystal structures of the complete set of type I (IL-4R{alpha}/{gamma}{sub c}/IL-4) and type II (IL-4R/IL-13R{alpha}1/IL-4, IL-4R{alpha}/IL-13R{alpha}1/IL-13) ternary signaling complexes. The type I complex reveals a structural basis for {gamma}{sub c}'s ability to recognize six different {gamma}{sub c}-cytokines. The two type II complexes utilize an unusual top-mounted Ig-like domain on IL-13R{alpha}1 for a novel mode of cytokine engagement that contributes to a reversal in the IL-4 versus IL-13 ternary complex assembly sequences, which are mediated through substantially different recognition chemistries. We also show that the type II receptor heterodimer signals with different potencies in response to IL-4 versus IL-13 and suggest that the extracellular cytokine-receptor interactions are modulating intracellular membrane-proximal signaling events.

  9. The crystal structure of a bimorphinan with highly selective kappa opioid receptor antagonist activity (United States)

    Urbańczyk-Lipkowska, Zofia; Etter, Margaret C.; Lipkowski, Andrzej W.; Portoghese, Philip S.


    The crystal structure of the dihydrobromide heptahydrate of nor-binaltorphimine (17, 17'-bis(cyclopropylmethyl)-6,6',7,7'-tetrahydro-4,5α: 4',5'α-diepoxy-6,6'-imino[7,7' bimorphinan]-3,3',14,14'-tetraol)is presented. This structure is the first reported structure of a rigid bivalent opioid ligand. Two morphinan pharmacophores are connected by a rigid spacer, the pyrrole ring. The nor-binaltorphimine structure itself shows unique, high selectivity as a kappa opioid receptor antagonist. Crystal data: P3 2, Z = 3, a = b = 20.223 (4), c = 9.541(7) Å, α = β = 90°, γ = 120°; R = 0.079 (1765 reflections, Fobs > 1σ( F)).

  10. Structure modeling of all identified G protein-coupled receptors in the human genome.

    Directory of Open Access Journals (Sweden)

    Yang Zhang


    Full Text Available G protein-coupled receptors (GPCRs, encoded by about 5% of human genes, comprise the largest family of integral membrane proteins and act as cell surface receptors responsible for the transduction of endogenous signal into a cellular response. Although tertiary structural information is crucial for function annotation and drug design, there are few experimentally determined GPCR structures. To address this issue, we employ the recently developed threading assembly refinement (TASSER method to generate structure predictions for all 907 putative GPCRs in the human genome. Unlike traditional homology modeling approaches, TASSER modeling does not require solved homologous template structures; moreover, it often refines the structures closer to native. These features are essential for the comprehensive modeling of all human GPCRs when close homologous templates are absent. Based on a benchmarked confidence score, approximately 820 predicted models should have the correct folds. The majority of GPCR models share the characteristic seven-transmembrane helix topology, but 45 ORFs are predicted to have different structures. This is due to GPCR fragments that are predominantly from extracellular or intracellular domains as well as database annotation errors. Our preliminary validation includes the automated modeling of bovine rhodopsin, the only solved GPCR in the Protein Data Bank. With homologous templates excluded, the final model built by TASSER has a global C(alpha root-mean-squared deviation from native of 4.6 angstroms, with a root-mean-squared deviation in the transmembrane helix region of 2.1 angstroms. Models of several representative GPCRs are compared with mutagenesis and affinity labeling data, and consistent agreement is demonstrated. Structure clustering of the predicted models shows that GPCRs with similar structures tend to belong to a similar functional class even when their sequences are diverse. These results demonstrate the usefulness

  11. Structure-based drug design targeting the cell membrane receptor GPBAR1: exploiting the bile acid scaffold towards selective agonism (United States)

    di Leva, Francesco Saverio; Festa, Carmen; Renga, Barbara; Sepe, Valentina; Novellino, Ettore; Fiorucci, Stefano; Zampella, Angela; Limongelli, Vittorio


    Bile acids can regulate nutrient metabolism through the activation of the cell membrane receptor GPBAR1 and the nuclear receptor FXR. Developing an exogenous control over these receptors represents an attractive strategy for the treatment of enterohepatic and metabolic disorders. A number of dual GPBAR1/FXR agonists are known, however their therapeutic use is limited by multiple unwanted effects due to activation of the diverse downstream signals controlled by the two receptors. On the other hand, designing selective GPBAR1 and FXR agonists is challenging since the two proteins share similar structural requisites for ligand binding. Here, taking advantage of our knowledge of the two targets, we have identified through a rational drug design study a series of amine lithocholic acid derivatives as selective GPBAR1 agonists. The presence of the 3α-NH2 group on the steroidal scaffold is responsible for the selectivity over FXR unveiling unprecedented structural insights into bile acid receptors activity modulation.

  12. Novel highly potent serotonin 5-HT7 receptor ligands: structural modifications to improve pharmacokinetic properties. (United States)

    Lacivita, Enza; Di Pilato, Pantaleo; Stama, Madia Letizia; Colabufo, Nicola Antonio; Berardi, Francesco; Perrone, Roberto; De Filippis, Bianca; Laviola, Giovanni; Adriani, Walter; Niso, Mauro; Leopoldo, Marcello


    Here we report the synthesis, pharmacological and pharmacokinetic evaluation of a pilot set of compounds structurally related to the potent and selective 5-HT7 ligand LP-211. Among the studied compounds, N-pyridin-3-ylmethyl-3-[4-[2-(4-methoxyphenyl)phenyl]piperazin-1-yl]ethoxy]propanamide (4b) showed high affinity for 5-HT7 receptors (K(i)=23.8 nM), selectivity over 5-HT1A receptors (>50-fold), in vitro metabolic stability (82%) and weak interaction with P-glycoprotein (BA/AB=3.3). Compound 4b was injected ip in mice to preliminarily evaluate its distribution between blood and brain.

  13. Structural basis for chemokine recognition and activation of a viral G protein-coupled receptor

    Energy Technology Data Exchange (ETDEWEB)

    Burg, John S.; Ingram, Jessica R.; Venkatakrishnan, A.J.; Jude, Kevin M.; Dukkipati, Abhiram; Feinberg, Evan N.; Angelini, Alessandro; Waghray, Deepa; Dror, Ron O.; Ploegh, Hidde L.; Garcia, K. Christopher (Stanford); (Stanford-MED); (Whitehead); (MIT)


    Chemokines are small proteins that function as immune modulators through activation of chemokine G protein-coupled receptors (GPCRs). Several viruses also encode chemokines and chemokine receptors to subvert the host immune response. How protein ligands activate GPCRs remains unknown. We report the crystal structure at 2.9 angstrom resolution of the human cytomegalovirus GPCR US28 in complex with the chemokine domain of human CX3CL1 (fractalkine). The globular body of CX3CL1 is perched on top of the US28 extracellular vestibule, whereas its amino terminus projects into the central core of US28. The transmembrane helices of US28 adopt an active-state-like conformation. Atomic-level simulations suggest that the agonist-independent activity of US28 may be due to an amino acid network evolved in the viral GPCR to destabilize the receptor’s inactive state.

  14. Transient Receptor Potential Channels Contribute to Pathological Structural and Functional Remodeling After Myocardial Infarction (United States)

    Davis, Jennifer; Correll, Robert N.; Trappanese, Danielle M.; Hoffman, Nicholas E.; Troupes, Constantine D.; Berretta, Remus M.; Kubo, Hajime; Madesh, Muniswamy; Chen, Xiongwen; Gao, Erhe; Molkentin, Jeffery D.; Houser, Steven R.


    Rationale The cellular and molecular basis for post myocardial infarction (MI) structural and functional remodeling is not well understood. Objective To determine if Ca2+ influx through transient receptor potential (canonical) (TRPC) channels contributes to post-MI structural and functional remodeling. Methods and Results TRPC1/3/4/6 channel mRNA increased after MI in mice and was associated with TRPC-mediated Ca2+ entry. Cardiac myocyte specific expression of a dominant negative (dn: loss of function) TRPC4 channel increased basal myocyte contractility and reduced hypertrophy and cardiac structural and functional remodeling after MI while increasing survival. We used adenovirus-mediated expression of TRPC3/4/6 channels in cultured adult feline myocytes (AFMs) to define mechanistic aspects of these TRPC-related effects. TRPC3/4/6 over expression in AFMs induced calcineurin (Cn)-Nuclear Factor of Activated T cells (NFAT) mediated hypertrophic signaling, which was reliant on caveolae targeting of TRPCs. TRPC3/4/6 expression in AFMs increased rested state contractions and increased spontaneous sarcoplasmic reticulum (SR) Ca2+ sparks mediated by enhanced phosphorylation of the ryanodine receptor. TRPC3/4/6 expression was associated with reduced contractility and response to catecholamines during steady state pacing, likely due to enhanced SR Ca2+ leak. Conclusions Ca2+ influx through TRPC channels expressed after MI activates pathological cardiac hypertrophy and reduces contractility reserve. Blocking post-MI TRPC activity improved post-MI cardiac structure and function. PMID:25047165

  15. Structure of the human [kappa]-opioid receptor in complex with JDTic

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Huixian; Wacker, Daniel; Mileni, Mauro; Katritch, Vsevolod; Han, Gye Won; Vardy, Eyal; Liu, Wei; Thompson, Aaron A.; Huang, Xi-Ping; Carroll, F. Ivy; Mascarella, S. Wayne; Westkaemper, Richard B.; Mosier, Philip D.; Roth, Bryan L.; Cherezov, Vadim; Stevens, Raymond C. (VCU); (Scripps); (UNC); (Res. Tri. Inst.)


    Opioid receptors mediate the actions of endogenous and exogenous opioids on many physiological processes, including the regulation of pain, respiratory drive, mood, and - in the case of {kappa}-opioid receptor ({kappa}-OR) - dysphoria and psychotomimesis. Here we report the crystal structure of the human {kappa}-OR in complex with the selective antagonist JDTic, arranged in parallel dimers, at 2.9 {angstrom} resolution. The structure reveals important features of the ligand-binding pocket that contribute to the high affinity and subtype selectivity of JDTic for the human {kappa}-OR. Modelling of other important {kappa}-OR-selective ligands, including the morphinan-derived antagonists norbinaltorphimine and 5'-guanidinonaltrindole, and the diterpene agonist salvinorin A analogue RB-64, reveals both common and distinct features for binding these diverse chemotypes. Analysis of site-directed mutagenesis and ligand structure-activity relationships confirms the interactions observed in the crystal structure, thereby providing a molecular explanation for {kappa}-OR subtype selectivity, and essential insights for the design of compounds with new pharmacological properties targeting the human {kappa}-OR.

  16. High-resolution crystal structure of an engineered human beta2-adrenergic G protein-coupled receptor

    DEFF Research Database (Denmark)

    Cherezov, Vadim; Rosenbaum, Daniel M; Hanson, Michael A;


    Heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors constitute the largest family of eukaryotic signal transduction proteins that communicate across the membrane. We report the crystal structure of a human beta2-adrenergic receptor-T4 lysozyme fusion protein bound...... to the partial inverse agonist carazolol at 2.4 angstrom resolution. The structure provides a high-resolution view of a human G protein-coupled receptor bound to a diffusible ligand. Ligand-binding site accessibility is enabled by the second extracellular loop, which is held out of the binding cavity by a pair...

  17. 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


    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.

  18. Forster Resonance Energy Transfer (FRET) Analysis of Dual CFP/YFP Labeled AMPA Receptors Reveals Structural Rearrangement within the C-Terminal Domain during Receptor Activation

    DEFF Research Database (Denmark)

    Zachariassen, Linda Grønborg; Katchan, Mila; Plested, Andrew;


    AMPA receptors (AMPARs) are glutamate-gated cation channels that mediate the majority of fast excitatory neurotransmission in the central nervous system. AMPARs are formed by homo- or heterotetramers of GluA1 to GluA4 sub- units. A recent X-ray crystal structure of a full-length homomeric GluA2 A...

  19. Structure-based virtual screening of the nociceptin receptor: hybrid docking and shape-based approaches for improved hit identification. (United States)

    Daga, Pankaj R; Polgar, Willma E; Zaveri, Nurulain T


    The antagonist-bound crystal structure of the nociceptin receptor (NOP), from the opioid receptor family, was recently reported along with those of the other opioid receptors bound to opioid antagonists. We recently reported the first homology model of the 'active-state' of the NOP receptor, which when docked with 'agonist' ligands showed differences in the TM helices and residues, consistent with GPCR activation after agonist binding. In this study, we explored the use of the active-state NOP homology model for structure-based virtual screening to discover NOP ligands containing new chemical scaffolds. Several NOP agonist and antagonist ligands previously reported are based on a common piperidine scaffold. Given the structure-activity relationships for known NOP ligands, we developed a hybrid method that combines a structure-based and ligand-based approach, utilizing the active-state NOP receptor as well as the pharmacophoric features of known NOP ligands, to identify novel NOP binding scaffolds by virtual screening. Multiple conformations of the NOP active site including the flexible second extracellular loop (EL2) loop were generated by simulated annealing and ranked using enrichment factor (EF) analysis and a ligand-decoy dataset containing known NOP agonist ligands. The enrichment factors were further improved by combining shape-based screening of this ligand-decoy dataset and calculation of consensus scores. This combined structure-based and ligand-based EF analysis yielded higher enrichment factors than the individual methods, suggesting the effectiveness of the hybrid approach. Virtual screening of the CNS Permeable subset of the ZINC database was carried out using the above-mentioned hybrid approach in a tiered fashion utilizing a ligand pharmacophore-based filtering step, followed by structure-based virtual screening using the refined NOP active-state models from the enrichment analysis. Determination of the NOP receptor binding affinity of a selected set

  20. Structure and Mechanism of Receptor Sharing by the IL-10R2 Common Chain

    Energy Technology Data Exchange (ETDEWEB)

    Yoon, Sung-il; Jones, Brandi C.; Logsdon, Naomi J.; Harris, Bethany D.; Deshpande, Ashlesha; Radaeva, Svetlana; Halloran, Brian A.; Gao, Bin; Walter, Mark R. (NIH); (UAB)


    IL-10R2 is a shared cell surface receptor required for the activation of five class 2 cytokines (IL-10, IL-22, IL-26, IL-28, and IL-29) that play critical roles in host defense. To define the molecular mechanisms that regulate its promiscuous binding, we have determined the crystal structure of the IL-10R2 ectodomain at 2.14 {angstrom} resolution. IL-10R2 residues required for binding were identified by alanine scanning and used to derive computational models of IL-10/IL-10R1/IL-10R2 and IL-22/IL-22R1/IL-10R2 ternary complexes. The models reveal a conserved binding epitope that is surrounded by two clefts that accommodate the structural and chemical diversity of the cytokines. These results provide a structural framework for interpreting IL-10R2 single nucleotide polymorphisms associated with human disease.

  1. Insights into the structural biology of G-protein coupled receptors impacts drug design for central nervous system neurodegenerative processes**

    Institute of Scientific and Technical Information of China (English)

    Farfán-García Eunice Dalet; Soriano-Ursúa Marvin Antonio


    In the last few years, there have been important new insights into the structural biology of G-protein coupled receptors. It is now known that al osteric binding sites are involved in the affinity and selec-tivity of ligands for G-protein coupled receptors, and that signaling by these receptors involves both G-protein dependent and independent pathways. The present review outlines the physiological and pharmacological implications of this perspective for the design of new drugs to treat disorders of the central nervous system. Specifical y, new possibilities are explored in relation to al osteric and orthosteric binding sites on dopamine receptors for the treatment of Parkinson’s disease, and on muscarinic receptors for Alzheimer’s disease. Future research can seek to identify ligands that can bind to more than one site on the same receptor, or simultaneously bind to two receptors and form a dimer. For example, the design of bivalent drugs that can reach homo/hetero-dimers of D2 dopa-mine receptor holds promise as a relevant therapeutic strategy for Parkinson’s disease. Regarding the treatment of Alzheimer’s disease, the design of dualsteric ligands for mono-oligomeric musca-rinic receptors could increase therapeutic effectiveness by generating potent compounds that could activate more than one signaling pathway.

  2. Structural basis by which alternative splicing confers specificity in fibroblast growth factor receptors. (United States)

    Yeh, Brian K; Igarashi, Makoto; Eliseenkova, Anna V; Plotnikov, Alexander N; Sher, Ifat; Ron, Dina; Aaronson, Stuart A; Mohammadi, Moosa


    Binding specificity between fibroblast growth factors (FGFs) and their receptors (FGFRs) is essential for mammalian development and is regulated primarily by two alternatively spliced exons, IIIb ("b") and IIIc ("c"), that encode the second half of Ig-like domain 3 (D3) of FGFRs. FGF7 and FGF10 activate only the b isoform of FGFR2 (FGFR2b). Here, we report the crystal structure of the ligand-binding portion of FGFR2b bound to FGF10. Unique contacts between divergent regions in FGF10 and two b-specific loops in D3 reveal the structural basis by which alternative splicing provides FGF10-FGFR2b specificity. Structure-based mutagenesis of FGF10 confirms the importance of the observed contacts for FGF10 biological activity. Interestingly, FGF10 binding induces a previously unobserved rotation of receptor Ig domain 2 (D2) to introduce specific contacts with FGF10. Hence, both D2 and D3 of FGFR2b contribute to the exceptional specificity between FGF10 and FGFR2b. We propose that ligand-induced conformational change in FGFRs may also play an important role in determining specificity for other FGF-FGFR complexes.

  3. Local and global ligand-induced changes in the structure of the GABA(A) receptor. (United States)

    Muroi, Yukiko; Czajkowski, Cynthia; Jackson, Meyer B


    Ligand-gated channels mediate synaptic transmission through conformational transitions triggered by the binding of neurotransmitters. These transitions are well-defined in terms of ion conductance, but their structural basis is poorly understood. To probe these changes in structure, GABA(A) receptors were expressed in Xenopus oocytes and labeled at selected sites with environment-sensitive fluorophores. With labels at two different residues in the alpha1 subunit in loop E of the GABA-binding pocket, GABA elicited fluorescence changes opposite in sign. This pattern of fluorescence changes is consistent with a closure of the GABA-binding cavity at the subunit interface. The competitive antagonist SR-95531 inverted this pattern of fluorescence change, but the noncompetitive antagonist picrotoxin failed to elicit optical signals. In response to GABA (but not SR-95531), labels at the homologous residues in the beta2 subunit showed the same pattern of fluorescence change as the alpha1-subunit labels, indicating a global transition with comparable movements in homologous regions of different subunits. Incorporation of the gamma2 subunit altered the fluorescence changes of alpha1-subunit labels and eliminated them in beta2-subunit labels. Thus, the ligand-induced structural changes in the GABA(A) receptor can extend over considerable distances or remain highly localized, depending upon subunit composition and ligand.

  4. Macrophage Receptor with Collagenous Structure (MARCO Is Processed by either Macropinocytosis or Endocytosis-Autophagy Pathway.

    Directory of Open Access Journals (Sweden)

    Seishiro Hirano

    Full Text Available The Macrophage Receptor with COllagenous structure (MARCO protein is a plasma membrane receptor for un-opsonized or environmental particles on phagocytic cells. Here, we show that MARCO was internalized either by ruffling of plasma membrane followed by macropinocytosis or by endocytosis followed by fusion with autophagosome in CHO-K1 cells stably transfected with GFP-MARCO. The macropinocytic process generated large vesicles when the plasma membrane subsided. The endocytosis/autophagosome (amphisome generated small fluorescent puncta which were visible in the presence of glutamine, chloroquine, bafilomycin, ammonia, and other amines. The small puncta, but not the large vesicles, co-localized with LC3B and lysosomes. The LC3-II/LC3-I ratio increased in the presence of glutamine, ammonia, and chloroquine in various cells. The small puncta trafficked between the peri-nuclear region and the distal ends of cells back and forth at rates of up to 2-3 μm/sec; tubulin, but not actin, regulated the trafficking of the small puncta. Besides phagocytosis MARCO, an adhesive plasma membrane receptor, may play a role in incorporation of various extracellular materials into the cell via both macropinocytic and endocytic pathways.

  5. Structural analysis of the evolution of steroid specificity in the mineralocorticoid and glucocorticoid receptors

    Directory of Open Access Journals (Sweden)

    Ollikainen Noah


    Full Text Available Abstract Background The glucocorticoid receptor (GR and mineralocorticoid receptor (MR evolved from a common ancestor. Still not completely understood is how specificity for glucocorticoids (e.g. cortisol and mineralocorticoids (e.g. aldosterone evolved in these receptors. Results Our analysis of several vertebrate GRs and MRs in the context of 3D structures of human GR and MR indicates that with the exception of skate GR, a cartilaginous fish, there is a deletion in all GRs, at the position corresponding to Ser-949 in human MR. This deletion occurs in a loop before helix 12, which contains the activation function 2 (AF2 domain, which binds coactivator proteins and influences transcriptional activity of steroids. Unexpectedly, we find that His-950 in human MR, which is conserved in the MR in chimpanzee, orangutan and macaque, is glutamine in all teleost and land vertebrate MRs, including New World monkeys and prosimians. Conclusion Evolution of differences in the responses of the GR and MR to corticosteroids involved deletion in the GR of a residue corresponding to Ser-949 in human MR. A mutation corresponding to His-950 in human MR may have been important in physiological changes associated with emergence of Old World monkeys from prosimians.

  6. Steroid hormone receptors and prostate cancer: role of structural dynamics in therapeutic targeting (United States)

    Kumar, Raj


    Steroid hormone receptors (SHRs) act in cell type- and gene-specific manner through interactions with coregulatory proteins to regulate numerous physiological and pathological processes at the level of gene regulation. Binding of steroid receptor modulator (SRM) ligand leads to allosteric changes in SHR to exert positive or negative effects on the expression of target genes. Due, in part, to the fact that current SRMs generally target ligand binding domain (LBD)/AF2 and neglect intrinsically disordered (ID) N-terminal domain (NTD)/AF1, clinically relevant SRMs lack selectivity and are also prone to the development of resistance over time. Therefore, to maximize the efficacy of SHR-based therapeutics, the possibility of developing unique modulators that act to control AF1 activity must be considered. Recent studies targeting androgen receptor's (AR's) ID AF1 domain for the castration-resistant prostate cancer has provided the possibility of therapeutically targeting ID NTD/AF1 surfaces by allosteric modulations to achieve desired effects. In this review article, we discuss how inter- and intra- molecular allosteric regulations controlled by AR's structural flexibility and dynamics particularly the ID NTD/AF1 is an emerging area of investigation, which could be exploited for drug development and therapeutic targeting of prostate cancer. PMID:27364545

  7. The Structure of a High-Affinity Kainate Receptor: GluK4 Ligand-Binding Domain Crystallized with Kainate. (United States)

    Kristensen, Ole; Kristensen, Lise Baadsgaard; Møllerud, Stine; Frydenvang, Karla; Pickering, Darryl S; Kastrup, Jette Sandholm


    Ionotropic glutamate receptors play a key role in fast neurotransmission in the CNS and have been linked to several neurological diseases and disorders. One subfamily is the kainate receptors, which are grouped into low-affinity (GluK1-3) and high-affinity (GluK4-5) receptors based on their affinity for kainate. Although structures of the ligand-binding domain (LBD) of all low-affinity kainate receptors have been reported, no structures of the high-affinity receptor subunits are available. Here, we present the X-ray structure of GluK4-LBD with kainate at 2.05 Å resolution, together with thermofluor and radiolabel binding affinity data. Whereas binding-site residues in GluK4 are most similar to the AMPA receptor subfamily, the domain closure and D1-D2 interlobe contacts induced by kainate are similar to the low-affinity kainate receptor GluK1. These observations provide a likely explanation for the high binding affinity of kainate at GluK4-LBD.

  8. Structure of unliganded HSV gD reveals a mechanism for receptor-mediated activation of virus entry

    Energy Technology Data Exchange (ETDEWEB)

    Krummenacher, Claude; Supekar, Vinit M.; Whitbeck, J. Charles; Lazear, Eric; Connolly, Sarah A.; Eisenberg, Roselyn J.; Cohen, Gary H.; Wiley, Don C.; Carfi, Andrea (UPENN); (IRBM); (CHLMM)


    Herpes simplex virus (HSV) entry into cells requires binding of the envelope glycoprotein D (gD) to one of several cell surface receptors. The 50 C-terminal residues of the gD ectodomain are essential for virus entry, but not for receptor binding. We have determined the structure of an unliganded gD molecule that includes these C-terminal residues. The structure reveals that the C-terminus is anchored near the N-terminal region and masks receptor-binding sites. Locking the C-terminus in the position observed in the crystals by an intramolecular disulfide bond abolished receptor binding and virus entry, demonstrating that this region of gD moves upon receptor binding. Similarly, a point mutant that would destabilize the C-terminus structure was nonfunctional for entry, despite increased affinity for receptors. We propose that a controlled displacement of the gD C-terminus upon receptor binding is an essential feature of HSV entry, ensuring the timely activation of membrane fusion.

  9. Receptor Polymorphism and Genomic Structure Interact to Shape Bitter Taste Perception.

    Directory of Open Access Journals (Sweden)

    Natacha Roudnitzky

    Full Text Available The ability to taste bitterness evolved to safeguard most animals, including humans, against potentially toxic substances, thereby leading to food rejection. Nonetheless, bitter perception is subject to individual variations due to the presence of genetic functional polymorphisms in bitter taste receptor (TAS2R genes, such as the long-known association between genetic polymorphisms in TAS2R38 and bitter taste perception of phenylthiocarbamide. Yet, due to overlaps in specificities across receptors, such associations with a single TAS2R locus are uncommon. Therefore, to investigate more complex associations, we examined taste responses to six structurally diverse compounds (absinthin, amarogentin, cascarillin, grosheimin, quassin, and quinine in a sample of the Caucasian population. By sequencing all bitter receptor loci, inferring long-range haplotypes, mapping their effects on phenotype variation, and characterizing functionally causal allelic variants, we deciphered at the molecular level how a subjects' genotype for the whole-family of TAS2R genes shapes variation in bitter taste perception. Within each haplotype block implicated in phenotypic variation, we provided evidence for at least one locus harboring functional polymorphic alleles, e.g. one locus for sensitivity to amarogentin, one of the most bitter natural compounds known, and two loci for sensitivity to grosheimin, one of the bitter compounds of artichoke. Our analyses revealed also, besides simple associations, complex associations of bitterness sensitivity across TAS2R loci. Indeed, even if several putative loci harbored both high- and low-sensitivity alleles, phenotypic variation depended on linkage between these alleles. When sensitive alleles for bitter compounds were maintained in the same linkage phase, genetically driven perceptual differences were obvious, e.g. for grosheimin. On the contrary, when sensitive alleles were in opposite phase, only weak genotype

  10. Design of an insulin analog with enhanced receptor binding selectivity: rationale, structure, and therapeutic implications. (United States)

    Zhao, Ming; Wan, Zhu-li; Whittaker, Linda; Xu, Bin; Phillips, Nelson B; Katsoyannis, Panayotis G; Ismail-Beigi, Faramarz; Whittaker, Jonathan; Weiss, Michael A


    Insulin binds with high affinity to the insulin receptor (IR) and with low affinity to the type 1 insulin-like growth factor (IGF) receptor (IGFR). Such cross-binding, which reflects homologies within the insulin-IGF signaling system, is of clinical interest in relation to the association between hyperinsulinemia and colorectal cancer. Here, we employ nonstandard mutagenesis to design an insulin analog with enhanced affinity for the IR but reduced affinity for the IGFR. Unnatural amino acids were introduced by chemical synthesis at the N- and C-capping positions of a recognition alpha-helix (residues A1 and A8). These sites adjoin the hormone-receptor interface as indicated by photocross-linking studies. Specificity is enhanced more than 3-fold on the following: (i) substitution of Gly(A1) by D-Ala or D-Leu, and (ii) substitution of Thr(A8) by diaminobutyric acid (Dab). The crystal structure of [D-Ala(A1),Dab(A8)]insulin, as determined within a T(6) zinc hexamer to a resolution of 1.35 A, is essentially identical to that of human insulin. The nonstandard side chains project into solvent at the edge of a conserved receptor-binding surface shared by insulin and IGF-I. Our results demonstrate that modifications at this edge discriminate between IR and IGFR. Because hyperinsulinemia is typically characterized by a 3-fold increase in integrated postprandial insulin concentrations, we envisage that such insulin analogs may facilitate studies of the initiation and progression of cancer in animal models. Future development of clinical analogs lacking significant IGFR cross-binding may enhance the safety of insulin replacement therapy in patients with type 2 diabetes mellitus at increased risk of colorectal cancer.

  11. New G-protein-coupled receptor structures provide insights into the recognition of CXCL12 and HIV-1 gp120 by CXCR4

    Institute of Scientific and Technical Information of China (English)

    Chen Zhong; Jianping Ding


    The G protein-coupled receptor (GPCR) superfamily consists of thousands of integral membrane proteins that exert a wide variety of physiological functions and account for a large portion of the drag targets identified so far.However,structural knowledge of GPCRs is scarce, with crystal structures determined for only a few members including β1and β2 adrenergic receptors, adenosine receptor, rhodopsin,and dopamine D3 receptor [1].

  12. Structural determinants of agonist-specific kinetics at the ionotropic glutamate receptor 2. (United States)

    Holm, Mai Marie; Lunn, Marie-Louise; Traynelis, Stephen F; Kastrup, Jette S; Egebjerg, Jan


    Glutamate receptors (GluRs) are the most abundant mediators of the fast excitatory neurotransmission in the human brain. Agonists will, after activation of the receptors, induce different degrees of desensitization. The efficacy of agonists strongly correlates with the agonist-induced closure of the ligand-binding domain. However, the differences in desensitization properties are less well understood. By using high-resolution x-ray structure of the GluR2 flop (GluR2o) ligand-binding core protein in complex with the partial glutamate receptor agonist (S)-2-amino-3-(3-hydroxy-5-tert-butyl-4-isothiazolyl)propionic acid [(S)-thio-ATPA], we show that (S)-thio-ATPA induces an 18 degrees closure of the binding core similar to another partial agonist, (S)-2-amino-3-(4-bromo-3-hydroxy-5-isoxazolyl)propionic acid [(S)-Br-HIBO]. Despite the similar closure of the ligand-binding domain, we find in electrophysiological studies that (S)-thio-ATPA induced a 6.4-fold larger steady-state current than (RS)-Br-HIBO, and rapid agonist applications show that (S)-thio-ATPA induces a 3.6-fold higher steady-state/peak ratio and a 2.2-fold slower desensitization time constant than (RS)-Br-HIBO. Structural comparisons reveal that (S)-Br-HIBO, but not (S)-thio-ATPA, induces a twist of the ligand-binding core compared with the apostructure, and the agonist-specific conformation of Leu-650 correlates with the different kinetic profiles pointing at a key role in defining the desensitization kinetics. We conclude that, especially for intermediate efficacious agonists, the desensitization properties are influenced by additional ligand-induced factors beyond domain closure.

  13. Disulfide Trapping for Modeling and Structure Determination of Receptor:Chemokine Complexes (United States)

    Kufareva, Irina; Gustavsson, Martin; Holden, Lauren G.; Qin, Ling; Zheng, Yi; Handel, Tracy M.


    Despite the recent breakthrough advances in GPCR crystallography, structure determination of protein-protein complexes involving chemokine receptors and their endogenous chemokine ligands remains challenging. Here we describe disulfide trapping, a methodology for generating irreversible covalent binary protein complexes from unbound protein partners by introducing two cysteine residues, one per interaction partner, at selected positions within their interaction interface. Disulfide trapping can serve at least two distinct purposes: (i) stabilization of the complex to assist structural studies, and/or (ii) determination of pairwise residue proximities to guide molecular modeling. Methods for characterization of disulfide-trapped complexes are described and evaluated in terms of throughput, sensitivity, and specificity towards the most energetically favorable cross-links. Due to abundance of native disulfide bonds at receptor:chemokine interfaces, disulfide trapping of their complexes can be associated with intramolecular disulfide shuffling and result in misfolding of the component proteins; because of this, evidence from several experiments is typically needed to firmly establish a positive disulfide crosslink. An optimal pipeline that maximizes throughput and minimizes time and costs by early triage of unsuccessful candidate constructs is proposed. PMID:26921956

  14. Structural basis for integration of GluD receptors within synaptic organizer complexes. (United States)

    Elegheert, Jonathan; Kakegawa, Wataru; Clay, Jordan E; Shanks, Natalie F; Behiels, Ester; Matsuda, Keiko; Kohda, Kazuhisa; Miura, Eriko; Rossmann, Maxim; Mitakidis, Nikolaos; Motohashi, Junko; Chang, Veronica T; Siebold, Christian; Greger, Ingo H; Nakagawa, Terunaga; Yuzaki, Michisuke; Aricescu, A Radu


    Ionotropic glutamate receptor (iGluR) family members are integrated into supramolecular complexes that modulate their location and function at excitatory synapses. However, a lack of structural information beyond isolated receptors or fragments thereof currently limits the mechanistic understanding of physiological iGluR signaling. Here, we report structural and functional analyses of the prototypical molecular bridge linking postsynaptic iGluR δ2 (GluD2) and presynaptic β-neurexin 1 (β-NRX1) via Cbln1, a C1q-like synaptic organizer. We show how Cbln1 hexamers "anchor" GluD2 amino-terminal domain dimers to monomeric β-NRX1. This arrangement promotes synaptogenesis and is essential for D: -serine-dependent GluD2 signaling in vivo, which underlies long-term depression of cerebellar parallel fiber-Purkinje cell (PF-PC) synapses and motor coordination in developing mice. These results lead to a model where protein and small-molecule ligands synergistically control synaptic iGluR function.

  15. Structural basis for subtype-specific inhibition of the P2X7 receptor

    Energy Technology Data Exchange (ETDEWEB)

    Karasawa, Akira; Kawate, Toshimitsu


    The P2X7 receptor is a non-selective cation channel activated by extracellular adenosine triphosphate (ATP). Chronic activation of P2X7 underlies many health problems such as pathologic pain, yet we lack effective antagonists due to poorly understood mechanisms of inhibition. Here we present crystal structures of a mammalian P2X7 receptor complexed with five structurally-unrelated antagonists. Unexpectedly, these drugs all bind to an allosteric site distinct from the ATP-binding pocket in a groove formed between two neighboring subunits. This novel drug-binding pocket accommodates a diversity of small molecules mainly through hydrophobic interactions. Functional assays propose that these compounds allosterically prevent narrowing of the drug-binding pocket and the turret-like architecture during channel opening, which is consistent with a site of action distal to the ATP-binding pocket. These novel mechanistic insights will facilitate the development of P2X7-specific drugs for treating human diseases.

  16. Evaluation of hypothalamic murine and human melanocortin 3 receptor transcript structure. (United States)

    Taylor-Douglas, Dezmond C; Basu, Arunabha; Gardner, Ryan M; Aspelund, Sender; Wen, Xin; Yanovski, Jack A


    The melanocortin 3 receptor (MC3R) is involved in regulation of energy homeostasis. However, its transcript structure is not well understood. We therefore studied initiation and termination sites for hypothalamic murine Mc3r and human MC3R transcripts. Rapid Amplification of cDNA Ends (RACE) was performed for the 5' and 3' ends of murine and human hypothalamic RNA. 5' RACE experiments using hypothalamic murine RNA indicated mouse hypothalamus expresses two major Mc3r transcription start sites: one with a 5' UTR approximately 368 bases in length and another previously unknown transcript with a 5' UTR approximately 440 bases in length. 5' RACE experiments using human hypothalamic RNA identified a 5' UTR beginning 533 bases upstream of the start codon with a 248 base splice. 3' RACE experiments using hypothalamic murine RNA indicated the 3' UTR terminates approximately 1286 bases after the translational stop codon, with a previously unknown 787 base splice between consensus splice donor and acceptor sites. 3' RACE experiments using human MC3R transcript indicated the 3' UTR terminates approximately 115-160 bases after the translational stop codon. These data provide insight into melanocortin 3 receptor transcript structure.

  17. Toward a structure-based model of salvinorin A recognition of the kappa-opioid receptor. (United States)

    Kane, Brian E; McCurdy, Christopher R; Ferguson, David M


    The structural basis to salvinorin A recognition of the kappa-opioid receptor is evaluated using a combination of site-directed mutagenesis and molecular-modeling techniques. The results show that salvinorin A recognizes a collection of residues in transmembrane II and VII, including Q115, Y119, Y313, I316, and Y320. The mutation of one hydrophobic residue in particular, I316, was found to completely abolish salvinorin A binding. As expected, none of the residues in transmembrane III or VI commonly associated with opiate recognition (such as D138 or E297) appear to be required for ligand binding. On the basis of the results presented here and elsewhere, a binding site model is proposed that aligns salvinorin A vertically within a pocket spanning transmembrane II and VII, with the 2' substituent directed toward the extracellular domains. The model explains the role that hydrophobic contacts play in binding this lipophilic ligand and gives insight into the structural basis to the mu-opioid receptor selectivity of 2'-benzoyl salvinorin (herkinorin).

  18. Loss of Insulin Receptor in Osteoprogenitor Cells Impairs Structural Strength of Bone

    Directory of Open Access Journals (Sweden)

    Kathryn Thrailkill


    Full Text Available Type 1 diabetes mellitus (T1D is associated with decreased bone mineral density, a deficit in bone structure, and subsequently an increased risk of fragility fracture. These clinical observations, paralleled by animal models of T1D, suggest that the insulinopenia of T1D has a deleterious effect on bone. To further examine the action of insulin signaling on bone development, we generated mice with an osteoprogenitor-selective (osterix-Cre ablation of the insulin receptor (IR, designated OIRKO. OIRKO mice exhibited an 80% decrease in IR in osteoblasts. Prenatal elimination of IR did not affect fetal survival or gross morphology. However, loss of IR in mouse osteoblasts resulted in a postnatal growth-constricted phenotype. By 10–12 weeks of age, femurs of OIRKO mice were more slender, with a thinner diaphyseal cortex and, consequently, a decrease in whole bone strength when subjected to bending. In male mice alone, decreased metaphyseal trabecular bone, with thinner and more rodlike trabeculae, was also observed. OIRKO mice did not, however, exhibit abnormal glucose tolerance. The skeletal phenotype of the OIRKO mouse appeared more severe than that of previously reported bone-specific IR knockdown models, and confirms that insulin receptor expression in osteoblasts is critically important for proper bone development and maintenance of structural integrity.

  19. Structural basis for receptor recognition by New World hemorrhagic fever arenaviruses

    Energy Technology Data Exchange (ETDEWEB)

    Abraham, Jonathan; Corbett, Kevin D.; Farzan, Michael; Choe, Hyeryun; Harrison, Stephen C. (Harvard-Med)


    New World hemorrhagic fever arenaviruses are rodent-borne agents that cause severe human disease. The GP1 subunit of the surface glycoprotein mediates cell attachment through transferrin receptor 1 (TfR1). We report the structure of Machupo virus (MACV) GP1 bound with human TfR1. Atomic details of the GP1-TfR1 interface clarify the importance of TfR1 residues implicated in New World arenavirus host specificity. Analysis of sequence variation among New World arenavirus GP1s and their host-species receptors, in light of the molecular structure, indicates determinants of viral zoonotic transmission. Infectivities of pseudoviruses in cells expressing mutated TfR1 confirm that contacts at the tip of the TfR1 apical domain determine the capacity of human TfR1 to mediate infection by particular New World arenaviruses. We propose that New World arenaviruses that are pathogenic to humans fortuitously acquired affinity for human TfR1 during adaptation to TfR1 of their natural hosts.

  20. 8-epi-Salvinorin B: crystal structure and affinity at the κ opioid receptor

    Directory of Open Access Journals (Sweden)

    Béguin Cécile


    Full Text Available Abstract There have been many reports of epimerization of salvinorins at C-8 under basic conditions, but little evidence has been presented to establish the structure of these compounds. We report here the first crystal structure of an 8-epi-salvinorin or derivative: the title compound, 2b. The lactone adopts a boat conformation with the furan equatorial. Several lines of evidence suggest that epimerization proceeds via enolization of the lactone rather than a previously proposed indirect mechanism. Consistent with the general trend in related compounds, the title compound showed lower affinity at the kappa opioid receptor than the natural epimer salvinorin B (2a. The related 8-epi-acid 4b showed no affinity.

  1. Structure-kinetics relationships of Capadenoson derivatives as adenosine A1 receptor agonists. (United States)

    Louvel, Julien; Guo, Dong; Soethoudt, Marjolein; Mocking, Tamara A M; Lenselink, Eelke B; Mulder-Krieger, Thea; Heitman, Laura H; IJzerman, Adriaan P


    We report the synthesis and biological evaluation of new derivatives of Capadenoson, a former drug candidate that was previously advanced to phase IIa clinical trials. 19 of the 20 ligands show an affinity below 100 nM at the human adenosine A1 receptor (hA1AR) and display a wide range of residence times at this target (from approx. 5 min (compound 10) up to 132 min (compound 5)). Structure-affinity and structure-kinetics relationships were established, and computational studies of a homology model of the hA1AR revealed crucial interactions for both the affinity and dissociation kinetics of this family of ligands. These results were also combined with global metrics (Ligand Efficiency, cLogP), showing the importance of binding kinetics as an additional way to better select a drug candidate amongst seemingly similar leads.

  2. Structure and interactions of the human programmed cell death 1 receptor. (United States)

    Cheng, Xiaoxiao; Veverka, Vaclav; Radhakrishnan, Anand; Waters, Lorna C; Muskett, Frederick W; Morgan, Sara H; Huo, Jiandong; Yu, Chao; Evans, Edward J; Leslie, Alasdair J; Griffiths, Meryn; Stubberfield, Colin; Griffin, Robert; Henry, Alistair J; Jansson, Andreas; Ladbury, John E; Ikemizu, Shinji; Carr, Mark D; Davis, Simon J


    PD-1, a receptor expressed by T cells, B cells, and monocytes, is a potent regulator of immune responses and a promising therapeutic target. The structure and interactions of human PD-1 are, however, incompletely characterized. We present the solution nuclear magnetic resonance (NMR)-based structure of the human PD-1 extracellular region and detailed analyses of its interactions with its ligands, PD-L1 and PD-L2. PD-1 has typical immunoglobulin superfamily topology but differs at the edge of the GFCC' sheet, which is flexible and completely lacks a C" strand. Changes in PD-1 backbone NMR signals induced by ligand binding suggest that, whereas binding is centered on the GFCC' sheet, PD-1 is engaged by its two ligands differently and in ways incompletely explained by crystal structures of mouse PD-1 · ligand complexes. The affinities of these interactions and that of PD-L1 with the costimulatory protein B7-1, measured using surface plasmon resonance, are significantly weaker than expected. The 3-4-fold greater affinity of PD-L2 versus PD-L1 for human PD-1 is principally due to the 3-fold smaller dissociation rate for PD-L2 binding. Isothermal titration calorimetry revealed that the PD-1/PD-L1 interaction is entropically driven, whereas PD-1/PD-L2 binding has a large enthalpic component. Mathematical simulations based on the biophysical data and quantitative expression data suggest an unexpectedly limited contribution of PD-L2 to PD-1 ligation during interactions of activated T cells with antigen-presenting cells. These findings provide a rigorous structural and biophysical framework for interpreting the important functions of PD-1 and reveal that potent inhibitory signaling can be initiated by weakly interacting receptors.

  3. Role of Structural Dynamics at the Receptor G Protein Interface for Signal Transduction.

    Directory of Open Access Journals (Sweden)

    Alexander S Rose

    Full Text Available GPCRs catalyze GDP/GTP exchange in the α-subunit of heterotrimeric G proteins (Gαßγ through displacement of the Gα C-terminal α5 helix, which directly connects the interface of the active receptor (R* to the nucleotide binding pocket of G. Hydrogen-deuterium exchange mass spectrometry and kinetic analysis of R* catalysed G protein activation have suggested that displacement of α5 starts from an intermediate GDP bound complex (R*•GGDP. To elucidate the structural basis of receptor-catalysed displacement of α5, we modelled the structure of R*•GGDP. A flexible docking protocol yielded an intermediate R*•GGDP complex, with a similar overall arrangement as in the X-ray structure of the nucleotide free complex (R*•Gempty, however with the α5 C-terminus (GαCT forming different polar contacts with R*. Starting molecular dynamics simulations of GαCT bound to R* in the intermediate position, we observe a screw-like motion, which restores the specific interactions of α5 with R* in R*•Gempty. The observed rotation of α5 by 60° is in line with experimental data. Reformation of hydrogen bonds, water expulsion and formation of hydrophobic interactions are driving forces of the α5 displacement. We conclude that the identified interactions between R* and G protein define a structural framework in which the α5 displacement promotes direct transmission of the signal from R* to the GDP binding pocket.

  4. Role of Structural Dynamics at the Receptor G Protein Interface for Signal Transduction. (United States)

    Rose, Alexander S; Zachariae, Ulrich; Grubmüller, Helmut; Hofmann, Klaus Peter; Scheerer, Patrick; Hildebrand, Peter W


    GPCRs catalyze GDP/GTP exchange in the α-subunit of heterotrimeric G proteins (Gαßγ) through displacement of the Gα C-terminal α5 helix, which directly connects the interface of the active receptor (R*) to the nucleotide binding pocket of G. Hydrogen-deuterium exchange mass spectrometry and kinetic analysis of R* catalysed G protein activation have suggested that displacement of α5 starts from an intermediate GDP bound complex (R*•GGDP). To elucidate the structural basis of receptor-catalysed displacement of α5, we modelled the structure of R*•GGDP. A flexible docking protocol yielded an intermediate R*•GGDP complex, with a similar overall arrangement as in the X-ray structure of the nucleotide free complex (R*•Gempty), however with the α5 C-terminus (GαCT) forming different polar contacts with R*. Starting molecular dynamics simulations of GαCT bound to R* in the intermediate position, we observe a screw-like motion, which restores the specific interactions of α5 with R* in R*•Gempty. The observed rotation of α5 by 60° is in line with experimental data. Reformation of hydrogen bonds, water expulsion and formation of hydrophobic interactions are driving forces of the α5 displacement. We conclude that the identified interactions between R* and G protein define a structural framework in which the α5 displacement promotes direct transmission of the signal from R* to the GDP binding pocket.

  5. Structure and Function of the Intracellular Region of the Plexin-B1 Transmembrane Receptor

    Energy Technology Data Exchange (ETDEWEB)

    Tong, Yufeng; Hota, Prasanta K.; Penachioni, Junia Y.; Hamaneh, Mehdi B.; Kim, SoonJeung; Alviani, Rebecca S.; Shen, Limin; He, Hao; Tempel, Wolfram; Tamagnone, Luca; Park, Hee-Won; Buck, Matthias; (Torino); (Toronto); (Case Western U.-Med)


    Members of the plexin family are unique transmembrane receptors in that they interact directly with Rho family small GTPases; moreover, they contain a GTPase-activating protein (GAP) domain for R-Ras, which is crucial for plexin-mediated regulation of cell motility. However, the functional role and structural basis of the interactions between the different intracellular domains of plexins remained unclear. Here we present the 2.4 {angstrom} crystal structure of the complete intracellular region of human plexin-B1. The structure is monomeric and reveals that the GAP domain is folded into one structure from two segments, separated by the Rho GTPase binding domain (RBD). The RBD is not dimerized, as observed previously. Instead, binding of a conserved loop region appears to compete with dimerization and anchors the RBD to the GAP domain. Cell-based assays on mutant proteins confirm the functional importance of this coupling loop. Molecular modeling based on structural homology to p120{sup GAP} {center_dot}H-Ras suggests that Ras GTPases can bind to the plexin GAP region. Experimentally, we show that the monomeric intracellular plexin-B1 binds R-Ras but not H-Ras. These findings suggest that the monomeric form of the intracellular region is primed for GAP activity and extend a model for plexin activation.

  6. Insulin/receptor binding: the last piece of the puzzle? What recent progress on the structure of the insulin/receptor complex tells us (or not) about negative cooperativity and activation. (United States)

    De Meyts, Pierre


    Progress in solving the structure of insulin bound to its receptor has been slow and stepwise, but a milestone has now been reached with a refined structure of a complex of insulin with a "microreceptor" that contains the primary binding site. The insulin receptor is a dimeric allosteric enzyme that belongs to the family of receptor tyrosine kinases. The insulin binding process is complex and exhibits negative cooperativity. Biochemical evidence suggested that insulin, through two distinct binding sites, crosslinks two receptor sites located on each α subunit. The structure of the unliganded receptor ectodomain showed a symmetrical folded-over conformation with an antiparallel disposition. Further work resolved the detailed structure of receptor site 1, both without and with insulin. Recently, a missing piece in the puzzle was added: the C-terminal portion of insulin's B-chain known to be critical for binding and negative cooperativity. Here I discuss these findings and their implications.

  7. Concomitant action of structural elements and receptor phosphorylation determines arrestin-3 interaction with the free fatty acid receptor FFA4

    DEFF Research Database (Denmark)

    Butcher, Adrian J; Hudson, Brian D; Shimpukade, Bharat;


    In addition to being nutrients, free fatty acids act as signaling molecules by activating a family of G protein-coupled receptors. Among these is FFA4, previously called GPR120, which responds to medium and long chain fatty acids, including health-promoting ω-3 fatty acids, which have been implic...

  8. Structure and evolution of the sea star egg receptor for sperm bindin. (United States)

    Hart, Michael W


    Selection on coevolving sperm- and egg-recognition molecules is a potent engine of population divergence leading to reproductive isolation and speciation. The study of receptor-ligand pairs can reveal co-evolution of male- and female-expressed genes or differences between their evolution in response to selective factors such as sperm competition and sexual conflict. Phylogeographical studies of these patterns have been limited by targeted gene methods that favour short protein-coding sequences amplifiable by PCR. Here, I use high-throughput transcriptomic methods to characterize the structure and divergence of full-length coding sequences for the gene encoding the protein component of a large complex egg surface glycopeptide receptor for the sperm acrosomal protein bindin from the sea star Patiria miniata. I used a simple but effective method for resolving nucleotide polymorphisms into haplotypes for phylogeny-based analyses of selection. The protein domain organization of sea star egg bindin receptor (EBR1) was similar to sea urchins and included a pair of protein-recognition domains plus a series of tandem repeat domains of two types. Two populations separated by a well-characterized phylogeographical break included lineages of EBR1 alleles under positive selection at several codons (similar to selection on sperm bindin in the same populations). However, these populations shared the same alleles that were under selection for amino acid differences at multiple codons (unlike the pattern of selection for population divergence in sperm bindin). The significance of positively selected EBR1 domains and alleles could be tested in functional analyses of fertilization rates associated with EBR1 (and bindin) polymorphisms.

  9. Loss of progesterone receptor-mediated actions induce preterm cellular and structural remodeling of the cervix and premature birth.

    Directory of Open Access Journals (Sweden)

    Steven M Yellon

    Full Text Available A decline in serum progesterone or antagonism of progesterone receptor function results in preterm labor and birth. Whether characteristics of premature remodeling of the cervix after antiprogestins or ovariectomy are similar to that at term was the focus of the present study. Groups of pregnant rats were treated with vehicle, a progesterone receptor antagonist (onapristone or mifepristone, or ovariectomized on day 17 postbreeding. As expected, controls given vehicle delivered at term while rats delivered preterm after progesterone receptor antagonist treatment or ovariectomy. Similar to the cervix before term, the preterm cervix of progesterone receptor antagonist-treated rats was characterized by reduced cell nuclei density, decreased collagen content and structure, as well as a greater presence of macrophages per unit area. Thus, loss of nuclear progesterone receptor-mediated actions promoted structural remodeling of the cervix, increased census of resident macrophages, and preterm birth much like that found in the cervix at term. In contrast to the progesterone receptor antagonist-induced advance in characteristics associated with remodeling, ovariectomy-induced loss of systemic progesterone did not affect hypertrophy, extracellular collagen, or macrophage numbers in the cervix. Thus, the structure and macrophage census in the cervix appear sufficient for premature ripening and birth to occur well before term. With progesterone receptors predominantly localized on cells other than macrophages, the findings suggest that interactions between cells may facilitate the loss of progesterone receptor-mediated actions as part of a final common mechanism that remodels the cervix in certain etiologies of preterm and with parturition at term.

  10. NRLiSt BDB, the manually curated nuclear receptors ligands and structures benchmarking database. (United States)

    Lagarde, Nathalie; Ben Nasr, Nesrine; Jérémie, Aurore; Guillemain, Hélène; Laville, Vincent; Labib, Taoufik; Zagury, Jean-François; Montes, Matthieu


    Nuclear receptors (NRs) constitute an important class of drug targets. We created the most exhaustive NR-focused benchmarking database to date, the NRLiSt BDB (NRs ligands and structures benchmarking database). The 9905 compounds and 339 structures of the NRLiSt BDB are ready for structure-based and ligand-based virtual screening. In the present study, we detail the protocol used to generate the NRLiSt BDB and its features. We also give some examples of the errors that we found in ChEMBL that convinced us to manually review all original papers. Since extensive and manually curated experimental data about NR ligands and structures are provided in the NRLiSt BDB, it should become a powerful tool to assess the performance of virtual screening methods on NRs, to assist the understanding of NR's function and modulation, and to support the discovery of new drugs targeting NRs. NRLiSt BDB is freely available online at .

  11. Accessing Structurally Diverse Near-Infrared Cyanine Dyes for Folate Receptor-Targeted Cancer Cell Staining. (United States)

    König, Sandra G; Krämer, Roland


    Folate receptor (FR) targeting is one of the most promising strategies for the development of small-molecule based cancer imaging agents since the FR is highly overexpressed on the surface of many cancer cell types. FR-targeted conjugates of NIR emissive cyanine dyes are in advanced clinical trials for fluorescence-guided surgery and are valuable research tools for optical molecular imaging in animal models. Only a small number of promising conjugates has been evaluated so far. Analysis of structure-performance relations to identify critical factors modulating the performance of targeted conjugates is essential for successful further optimization. This contribution addresses the need for convenient synthetic access to structurally diverse NIR-emissive cyanine dyes for conjugation with folic acid. Structural variations were introduced to readily available cyanine precursors in particular via C-C-coupling reactions including Suzuki- and (for the first time with these types of dyes) Sonogashira cross couplings. Photophysical properties such as absorbance maxima, brightness, and photostability are highly dependent on the molecular structure. Selected modified cyanines were conjugated to folic acid for cancer cell targeting. Several conjugates display a favorable combination of high fluorescence brightness and photostability with high affinity to FR positive cancer cells, and enable the selective imaging of these cells with low background.

  12. Structural insights into human peroxisome proliferator activated receptor delta (PPAR-delta selective ligand binding.

    Directory of Open Access Journals (Sweden)

    Fernanda A H Batista

    Full Text Available Peroxisome proliferator activated receptors (PPARs δ, α and γ are closely related transcription factors that exert distinct effects on fatty acid and glucose metabolism, cardiac disease, inflammatory response and other processes. Several groups developed PPAR subtype specific modulators to trigger desirable effects of particular PPARs without harmful side effects associated with activation of other subtypes. Presently, however, many compounds that bind to one of the PPARs cross-react with others and rational strategies to obtain highly selective PPAR modulators are far from clear. GW0742 is a synthetic ligand that binds PPARδ more than 300-fold more tightly than PPARα or PPARγ but the structural basis of PPARδ:GW0742 interactions and reasons for strong selectivity are not clear. Here we report the crystal structure of the PPARδ:GW0742 complex. Comparisons of the PPARδ:GW0742 complex with published structures of PPARs in complex with α and γ selective agonists and pan agonists suggests that two residues (Val312 and Ile328 in the buried hormone binding pocket play special roles in PPARδ selective binding and experimental and computational analysis of effects of mutations in these residues confirms this and suggests that bulky substituents that line the PPARα and γ ligand binding pockets as structural barriers for GW0742 binding. This analysis suggests general strategies for selective PPARδ ligand design.

  13. Novel computational methodologies for structural modeling of spacious ligand binding sites of G-protein-coupled receptors: development and application to human leukotriene B4 receptor. (United States)

    Ishino, Yoko; Harada, Takanori


    This paper describes a novel method to predict the activated structures of G-protein-coupled receptors (GPCRs) with high accuracy, while aiming for the use of the predicted 3D structures in in silico virtual screening in the future. We propose a new method for modeling GPCR thermal fluctuations, where conformation changes of the proteins are modeled by combining fluctuations on multiple time scales. The core idea of the method is that a molecular dynamics simulation is used to calculate average 3D coordinates of all atoms of a GPCR protein against heat fluctuation on the picosecond or nanosecond time scale, and then evolutionary computation including receptor-ligand docking simulations functions to determine the rotation angle of each helix of a GPCR protein as a movement on a longer time scale. The method was validated using human leukotriene B4 receptor BLT1 as a sample GPCR. Our study demonstrated that the proposed method was able to derive the appropriate 3D structure of the active-state GPCR which docks with its agonists.

  14. Efficacité du traitement par calcimimétiques dans l'hyperparathyroïdie néonatale sévère, associée à des mutations du calcium sensing receptor (CaSR)


    Wilhelm-Bals, Alexandra Marina


    L'Hyperparathyroïdie néonatale sévère (NPHT) est associée à des mutations homozygotes inactivatrices du récepteur du calcium (CaSR). Le CaSR est exprimé majoritairement dans les glandes parathyroïdes et les reins où il régule l'homéostasie du calcium en modulant la sécrétion de PTH et la réabsorption rénale du calcium. L'NPHT se présente avec une hypercalcémie sévère mettant en jeu le pronostic vital. La chirurgie est le traitement de choix. Les bisphosphonates offrent une bonne alternative d...

  15. Multiplex ligation-dependent probe amplification (MLPA) screening for exon copy number variation in the calcium sensing receptor gene: no large rearrangements identified in patients with calcium metabolic disorders

    DEFF Research Database (Denmark)

    Nissen, Peter H; Christensen, Signe E; Wallace, Andrew;


    samples were previously found negative for CASR mutations. Multiplex ligation-dependent probe amplification was used to screen the patients for exon copy number variations. Results. All exons were amplified with mean normalised ratios between 0.98 and 1.06. We did not identify any exon copy number......Summary Background. Mutation screening of the CASR by DNA sequencing is commonly used in the diagnosis of disorders of calcium metabolism, such as familial hypocalciuric hypercalcaemia (FHH). Exon copy number variation is not detected by currently used molecular genetic screening methods, and might...... be a genetic cause of inherited forms of hyper- or hypocalcaemia caused by the CASR. Objective. We wanted to further evaluate possible genetic causes for disorders of calcium metabolism, by investigating the prevalence of exon copy number variations, such as large deletions or duplications of the CASR...

  16. Structure and dynamics of IgE-receptor interactions: FcεRI and CD23/FcεRII. (United States)

    Sutton, Brian J; Davies, Anna M


    Immunoglobulin E (IgE) is well known for its role in allergic disease, the manifestations of which are mediated through its two Fc receptors, FcεRI and CD23 (FcεRII). IgE and its interactions with these receptors are therefore potential targets for therapeutic intervention, and exciting progress has been made in this direction. Furthermore, recent structural studies of IgE-Fc, the two receptors, and of their complexes, have revealed a remarkable degree of plasticity at the IgE-CD23 interface and an even more remarkable degree of dynamic flexibility within the IgE molecule. Indeed, there is allosteric communication between the two receptor-binding sites, which we now know are located at some distance from each other in IgE-Fc (at opposite ends of the Cε3 domain). The conformational changes associated with FcεRI and CD23 binding to IgE-Fc ensure that their interactions are mutually incompatible, and it may be that this functional imperative has driven IgE to evolve such a dynamic structure. Appreciation of these new structural data has revised our view of IgE structure, shed light on the co-evolution of antibodies and their receptors, and may open up new therapeutic opportunities.

  17. [The high-affinity IgE receptor: lessons from structural analysis]. (United States)

    Blank, Ulrich; Jouvin, Marie-Hélène; Guérin-Marchand, Claudine; Kinet, Jean-Pierre


    The high affinity receptor for IgE, FcERI, is at the core of the allergic reaction. This receptor is expressed mainly on mast cells and basophils. Interaction of an allergen with its specific IgE bound to FcERI triggers cell activation, which induces the release of numerous mediators that are responsible for allergic manifestations. The recent increase in the prevalence of allergic diseases in developed countries has resulted in renewed efforts towards the development of new drugs. One of these is a humanised antibody directed against the IgE ligand. This antibody recognises specifically free but not FcERI-bound IgE thus preventing ligand binding and subsequent cell activation. This antibody has shown some efficacy in clinical trials involving patients with asthma and allergic rhinitis. The recent elucidation of the tridimensional structure of the complex between IgE and FcERI provides unexpected information regarding the mechanism of assembly of the complex, which now can be used to design small chemical compounds capable of specifically inhibiting this interaction.

  18. Steroid hormone receptors and prostate cancer: role of structural dynamics in therapeutic targeting

    Directory of Open Access Journals (Sweden)

    Raj Kumar


    Full Text Available Steroid hormone receptors (SHRs act in cell type- and gene-specific manner through interactions with coregulatory proteins to regulate numerous physiological and pathological processes at the level of gene regulation. Binding of steroid receptor modulator (SRM ligand leads to allosteric changes in SHR to exert positive or negative effects on the expression of target genes. Due, in part, to the fact that current SRMs generally target ligand binding domain (LBD/AF2 and neglect intrinsically disordered (ID N-terminal domain (NTD/AF1, clinically relevant SRMs lack selectivity and are also prone to the development of resistance over time. Therefore, to maximize the efficacy of SHR-based therapeutics, the possibility of developing unique modulators that act to control AF1 activity must be considered. Recent studies targeting androgen receptor′s (AR′s ID AF1 domain for the castration-resistant prostate cancer has provided the possibility of therapeutically targeting ID NTD/AF1 surfaces by allosteric modulations to achieve desired effects. In this review article, we discuss how inter- and intra- molecular allosteric regulations controlled by AR′s structural flexibility and dynamics particularly the ID NTD/AF1 is an emerging area of investigation, which could be exploited for drug development and therapeutic targeting of prostate cancer.

  19. Imidazo-thiazine, -diazinone and -diazepinone derivatives. Synthesis, structure and benzodiazepine receptor binding. (United States)

    Kieć-Kononowicz, K; Karolak-Wojciechowska, J; Müller, C E; Schumacher, B; Pekala, E; Szymańska, E


    In our search for new compounds acting on benzodiazepine receptors among the fused 2-thiohydantoin derivatives, a series of arylidene imidazo[2,1-b]thiazines was synthesized. The 1,2- and 2,3- cyclized derivatives of mono- and di-substituted Z-5-arylidene-2-thiohydantoins were examined (the X-ray crystal structure of Z-2-cinnamylidene-6,7-dihydro-5H-imidazo[2,1-b][1,3]thiazin-3(2H)-one was determined) and compared with the diphenyl derivatives. To investigate the influence of the type of annelated ring on the biological activity, imidazo[2,1-b]pyrimidinone and imidazo[2,1-b]diazepinone derivatives were obtained. The method used in annelation (1,2- and 2,3-cyclized isomers with the exception of fused arylidene imidazothiazines), the substitution pattern (arylidene towards diphenyl) as well as the character of the annelated ring had minor influence on the benzodiazepine receptor affinity of the investigated compounds. It appears that the greatest influence on the biological activity has the character and position of the substituents on the arylidene ring.

  20. Correlation between TCDD acute toxicity and aryl hydrocarbon receptor structure for different mammals. (United States)

    Wang, Yonghua; Wang, Qiuying; Wu, Bing; Li, Yi; Lu, Guanghua


    The 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxicity has large species differences, and TCDD exerts its toxicity by binding into aryl hydrocarbon receptor (AHR). In this study, we applied bioinformatics approaches to quantitatively analyze the correlation between TCDD acute toxicity and AHRs. Seven mammalian AHRs were chosen as target receptors. Low conserved functional domains of AHRs were identified and quantitatively characterized. Linear regression was applied to determine the relationships of different mammalian AHRs and TCDD LD(50) values. The results indicated that ligand binding domain and glutamine-rich domain of mammalian AHRs showed a low degree of conservation. Based on previous literatures, the number of glutamine residues (NOQ) and binding free energy with TCDD were applied to quantitatively represent the differences of glutamine-rich domain and ligand binding domain, respectively. Then, regression equations between studied mammalian AHR structures and TCDD LD(50) were constructed, and high linear correlation was found (R(2)=0.986). This study indicated that mammalian differences of TCDD acute toxicity might be partly determined by the differences of glutamine-rich domain and ligand binding domain of AHR, which provides a potential insight to analyze the species differences of TCDD toxicity.

  1. Structure-activity relationships for the irreversible blockade of nicotinic receptor agonist sites by lophotoxin and congeneric diterpene lactones

    Energy Technology Data Exchange (ETDEWEB)

    Culver, P.; Burch, M.; Potenza, C.; Wasserman, L.; Fenical, W.; Taylor, P.


    Lophotoxin, a diterpene lactone paralytic toxin from gorgonian corals of the genus Lophogorgia, inhibits ( SVI)-alpha-toxin binding to surface nicotinic receptors of BC3H-1 cells by irreversible occupation of the primary agonist sites. In contrast, receptor-bearing membrane fragments or detergent-solubilized receptors prepared from BC3H-1 cells are not susceptible to lophotoxin block. Thus, lophotoxin inhibition requires intact cells. However, when intact cells were incubated with lophotoxin, subsequent membrane-fragment preparation or detergent solubilization of the receptors did not diminish lophotoxin occupation of ( SVI)-alpha-toxin-binding sites, indicating that lophotoxin binds very tightly to nicotinic receptors. These studies further demonstrate that both surface and nonsurface nicotinic receptors of BC3H-1 cells are susceptible to irreversible occupation by lophotoxin, indicating that the lipophilic toxin freely permeates intact cells. The authors also examined several structural analogs of lophotoxin, one of which was equipotent with lophotoxin for inhibition of ( SVI)-alpha-toxin binding to intact cells and, notably, also blocked alpha-toxin binding to detergent-extracted receptor.

  2. Grid inhomogeneous solvation theory: hydration structure and thermodynamics of the miniature receptor cucurbit[7]uril. (United States)

    Nguyen, Crystal N; Young, Tom Kurtzman; Gilson, Michael K


    The displacement of perturbed water upon binding is believed to play a critical role in the thermodynamics of biomolecular recognition, but it is nontrivial to unambiguously define and answer questions about this process. We address this issue by introducing grid inhomogeneous solvation theory (GIST), which discretizes the equations of inhomogeneous solvation theory (IST) onto a three-dimensional grid situated in the region of interest around a solute molecule or complex. Snapshots from explicit solvent simulations are used to estimate localized solvation entropies, energies, and free energies associated with the grid boxes, or voxels, and properly summing these thermodynamic quantities over voxels yields information about hydration thermodynamics. GIST thus provides a smoothly varying representation of water properties as a function of position, rather than focusing on hydration sites where solvent is present at high density. It therefore accounts for full or partial displacement of water from sites that are highly occupied by water, as well as for partly occupied and water-depleted regions around the solute. GIST can also provide a well-defined estimate of the solvation free energy and therefore enables a rigorous end-states analysis of binding. For example, one may not only use a first GIST calculation to project the thermodynamic consequences of displacing water from the surface of a receptor by a ligand, but also account, in a second GIST calculation, for the thermodynamics of subsequent solvent reorganization around the bound complex. In the present study, a first GIST analysis of the molecular host cucurbit[7]uril is found to yield a rich picture of hydration structure and thermodynamics in and around this miniature receptor. One of the most striking results is the observation of a toroidal region of high water density at the center of the host's nonpolar cavity. Despite its high density, the water in this toroidal region is disfavored energetically and

  3. Structure-activity relationships of fatty acid amide ligands in activating and desensitizing G protein-coupled receptor 119. (United States)

    Kumar, Pritesh; Kumar, Akhilesh; Song, Zhao-Hui


    The purpose of the current study was to apply a high throughput assay to investigate the structure-activity relationships of fatty acid amides for activating and desensitizing G protein-coupled receptor 119, a promising therapeutic target for both type 2 diabetes and obesity. A cell-based, homogenous time resolved fluorescence (HTRF) method for measuring G protein-coupled receptor 119-mediated increase of cyclic adenosine monophosphate (cAMP) levels was validated and applied in this study. Using novel fatty acid amides and detailed potency and efficacy analyses, we have demonstrated that degree of saturation in acyl chain and charged head groups of fatty acid amides have profound effects on the ability of these compounds to activate G protein-coupled receptor 119. In addition, we have demonstrated for the first time that pretreatments with G protein-coupled receptor 119 agonists desensitize the receptor and the degrees of desensitization caused by fatty acid amides correlate well with their structure-activity relationships in activating the receptor.

  4. Synthesis and evaluation of structurally constrained quinazolinone derivatives as potent and selective histamine H3 receptor inverse agonists. (United States)

    Nagase, Tsuyoshi; Mizutani, Takashi; Sekino, Etsuko; Ishikawa, Shiho; Ito, Sayaka; Mitobe, Yuko; Miyamoto, Yasuhisa; Yoshimoto, Ryo; Tanaka, Takeshi; Ishihara, Akane; Takenaga, Norihiro; Tokita, Shigeru; Sato, Nagaaki


    A series of structurally constrained derivatives of the potent H 3 inverse agonist 1 was designed, synthesized, and evaluated as histamine H 3 receptor inverse agonists. As a result, the N-cyclobutylpiperidin-4-yloxy group as in 2f was identified as an optimal surrogate structure for the flexible 1-pyrrolidinopropoxy group of 1. Subsequent optimization of the quinazolinone core of 2f revealed that substitution at the 5-position of the quinazolinone ring influences potency. Representative derivatives 5a and 5s showed improved potency in a histamine release assay in rats and a receptor occupancy assay in mice.

  5. cDNA heterogeneity suggests structural variants related to the high-affinity IgE receptor.


    Liu, F T; Albrandt, K; Robertson, M W


    The high-affinity IgE receptor present on mast cells and basophils is responsible for the IgE-mediated activation of these cells. The current model for this receptor depicts a four-subunit structure, alpha beta gamma 2. A cDNA for the alpha subunit was recently cloned and predicts a structure consisting of two homologous extracellular domains, a transmembrane segment, and a cytoplasmic tail. Using a synthetic oligonucleotide corresponding to the amino-terminal sequence of the alpha subunit, w...

  6. Two distinct conformations of helix 6 observed in antagonist-bound structures of a β1-adrenergic receptor



    The β1-adrenergic receptor (β1AR) is a G-protein-coupled receptor whose inactive state structure was determined using a thermostabilized mutant (β1AR–M23). However, it was not thought to be in a fully inactivated state because there was no salt bridge between Arg139 and Glu285 linking the cytoplasmic ends of transmembrane helices 3 and 6 (the R3.50 - D/E6.30 “ionic lock”). Here we compare eight new structures of β1AR–M23, determined from crystallographically independent molecules in four diff...

  7. Structural basis for EGF receptor inhibition by the therapeutic antibody IMC-11F8. (United States)

    Li, Shiqing; Kussie, Paul; Ferguson, Kathryn M


    Therapeutic anticancer strategies that target and inactivate the epidermal growth factor receptor (EGFR) are under intense study in the clinic. Here we describe the mechanism of EGFR inhibition by an antibody drug IMC-11F8. IMC-11F8 is a fully human antibody that has similar antitumor potency as the chimeric cetuximab/Erbitux and might represent a safer therapeutic alternative. We report the X-ray crystal structure of the Fab fragment of IMC-11F8 (Fab11F8) in complex with the entire extracellular region and with isolated domain III of EGFR. We compare this to our previous study of the cetuximab/EGFR interaction. Fab11F8 interacts with a remarkably similar epitope, but through a completely different set of interactions. Both the similarities and differences in binding of these two antibodies have important implications for the development of inhibitors that could exploit this same mechanism of EGFR inhibition.

  8. [Beta-3 adrenergic receptor--structure and role in obesity and metabolic disorders]. (United States)

    Wiejak, J; Wyroba, E


    Structure and essential motifs of beta 3-adrenergic receptor (known previously as atypical beta-AR), which plays a central role in regulation of lipid metabolism have been described. Obesity results from an imbalance between caloric intake and energy expenditure. The consequence of catecholamine activation of beta 3-AR is increased mobilization of fatty acids from triglyceride stores (lipolysis) in brown and white adipose tissue as well as increased fatty acid beta-oxidation and heat-production via UCP-1 (thermogenesis) in brown adipose tissue. A pharmacokinetic effects of beta 3-agonists and putative involvement of Trp/Arg mutation in beta 3-AR gene in obesity and another metabolic disorders have been discussed.

  9. Relationship of Structure and Function of DNA-Binding Domain in Vitamin D Receptor

    Directory of Open Access Journals (Sweden)

    Lin-Yan Wan


    Full Text Available While the structure of the DNA-binding domain (DBD of the vitamin D receptor (VDR has been determined in great detail, the roles of its domains and how to bind the motif of its target genes are still under debate. The VDR DBD consists of two zinc finger modules and a C-terminal extension (CTE, at the end of the C-terminal of each structure presenting α-helix. For the first zinc finger structure, N37 and S-box take part in forming a dimer with 9-cis retinoid X receptor (RXR, while V26, R50, P-box and S-box participate in binding with VDR response elements (VDRE. For the second zinc finger structure, P61, F62 and H75 are essential in the structure of the VDR homodimer with the residues N37, E92 and F93 of the downstream of partner VDR, which form the inter-DBD interface. T-box of the CTE, especially the F93 and I94, plays a critical role in heterodimerization and heterodimers–VDRE binding. Six essential residues (R102, K103, M106, I107, K109, and R110 of the CTE α-helix of VDR construct one interaction face, which packs against the DBD core of the adjacent symmetry mate. In 1,25(OH2D3-activated signaling, the VDR-RXR heterodimer may bind to DR3-type VDRE and ER9-type VDREs of its target gene directly resulting in transactivation and also bind to DR3-liked nVDRE of its target gene directly resulting in transrepression. Except for this, 1α,25(OH2D3 ligand VDR-RXR may bind to 1αnVDRE indirectly through VDIR, resulting in transrepression of the target gene. Upon binding of 1α,25(OH2D3, VDR can transactivate and transrepress its target genes depending on the DNA motif that DBD binds.

  10. Pathogenic Cysteine Removal Mutations in FGFR Extracellular Domains Stabilize Receptor Dimers and Perturb the TM Dimer Structure. (United States)

    Sarabipour, Sarvenaz; Hristova, Kalina


    Missense mutations that introduce or remove cysteine residues in receptor tyrosine kinases are believed to cause pathologies by stabilizing the active receptor tyrosine kinase dimers. However, the magnitude of this stabilizing effect has not been measured for full-length receptors. Here, we characterize the dimer stabilities of three full-length fibroblast growth factor receptor (FGFR) mutants harboring pathogenic cysteine substitutions: the C178S FGFR1 mutant, the C342R FGFR2 mutant, and the C228R FGFR3 mutant. We find that the three mutations stabilize the FGFR dimers. We further see that the mutations alter the configuration of the FGFR transmembrane dimers. Thus, both aberrant dimerization and perturbed dimer structure likely contribute to the pathological phenotypes arising due to these mutations.

  11. Structure-activity relationships of constrained phenylethylamine ligands for the serotonin 5-HT2 receptors.

    Directory of Open Access Journals (Sweden)

    Vignir Isberg

    Full Text Available Serotonergic ligands have proven effective drugs in the treatment of migraine, pain, obesity, and a wide range of psychiatric and neurological disorders. There is a clinical need for more highly 5-HT2 receptor subtype-selective ligands and the most attention has been given to the phenethylamine class. Conformationally constrained phenethylamine analogs have demonstrated that for optimal activity the free lone pair electrons of the 2-oxygen must be oriented syn and the 5-oxygen lone pairs anti relative to the ethylamine moiety. Also the ethyl linker has been constrained providing information about the bioactive conformation of the amine functionality. However, combined 1,2-constriction by cyclization has only been tested with one compound. Here, we present three new 1,2-cyclized phenylethylamines, 9-11, and describe their synthetic routes. Ligand docking in the 5-HT2B crystal structure showed that the 1,2-heterocyclized compounds can be accommodated in the binding site. Conformational analysis showed that 11 can only bind in a higher-energy conformation, which would explain its absent or low affinity. The amine and 2-oxygen interactions with D3.32 and S3.36, respectively, can form but shift the placement of the core scaffold. The constraints in 9-11 resulted in docking poses with the 4-bromine in closer vicinity to 5.46, which is polar only in the human 5-HT2A subtype, for which 9-11 have the lowest affinity. The new ligands, conformational analysis and docking expand the structure-activity relationships of constrained phenethylamines and contributes towards the development of 5-HT2 receptor subtype-selective ligands.

  12. The natural product magnolol as a lead structure for the development of potent cannabinoid receptor agonists.

    Directory of Open Access Journals (Sweden)

    Alexander Fuchs

    Full Text Available Magnolol (4-allyl-2-(5-allyl-2-hydroxyphenylphenol, the main bioactive constituent of the medicinal plant Magnolia officinalis, and its main metabolite tetrahydromagnolol were recently found to activate cannabinoid (CB receptors. We now investigated the structure-activity relationships of (tetrahydromagnolol analogs with variations of the alkyl chains and the phenolic groups and could considerably improve potency. Among the most potent compounds were the dual CB1/CB2 full agonist 2-(2-methoxy-5-propyl-phenyl-4-hexylphenol (61a, K(i CB1:0.00957 µM; K(i CB2:0.0238 µM, and the CB2-selective partial agonist 2-(2-hydroxy-5-propylphenyl-4-pentylphenol (60, K(i CB1:0.362 µM; K(i CB2:0.0371 µM, which showed high selectivity versus GPR18 and GPR55. Compound 61b, an isomer of 61a, was the most potent GPR55 antagonist with an IC50 value of 3.25 µM but was non-selective. The relatively simple structures, which possess no stereocenters, are easily accessible in a four- to five-step synthetic procedure from common starting materials. The central reaction step is the well-elaborated Suzuki-Miyaura cross-coupling reaction, which is suitable for a combinatorial chemistry approach. The scaffold is versatile and may be fine-tuned to obtain a broad range of receptor affinities, selectivities and efficacies.

  13. Distinct Structural Pathways Coordinate the Activation of AMPA Receptor-Auxiliary Subunit Complexes. (United States)

    Dawe, G Brent; Musgaard, Maria; Aurousseau, Mark R P; Nayeem, Naushaba; Green, Tim; Biggin, Philip C; Bowie, Derek


    Neurotransmitter-gated ion channels adopt different gating modes to fine-tune signaling at central synapses. At glutamatergic synapses, high and low activity of AMPA receptors (AMPARs) is observed when pore-forming subunits coassemble with or without auxiliary subunits, respectively. Whether a common structural pathway accounts for these different gating modes is unclear. Here, we identify two structural motifs that determine the time course of AMPAR channel activation. A network of electrostatic interactions at the apex of the AMPAR ligand-binding domain (LBD) is essential for gating by pore-forming subunits, whereas a conserved motif on the lower, D2 lobe of the LBD prolongs channel activity when auxiliary subunits are present. Accordingly, channel activity is almost entirely abolished by elimination of the electrostatic network but restored via auxiliary protein interactions at the D2 lobe. In summary, we propose that activation of native AMPAR complexes is coordinated by distinct structural pathways, favored by the association/dissociation of auxiliary subunits.

  14. Kinked structures of isolated nicotinic receptor M2 helices: a molecular dynamics study. (United States)

    Sankararamakrishnan, R; Samsom, M S


    The pore-lining M2 helix of the nicotinic acetylcholine receptor exhibits a pronounced kink when the corresponding ion channel is in a closed conformation [N. Unwin (1993) Journal of Molecular Biology, Vol. 229, pp. 1101-1124]. We have performed molecular dynamics simulations of isolated 22-residue M2 helices in order to identify a possible molecular origin of this kink. In order to sample a wide range of conformational space, a simulated annealing protocol was used to generate five initial M2 helix structures, each of which was subsequently used as the basis of 300 ps MD simulations. Two helix sequences (M2 alpha and M2 delta) were studied in this manner, resulting in a total of ten 300 ps trajectories. Kinked helices present in the trajectories were identified and energy minimized to yield a total of five different stable kinked structures. For comparison, a similar molecular dynamics simulation of a Leu23 helix yielded no stable kinked structures. In four of the five kinked helices, the kink was stabilized by H bonds between the helix backbone and polar side-chain atoms. Comparison with data from the literature on site-directed mutagenesis of M2 residues suggests that such polar side-chain to main-chain H bonds may also contribute to kinking of M2 helices in the intact channel protein.

  15. Structure of a pheromone receptor-associated MHC molecule with an open and empty groove.

    Directory of Open Access Journals (Sweden)


    Full Text Available Neurons in the murine vomeronasal organ (VNO express a family of class Ib major histocompatibility complex (MHC proteins (M10s that interact with the V2R class of VNO receptors. This interaction may play a direct role in the detection of pheromonal cues that initiate reproductive and territorial behaviors. The crystal structure of M10.5, an M10 family member, is similar to that of classical MHC molecules. However, the M10.5 counterpart of the MHC peptide-binding groove is open and unoccupied, revealing the first structure of an empty class I MHC molecule. Similar to empty MHC molecules, but unlike peptide-filled MHC proteins and non-peptide-binding MHC homologs, M10.5 is thermally unstable, suggesting that its groove is normally occupied. However, M10.5 does not bind endogenous peptides when expressed in mammalian cells or when offered a mixture of class I-binding peptides. The F pocket side of the M10.5 groove is open, suggesting that ligands larger than 8-10-mer class I-binding peptides could fit by extending out of the groove. Moreover, variable residues point up from the groove helices, rather than toward the groove as in classical MHC structures. These data suggest that M10s are unlikely to provide specific recognition of class I MHC-binding peptides, but are consistent with binding to other ligands, including proteins such as the V2Rs.

  16. Structure of a Pheromone Receptor-Associated Mhc Molecule With An Open And Empty Groove

    Energy Technology Data Exchange (ETDEWEB)

    Olson, R.; Huey-Tubman, K.E.; Dulac, C.; Bjorkman, P.J.; /Caltech /Harvard U.


    Neurons in the murine vomeronasal organ (VNO) express a family of class Ib major histocompatibility complex (MHC) proteins (M10s) that interact with the V2R class of VNO receptors. This interaction may play a direct role in the detection of pheromonal cues that initiate reproductive and territorial behaviors. The crystal structure of M10.5, an M10 family member, is similar to that of classical MHC molecules. However, the M10.5 counterpart of the MHC peptide-binding groove is open and unoccupied, revealing the first structure of an empty class I MHC molecule. Similar to empty MHC molecules, but unlike peptide-filled MHC proteins and non-peptide-binding MHC homologs, M10.5 is thermally unstable, suggesting that its groove is normally occupied. However, M10.5 does not bind endogenous peptides when expressed in mammalian cells or when offered a mixture of class I-binding peptides. The F pocket side of the M10.5 groove is open, suggesting that ligands larger than 8-10-mer class I-binding peptides could fit by extending out of the groove. Moreover, variable residues point up from the groove helices, rather than toward the groove as in classical MHC structures. These data suggest that M10s are unlikely to provide specific recognition of class I MHC-binding peptides, but are consistent with binding to other ligands, including proteins such as the V2Rs.

  17. A Combined Quantitative Structure-Activity Relationship Research of Quinolinone Derivatives as Androgen Receptor Antagonists. (United States)

    Wang, Yuwei; Bai, Fang; Cao, Hong; Li, Jiazhong; Liu, Huanxiang; Gramatica, Paola


    Antiandrogens bicalutamide, flutamide and enzalutamide etc. have been used in clinical trials to treat prostate cancer by binding to and antagonizing androgen receptor (AR). Although initially effective, the drug resistance problem will emerge eventually, which results in a high medical need for novel AR antagonist exploitation. Here in this work, to facilitate the rational design of novel AR antagonists, we studied the structure-activity relationships of a series of 2-quinolinone derivatives and investigated the structural requirements for their antiandrogenic activities. Different modeling methods, including 2D MLR, 3D CoMFA and CoMSIA, were implemented to evolve QSAR models. All these models, thoroughly validated, demonstrated satisfactory results especially for the good predictive abilities. The contour maps from 3D CoMFA and CoMSIA models provide visualized explanation of key structural characteristics relevant to the antiandrogenic activities, which is summarized to a position-specific conclusion at the end. The obtained results from this research are practically useful for rational design and screening of promising chemicals with high antiandrogenic activities.

  18. Solution structure of the transmembrane domain of the insulin receptor in detergent micelles. (United States)

    Li, Qingxin; Wong, Ying Lei; Kang, CongBao


    The insulin receptor (IR) binds insulin and plays important roles in glucose homeostasis by regulating the tyrosine kinase activity at its C-terminus. Its transmembrane domain (TMD) is shown to be important for transferring conformational changes induced by insulin across the cell membrane to regulate kinase activity. In this study, a construct IR(940-988) containing the TMD was expressed and purified for structural studies. Its solution structure in dodecylphosphocholine (DPC) micelles was determined. The sequence containing residues L962 to Y976 of the TMD of the IR in micelles adopts a well-defined helical structure with a kink formed by glycine and proline residues present at its N-terminus, which might be important for its function. Paramagnetic relaxation enhancement (PRE) and relaxation experimental results suggest that residues following the TMD are flexible and expose to aqueous solution. Although purified IR(940-988) in micelles existed mainly as a monomeric form verified by gel filtration and relaxation analysis, cross-linking study suggests that it may form a dimer or oligomers under micelle conditions.

  19. Structural Studies of the Parainfluenza Virus 5 Hemagglutinin-Neuraminidase Tetramer in Complex with Its Receptor, Sialyllactose

    Energy Technology Data Exchange (ETDEWEB)

    Yuan, Ping; Thompson, Thomas B.; Wurzburg, Beth A.; Paterson, Reay G.; Lamb, Robert A.; Jardetzky, Theodore S. (NWU)


    The paramyxovirus hemagglutinin-neuraminidase (HN) functions in virus attachment to cells, cleavage of sialic acid from oligosaccharides, and stimulating membrane fusion during virus entry into cells. The structural basis for these diverse functions remains to be fully understood. We report the crystal structures of the parainfluenza virus 5 (SV5) HN and its complexes with sialic acid, the inhibitor DANA, and the receptor sialyllactose. SV5 HN shares common structural features with HN of Newcastle disease virus (NDV) and human parainfluenza 3 (HPIV3), but unlike the previously determined HN structures, the SV5 HN forms a tetramer in solution, which is thought to be the physiological oligomer. The sialyllactose complex reveals intact receptor within the active site, but no major conformational changes in the protein. The SV5 HN structures do not support previously proposed models for HN action in membrane fusion and suggest alternative mechanisms by which HN may promote virus entry into cells.

  20. Structure and functional interaction of the extracellular domain of human GABA[subscript B] receptor GBR2

    Energy Technology Data Exchange (ETDEWEB)

    Geng, Yong; Xiong, Dazhi; Mosyak, Lidia; Malito, David L.; Kniazeff, Julie; Chen, Yan; Burmakina, Svetlana; Quick, Matthias; Bush, Martin; Javitch, Jonathan A.; Pin, Jean-Philippe; Fan, Qing R. (CNRS-UMR); (Columbia)


    Inhibitory neurotransmission is mediated primarily by GABA. The metabotropic GABA{sub B} receptor is a G protein-coupled receptor central to mammalian brain function. Malfunction of GABA{sub B} receptor has been implicated in several neurological disorders. GABA{sub B} receptor functions as a heterodimeric assembly of GBR1 and GBR2 subunits, where GBR1 is responsible for ligand-binding and GBR2 is responsible for G protein coupling. Here we demonstrate that the GBR2 ectodomain directly interacts with the GBR1 ectodomain to increase agonist affinity by selectively stabilizing the agonist-bound conformation of GBR1. We present the crystal structure of the GBR2 ectodomain, which reveals a polar heterodimeric interface. We also identify specific heterodimer contacts from both subunits, and GBR1 residues involved in ligand recognition. Lastly, our structural and functional data indicate that the GBR2 ectodomain adopts a constitutively open conformation, suggesting a structural asymmetry in the active state of GABA{sub B} receptor that is unique to the GABAergic system.

  1. Dopamine receptor ligands. Part 18: (1) modification of the structural skeleton of indolobenzazecine-type dopamine receptor antagonists. (United States)

    Robaa, Dina; Enzensperger, Christoph; Abul Azm, Shams El Din; El Khawass, El Sayeda; El Sayed, Ola; Lehmann, Jochen


    On the basis of the D(1/5)-selective dopamine antagonist LE 300 (1), an indolo[3,2-f]benzazecine derivative, we changed the annulation pattern of the heterocycles. The target compounds represent novel heterocyclic ring systems. The most constrained indolo[4,3a,3-ef]benzazecine 2 was inactive, but the indolo[4,3a,3-fg]benzazacycloundecene 3 showed antagonistic properties (functional Ca(2+) assay) with nanomolar affinities (radioligand binding) for all dopamine receptor subtypes, whereas the indolo[2,3-f]benzazecine 4 displayed a selectivity profile similar to 3 but with decreased affinities.

  2. Chalcones as positive allosteric modulators of α7 nicotinic acetylcholine receptors: a new target for a privileged structure. (United States)

    Balsera, Beatriz; Mulet, José; Fernández-Carvajal, Asia; de la Torre-Martínez, Roberto; Ferrer-Montiel, Antonio; Hernández-Jiménez, José G; Estévez-Herrera, Judith; Borges, Ricardo; Freitas, Andiara E; López, Manuela G; García-López, M Teresa; González-Muñiz, Rosario; Pérez de Vega, María Jesús; Valor, Luis M; Svobodová, Lucie; Sala, Salvador; Sala, Francisco; Criado, Manuel


    The α7 acetylcholine nicotine receptor is a ligand-gated ion channel that is involved in cognition disorders, schizophrenia, pain and inflammation among other diseases. Therefore, the development of new agents that target this receptor has great significance. Positive allosteric modulators might be advantageous, since they facilitate receptor responses without directly interacting with the agonist binding site. Here we report the search for and further design of new positive allosteric modulators having the relatively simple chalcone structure. From the natural product isoliquiritigenin as starting point, chalcones substituted with hydroxyl groups at defined locations were identified as optimal and specific promoters of α7 nicotinic function. The most potent compound (2,4,2',5'-tetrahydroxychalcone, 111) was further characterized showing its potential as neuroprotective, analgesic and cognitive enhancer, opening the way for future developments around the chalcone structure.

  3. Structural/functional relationships between internal and external MSH receptors: modulation of expression in Cloudman melanoma cells by UVB radiation

    Energy Technology Data Exchange (ETDEWEB)

    Chakraborty, A.K.; Orlow, S.J.; Bolognia, J.L.; Pawelek, J.M. (Department of Dermatology, Yale University School of Medicine, New Haven, CT (USA))


    Expression of internal receptors for MSH is an important criterion for responsiveness to MSH by Cloudman melanoma cells. Here, we show that internal and external receptors for MSH are of identical molecular weights (50-53 kDa) and share common antigenic determinants, indicating a structural relationship between the 2 populations of molecules. The internal receptors co-purified with a sub-cellular fraction highly enriched for small vesicles, many of which were coated. Ultraviolet B light (UVB) acted synergistically with MSH to increase tyrosinase activity and melanin content of cultured Cloudman melanoma cells, consistent with previous findings in the skin of mice and guinea pigs. Preceding the rise in tyrosinase activity in cultured cells, UVB elicited a decrease in internal MSH binding sites and a concomitant increase in external sites. The time frame for the UVB effects on MSH receptors and melanogenesis, 48 hours, was similar to that for a response to solar radiation in humans. Together, the results indicate a key role for MSH receptors in the induction of melanogenesis by UVB and suggest a potential mechanism of action for UVB: redistribution of MSH receptors with a resultant increase in cellular responsiveness to MSH.

  4. Genetic Variation in TLR10, an Inhibitory Toll-Like Receptor, Influences Susceptibility to Complicated Skin and Skin Structure Infections.

    NARCIS (Netherlands)

    Stappers, M.H.T.; Oosting, M.; Ioana, M.; Reimnitz, P.; Mouton, J.W.; Netea, M.G.; Gyssens, I.C.J.; Joosten, L.A.B.


    BACKGROUND: Toll-like receptors (TLRs) play a central role in the innate immune response to complicated skin and skin structure infections (cSSSIs), with TLR10 being the first family member known to have an inhibitory function. This study assessed the role of TLR10 in recognition of cSSSI-related pa

  5. Disease Mutations in the Ryanodine Receptor Central Region: Crystal Structures of a Phosphorylation Hot Spot Domain

    Energy Technology Data Exchange (ETDEWEB)

    Yuchi, Zhiguang; Lau, Kelvin; Van Petegem, Filip (UBC)


    Ryanodine Receptors (RyRs) are huge Ca{sup 2+} release channels in the endoplasmic reticulum membrane and form targets for phosphorylation and disease mutations. We present crystal structures of a domain in three RyR isoforms, containing the Ser2843 (RyR1) and Ser2808/Ser2814 (RyR2) phosphorylation sites. The RyR1 domain is the target for 11 disease mutations. Several of these are clustered near the phosphorylation sites, suggesting that phosphorylation and disease mutations may affect the same interface. The L2867G mutation causes a drastic thermal destabilization and aggregation at room temperature. Crystal structures for other disease mutants show that they affect surface properties and intradomain salt bridges. In vitro phosphorylation experiments show that up to five residues in one long loop of RyR2 can be phosphorylated by PKA or CaMKII. Docking into cryo-electron microscopy maps suggests a putative location in the clamp region, implying that mutations and phosphorylation may affect the allosteric motions within this area.

  6. Bioinformatics analysis of the structural and evolutionary characteristics for toll-like receptor 15

    Directory of Open Access Journals (Sweden)

    Jinlan Wang


    Full Text Available Toll-like receptors (TLRs play important role in the innate immune system. TLR15 is reported to have a unique role in defense against pathogens, but its structural and evolution characterizations are still poorly understood. In this study, we identified 57 completed TLR15 genes from avian and reptilian genomes. TLR15 clustered into an individual clade and was closely related to family 1 on the phylogenetic tree. Unlike the TLRs in family 1 with the broken asparagine ladders in the middle, TLR15 ectodomain had an intact asparagine ladder that is critical to maintain the overall shape of ectodomain. The conservation analysis found that TLR15 ectodomain had a highly evolutionarily conserved region on the convex surface of LRR11 module, which is probably involved in TLR15 activation process. Furthermore, the protein–protein docking analysis indicated that TLR15 TIR domains have the potential to form homodimers, the predicted interaction interface of TIR dimer was formed mainly by residues from the BB-loops and αC-helixes. Although TLR15 mainly underwent purifying selection, we detected 27 sites under positive selection for TLR15, 24 of which are located on its ectodomain. Our observations suggest the structural features of TLR15 which may be relevant to its function, but which requires further experimental validation.

  7. Low Resolution Structure and Dynamics of a Colicin-Receptor Complex Determined by Neutron Scattering

    Energy Technology Data Exchange (ETDEWEB)

    Clifton, Luke A [ORNL; Johnson, Christopher L [ORNL; Solovyova, Alexandra [University of Newcastle upon Tyne; Callow, Phil [Institut Laue-Langevin (ILL); Weiss, Kevin L [ORNL; Ridley, Helen [University of Newcastle upon Tyne; Le Brun, Anton P [ORNL; Kinane, Christian [ISIS Facility, Rutherford Appleton Laboratory; Webster, John [ISIS Facility, Rutherford Appleton Laboratory; Holt, Stephen A [ORNL; Lakey, Jeremy H [ORNL


    Proteins that translocate across cell membranes need to overcome a significant hydrophobic barrier. This is usually accomplished via specialized protein complexes, which provide a polar transmembrane pore. Exceptions to this include bacterial toxins, which insert into and cross the lipid bilayer itself. We are studying the mechanism by which large antibacterial proteins enter Escherichia coli via specific outer membrane proteins. Here we describe the use of neutron scattering to investigate the interaction of colicin N with its outer membrane receptor protein OmpF. The positions of lipids, colicin N, and OmpF were separately resolved within complex structures by the use of selective deuteration. Neutron reflectivity showed, in real time, that OmpF mediates the insertion of colicin N into lipid monolayers. This data were complemented by Brewster Angle Microscopy images, which showed a lateral association of OmpF in the presence of colicin N. Small angle neutron scattering experiments then defined the three-dimensional structure of the colicin N-OmpF complex. This revealed that colicin N unfolds and binds to the OmpF-lipid interface. The implications of this unfolding step for colicin translocation across membranes are discussed.

  8. The Structure-Function Relationships of Complement Receptor Type 2 (CR2; CD21). (United States)

    Hannan, Jonathan Paul


    Human complement receptor type 2 (CR2; CD21) is a surface-associated glycoprotein which binds to a variety of endogenous ligands, including the complement component C3 fragments iC3b, C3dg and C3d, the low-affinity IgE receptor CD23, and the type I cytokine, interferon-alpha. CR2 links the innate complement-mediated immune response to pathogens and foreign antigens with the adaptive immune response by binding to C3d that is covalently attached to targets, and which results in a cell signalling phenomenon that lowers the threshold for B cell activation. Variations or deletions of the CR2 gene in humans, or the Cr2 gene in mice associate with a variety of autoimmune and inflammatory conditions. A number of infectious agents including Epstein-Barr virus (EBV), Human Immunodeficiency Virus (HIV) and prions also bind to CR2 either directly or indirectly by means of C3d-targeted immune complexes. In this review we discuss the interactions that CR2 undertakes with its best characterized ligands C3d, CD23 and the EBV gp350/220 envelope protein. To date only a single physiologically relevant complex of CR2 with one of its ligands, C3d, has been elucidated. By contrast, the interactions with CD23 and EBV gp350/220, while being important from physiologic and disease-associated standpoints, respectively, are only incompletely understood. A detailed knowledge of the structure-function relationships that CR2 undergoes with its ligands is necessary to understand the implications of using recombinant CR2 in therapeutic or imaging agents, or alternatively targeting CR2 to down-regulate the antibody mediated immune response in cases of autoimmunity.

  9. Identification and Structure-Function Analysis of Subfamily Selective G Protein-Coupled Receptor Kinase Inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Homan, Kristoff T.; Larimore, Kelly M.; Elkins, Jonathan M.; Szklarz, Marta; Knapp, Stefan; Tesmer, John J.G. [Michigan; (Oxford)


    Selective inhibitors of individual subfamilies of G protein-coupled receptor kinases (GRKs) would serve as useful chemical probes as well as leads for therapeutic applications ranging from heart failure to Parkinson’s disease. To identify such inhibitors, differential scanning fluorimetry was used to screen a collection of known protein kinase inhibitors that could increase the melting points of the two most ubiquitously expressed GRKs: GRK2 and GRK5. Enzymatic assays on 14 of the most stabilizing hits revealed that three exhibit nanomolar potency of inhibition for individual GRKs, some of which exhibiting orders of magnitude selectivity. Most of the identified compounds can be clustered into two chemical classes: indazole/dihydropyrimidine-containing compounds that are selective for GRK2 and pyrrolopyrimidine-containing compounds that potently inhibit GRK1 and GRK5 but with more modest selectivity. The two most potent inhibitors representing each class, GSK180736A and GSK2163632A, were cocrystallized with GRK2 and GRK1, and their atomic structures were determined to 2.6 and 1.85 Å spacings, respectively. GSK180736A, developed as a Rho-associated, coiled-coil-containing protein kinase inhibitor, binds to GRK2 in a manner analogous to that of paroxetine, whereas GSK2163632A, developed as an insulin-like growth factor 1 receptor inhibitor, occupies a novel region of the GRK active site cleft that could likely be exploited to achieve more selectivity. However, neither compound inhibits GRKs more potently than their initial targets. This data provides the foundation for future efforts to rationally design even more potent and selective GRK inhibitors.

  10. Prostaglandin E2 receptor expression in the rat trigeminal-vascular system and other brain structures involved in pain

    DEFF Research Database (Denmark)

    Myren, Maja; Olesen, Jes; Gupta, Saurabh


    Prostaglandin E(2) (PGE(2)) is considered to be a key mediator in migraine pathophysiology. PGE(2) acts via four receptors (EP(1)-EP(4)) but their distribution in the brain districts implicated in migraine has yet to be delineated. We quantified amount of mRNA and protein expression for the EP...... receptors in both peripheral and central structures involved in pain transmission and perception in migraine: dura mater, cerebral arteries, trigeminal ganglion, trigeminal nucleus caudalis, periaqueductal grey, thalamus, hypothalamus, cortex, pituitary gland, hippocampus and cerebellum. In the trigeminal...

  11. Structure of the ligand-binding domain (LBD) of human androgen receptor in complex with a selective modulator LGD2226

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Feng; Liu, Xiao-qin; Li, He; Liang, Kai-ni [National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Chaoyang District, Beijing 100101 (China); Miner, Jeffrey N.; Hong, Mei; Kallel, E. Adam; Oeveren, Arjan van; Zhi, Lin [Discovery Research, Ligand Pharmaceuticals Inc., 10275 Science Center Drive, San Diego, California 92121 (United States); Jiang, Tao, E-mail: [National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Chaoyang District, Beijing 100101 (China)


    Crystal structure of the ligand-binding domain of androgen receptor in complex with LGD2226. The androgen receptor (AR) is a ligand-inducible steroid hormone receptor that mediates androgen action, determining male sexual phenotypes and promoting spermatogenesis. As the androgens play a dominant role in male sexual development and function, steroidal androgen agonists have been used clinically for some years. However, there is a risk of potential side effects and most steroidal androgens cannot be dosed orally, which limits the use of these substances. 1,2-Dihydro-6-N,N-bis(2,2,2-trifluoroethyl) amino-4-trifluoromethyl-2-quinolinone (LGD2226) is a synthetic nonsteroidal ligand and a novel selective AR modulator. The crystal structure of the complex of LGD2226 with the androgen receptor ligand-binding domain (AR LBD) at 2.1 Å was solved and compared with the structure of the AR LBD–R1881 complex. It is hoped that this will aid in further explaining the selectivity of LGD2226 observed in in vitro and in vivo assays and in developing more selective and effective therapeutic agents.

  12. [Study of the structure of receptor organs of the vestibular apparatus of rats after space flight on "Kosmos-1667"]. (United States)

    Lychakov, D V; Pashchinin, A N; Boiadzhieva-Mikhaĭlova, A; Khristov, I


    The receptor organs of the vestibular apparatus of rats flown for 7 days on Cosmos-1667 were examined. Serial sections were examined by light microscopy, some utriculus sections by electron microscopy, and otolith membranes by scanning electron microscopy. The fixation method used revealed a distinct structural heterogeneity of the receptor epithelium. In the striola area of the utriculus and sacculus as well as in the central apical area of cristae there are receptor cells surrounded by enlarged cup-like nerve endings. The nerve endings occupy over 70% of the cup-receptor cell complex. The area incorporating the enlarged nerve endings differs in size from animal to animal and from left to right ear in the same animal. The flown rat that was the first to be killed after recovery showed a very well pronounced asymmetry: in the right ear enlarged cups were seen all over the epithelium while in the left ear they were located in distinct spots. Since such changes were not identified in the remaining flown and control rats, it is concluded that they were produced by space flight effects but remained reversible and disappeared after recovery. This paper describes the causes responsible of the changes and their structural and functional relevances as well as other structural modifications that should be considered during vestibular studies.

  13. Molecular cloning, genomic organization, and developmental regulation of a novel receptor from Drosophila melanogaster structurally related to members of the thyroid-stimulating hormone, follicle-stimulating hormone, luteinizing hormone/choriogonadotropin receptor family from mammals

    DEFF Research Database (Denmark)

    Hauser, F; Nothacker, H P; Grimmelikhuijzen, C J


    Using oligonucleotide probes derived from consensus sequences for glycoprotein hormone receptors, we have cloned an 831-amino acid residue-long receptor from Drosophila melanogaster that shows a striking structural homology with members of the glycoprotein hormone (thyroid-stimulating hormone (TSH...... until after pupation. Adult male flies express high levels of receptor mRNA, but female flies express about 6 times less. The expression pattern in embryos and larvae suggests that the receptor is involved in insect development. This is the first report on the molecular cloning of a glycoprotein hormone...

  14. Structural change in dopamine D{sub 2} receptor gene in a patient with neuroleptic malignant syndrome

    Energy Technology Data Exchange (ETDEWEB)

    Ram, A.; Cao, Q.; Gershon, E.S. [National Institutes of Health, Bethesda, MD (United States)] [and others


    Dysfunction of the dopaminergic system has been suggested as a pathogenic mechanism in neuroleptic malignant syndrome. Therefore, we examined the complete coding sequences of the dopamine D{sub 2} receptor (DRD2) gene for structural abnormalities in 12 patients with a history of NMS, including two cases of familial NMS. Mutational analysis was performed by denaturing gradient gel electrophoresis (DGGE), a highly sensitive technique for detecting sequence differences. We found in one patient with a history of NMS a nucleotide substitution at codon 310 (CCG{r_arrow}TCG) of exon 7 of the DRD2 gene which predicts the replacement of proline to serine in the third cytoplasmic loop of the receptor, a part of the receptor that interacts with G-proteins. A larger series of patients with NMS needs to be investigated to establish whether this allele is associated with an increased susceptibility to NMS. 25 refs., 1 fig.

  15. Structure of the Human Dopamine D3 Receptor in Complex with a D2/D3 Selective Antagonist

    Energy Technology Data Exchange (ETDEWEB)

    Chien, Ellen Y.T.; Liu, Wei; Zhao, Qiang; Katritch, Vsevolod; Han, Gye Won; Hanson, Michael A.; Shi, Lei; Newman, Amy Hauck; Javitch, Jonathan A.; Cherezov, Vadim; Stevens, Raymond C. (Cornell); (Scripps); (NIDA); (Columbia); (UCSD); (Receptos)


    Dopamine modulates movement, cognition, and emotion through activation of dopamine G protein-coupled receptors in the brain. The crystal structure of the human dopamine D3 receptor (D3R) in complex with the small molecule D2R/D3R-specific antagonist eticlopride reveals important features of the ligand binding pocket and extracellular loops. On the intracellular side of the receptor, a locked conformation of the ionic lock and two distinctly different conformations of intracellular loop 2 are observed. Docking of R-22, a D3R-selective antagonist, reveals an extracellular extension of the eticlopride binding site that comprises a second binding pocket for the aryl amide of R-22, which differs between the highly homologous D2R and D3R. This difference provides direction to the design of D3R-selective agents for treating drug abuse and other neuropsychiatric indications.

  16. Somatostatin receptors

    DEFF Research Database (Denmark)

    Møller, Lars Neisig; Stidsen, Carsten Enggaard; Hartmann, Bolette;


    therefore been acknowledged to be a third endogenous ligand at SRIF receptors. This review goes through mechanisms of signal transduction, pharmacology, and anatomical distribution of SRIF receptors. Structurally, SRIF receptors belong to the superfamily of G protein-coupled (GPC) receptors, sharing......In 1972, Brazeau et al. isolated somatostatin (somatotropin release-inhibiting factor, SRIF), a cyclic polypeptide with two biologically active isoforms (SRIF-14 and SRIF-28). This event prompted the successful quest for SRIF receptors. Then, nearly a quarter of a century later, it was announced...... that a neuropeptide, to be named cortistatin (CST), had been cloned, bearing strong resemblance to SRIF. Evidence of special CST receptors never emerged, however. CST rather competed with both SRIF isoforms for specific receptor binding. And binding to the known subtypes with affinities in the nanomolar range, it has...

  17. Structural Basis of Natural Promoter Recognition by a Unique Nuclear Receptor, HNF4[alpha

    Energy Technology Data Exchange (ETDEWEB)

    Lu, Peng; Rha, Geun Bae; Melikishvili, Manana; Wu, Guangteng; Adkins, Brandon C.; Fried, Michael G.; Chi, Young-In (Kentucky)


    HNF4{alpha} (hepatocyte nuclear factor 4{alpha}) plays an essential role in the development and function of vertebrate organs, including hepatocytes and pancreatic {beta}-cells by regulating expression of multiple genes involved in organ development, nutrient transport, and diverse metabolic pathways. As such, HNF4{alpha} is a culprit gene product for a monogenic and dominantly inherited form of diabetes, known as maturity onset diabetes of the young (MODY). As a unique member of the nuclear receptor superfamily, HNF4{alpha} recognizes target genes containing two hexanucleotide direct repeat DNA-response elements separated by one base pair (DR1) by exclusively forming a cooperative homodimer. We describe here the 2.0 {angstrom} crystal structure of human HNF4{alpha} DNA binding domain in complex with a high affinity promoter element of another MODY gene, HNF1{alpha}, which reveals the molecular basis of unique target gene selection/recognition, DNA binding cooperativity, and dysfunction caused by diabetes-causing mutations. The predicted effects of MODY mutations have been tested by a set of biochemical and functional studies, which show that, in contrast to other MODY gene products, the subtle disruption of HNF4{alpha} molecular function can cause significant effects in afflicted MODY patients.

  18. The structure of myostatin:follistatin 288: insights into receptor utilization and heparin binding

    Energy Technology Data Exchange (ETDEWEB)

    Cash, Jennifer N.; Rejon, Carlis A.; McPherron, Alexandra C.; Bernard, Daniel J.; Thompson, Thomas B.; (UCIN); (McGill); (NIH)


    Myostatin is a member of the transforming growth factor-{beta} (TGF-{beta}) family and a strong negative regulator of muscle growth. Here, we present the crystal structure of myostatin in complex with the antagonist follistatin 288 (Fst288). We find that the prehelix region of myostatin very closely resembles that of TGF-{beta} class members and that this region alone can be swapped into activin A to confer signalling through the non-canonical type I receptor Alk5. Furthermore, the N-terminal domain of Fst288 undergoes conformational rearrangements to bind myostatin and likely acts as a site of specificity for the antagonist. In addition, a unique continuous electropositive surface is created when myostatin binds Fst288, which significantly increases the affinity for heparin. This translates into stronger interactions with the cell surface and enhanced myostatin degradation in the presence of either Fst288 or Fst315. Overall, we have identified several characteristics unique to myostatin that will be paramount to the rational design of myostatin inhibitors that could be used in the treatment of muscle-wasting disorders.

  19. Combined Ligand/Structure-Based Virtual Screening and Molecular Dynamics Simulations of Steroidal Androgen Receptor Antagonists

    Directory of Open Access Journals (Sweden)

    Yuwei Wang


    Full Text Available The antiandrogens, such as bicalutamide, targeting the androgen receptor (AR, are the main endocrine therapies for prostate cancer (PCa. But as drug resistance to antiandrogens emerges in advanced PCa, there presents a high medical need for exploitation of novel AR antagonists. In this work, the relationships between the molecular structures and antiandrogenic activities of a series of 7α-substituted dihydrotestosterone derivatives were investigated. The proposed MLR model obtained high predictive ability. The thoroughly validated QSAR model was used to virtually screen new dihydrotestosterones derivatives taken from PubChem, resulting in the finding of novel compounds CID_70128824, CID_70127147, and CID_70126881, whose in silico bioactivities are much higher than the published best one, even higher than bicalutamide. In addition, molecular docking, molecular dynamics (MD simulations, and MM/GBSA have been employed to analyze and compare the binding modes between the novel compounds and AR. Through the analysis of the binding free energy and residue energy decomposition, we concluded that the newly discovered chemicals can in silico bind to AR with similar position and mechanism to the reported active compound and the van der Waals interaction is the main driving force during the binding process.

  20. Antinociceptive profile of salvinorin A, a structurally unique kappa opioid receptor agonist. (United States)

    McCurdy, Christopher R; Sufka, Kenneth J; Smith, Grant H; Warnick, Jason E; Nieto, Marcelo J


    Salvinorin A, is a structurally unique, non-nitrogenous, kappa opioid receptor (KOP) agonist. Given the role of KOPs in analgesic processes, we set out to determine whether salvinorin A has antinociceptive activity in thermal and chemo-nociceptive assays. The tail-flick assay was employed to investigate 1) salvinorin A's (0.5, 1.0, 2.0, and 4.0 mg/kg) dose-response and time-course (10, 20, and 30 min) effects in a thermal nociceptive assay, and 2) the ability for the KOP antagonist norBNI (10.0 mg/kg) to prevent salvinorin A antinociception. The hotplate assay was utilized as a second thermal nociceptive measure to test salvinorin A's dose-response effects. The acetic acid abdominal constriction assay was used to study salvinorin A's dose-response and time-course (over 30 min) effects in a chemo-nociceptive assay. Together, these studies revealed that salvinorin A produces a dose-dependent antinociception that peaked at 10 min post-injection but rapidly returned to baseline. Additionally, pretreatment with the KOP antagonist norbinaltorphimine (norBNI) reversed salvinorin A-induced antinociception. These findings demonstrate that salvinorin A produces a KOP mediated antinociceptive effect with a short duration of action.

  1. Structure and calcium-binding studies of calmodulin-like domain of human non-muscle alpha-actinin-1


    Sara Drmota Prebil; Urška Slapšak; Miha Pavšič; Gregor Ilc; Vid Puž; Euripedes De Almeida Ribeiro; Dorothea Anrather; Markus Hartl; Lars Backman; Janez Plavec; Brigita Lenarčič; Kristina Djinović-Carugo


    The activity of several cytosolic proteins critically depends on the concentration of calcium ions. One important intracellular calcium-sensing protein is alpha-actinin-1, the major actin crosslinking protein in focal adhesions and stress fibers. The actin crosslinking activity of alpha-actinin-1 has been proposed to be negatively regulated by calcium, but the underlying molecular mechanisms are poorly understood. To address this, we determined the first high-resolution NMR structure of its f...

  2. Structure prediction of GPCRs using piecewise homologs and application to the human CCR5 chemokine receptor: validation through agonist and antagonist docking. (United States)

    Arumugam, Karthik; Crouzy, Serge; Chevigne, Andy; Seguin-Devaux, Carole; Schmit, Jean-Claude


    This article describes the construction and validation of a three-dimensional model of the human CC chemokine receptor 5 (CCR5) receptor using multiple homology modeling. A new methodology is presented where we built each secondary structural model of the protein separately from distantly related homologs of known structure. The reliability of our approach for G-protein coupled receptors was assessed through the building of the human C-X-C chemokine receptor type 4 (CXCR4) receptor of known crystal structure. The models are refined using molecular dynamics simulations and energy minimizations using CHARMM, a classical force field for proteins. Finally, docking models of both the natural agonists and the antagonists of the receptors CCR5 and CXCR4 are proposed. This study explores the possible binding process of ligands to the receptor cavity of chemokine receptors at molecular and atomic levels. We proposed few crucial residues in receptors binding to agonist/antagonist for further validation through experimental analysis. In particular, our study provides better understanding of the blockage mechanism of the chemokine receptors CCR5 and CXCR4, and may help the identification of new lead compounds for drug development in HIV infection, inflammatory diseases, and cancer metastasis.

  3. Structural determinants of agonist-specific kinetics at the ionotropic glutamate receptor 2

    DEFF Research Database (Denmark)

    Holm, Mai Marie; Lunn, Marie-Louise; Traynelis, Stephen F;


    Glutamate receptors (GluRs) are the most abundant mediators of the fast excitatory neurotransmission in the human brain. Agonists will, after activation of the receptors, induce different degrees of desensitization. The efficacy of agonists strongly correlates with the agonist-induced closure of ...

  4. G protein-coupled receptor mutations and human genetic disease. (United States)

    Thompson, Miles D; Hendy, Geoffrey N; Percy, Maire E; Bichet, Daniel G; Cole, David E C


    Genetic variations in G protein-coupled receptor genes (GPCRs) disrupt GPCR function in a wide variety of human genetic diseases. In vitro strategies and animal models have been used to identify the molecular pathologies underlying naturally occurring GPCR mutations. Inactive, overactive, or constitutively active receptors have been identified that result in pathology. These receptor variants may alter ligand binding, G protein coupling, receptor desensitization and receptor recycling. Receptor systems discussed include rhodopsin, thyrotropin, parathyroid hormone, melanocortin, follicle-stimulating hormone (FSH), luteinizing hormone, gonadotropin-releasing hormone (GNRHR), adrenocorticotropic hormone, vasopressin, endothelin-β, purinergic, and the G protein associated with asthma (GPRA or neuropeptide S receptor 1 (NPSR1)). The role of activating and inactivating calcium-sensing receptor (CaSR) mutations is discussed in detail with respect to familial hypocalciuric hypercalcemia (FHH) and autosomal dominant hypocalemia (ADH). The CASR mutations have been associated with epilepsy. Diseases caused by the genetic disruption of GPCR functions are discussed in the context of their potential to be selectively targeted by drugs that rescue altered receptors. Examples of drugs developed as a result of targeting GPCRs mutated in disease include: calcimimetics and calcilytics, therapeutics targeting melanocortin receptors in obesity, interventions that alter GNRHR loss from the cell surface in idiopathic hypogonadotropic hypogonadism and novel drugs that might rescue the P2RY12 receptor congenital bleeding phenotype. De-orphanization projects have identified novel disease-associated receptors, such as NPSR1 and GPR35. The identification of variants in these receptors provides genetic reagents useful in drug screens. Discussion of the variety of GPCRs that are disrupted in monogenic Mendelian disorders provides the basis for examining the significance of common

  5. Higher-Resolution Structure of the Human Insulin Receptor Ectodomain: Multi-Modal Inclusion of the Insert Domain. (United States)

    Croll, Tristan I; Smith, Brian J; Margetts, Mai B; Whittaker, Jonathan; Weiss, Michael A; Ward, Colin W; Lawrence, Michael C


    Insulin receptor (IR) signaling is critical to controlling nutrient uptake and metabolism. However, only a low-resolution (3.8 Å) structure currently exists for the IR ectodomain, with some segments ill-defined or unmodeled due to disorder. Here, we revise this structure using new diffraction data to 3.3 Å resolution that allow improved modeling of the N-linked glycans, the first and third fibronectin type III domains, and the insert domain. A novel haptic interactive molecular dynamics strategy was used to aid fitting to low-resolution electron density maps. The resulting model provides a foundation for investigation of structural transitions in IR upon ligand binding.

  6. Basic Pharmacological and Structural Evidence for Class A G-Protein-Coupled Receptor Heteromerization (United States)

    Franco, Rafael; Martínez-Pinilla, Eva; Lanciego, José L.; Navarro, Gemma


    Cell membrane receptors rarely work on isolation, often they form oligomeric complexes with other receptor molecules and they may directly interact with different proteins of the signal transduction machinery. For a variety of reasons, rhodopsin-like class A G-protein-coupled receptors (GPCRs) seem an exception to the general rule of receptor–receptor direct interaction. In fact, controversy surrounds their potential to form homo- hetero-dimers/oligomers with other class A GPCRs; in a sense, the field is going backward instead of forward. This review focuses on the convergent, complementary and telling evidence showing that homo- and heteromers of class A GPCRs exist in transfected cells and, more importantly, in natural sources. It is time to decide between questioning the occurrence of heteromers or, alternatively, facing the vast scientific and technical challenges that class A receptor-dimer/oligomer existence pose to Pharmacology and to Drug Discovery. PMID:27065866

  7. A Structural Model for the Membrane-Bound Form of the Juxtamembrane Domain of the Epidermal Growth Factor Receptor.

    Energy Technology Data Exchange (ETDEWEB)

    Choowongkomon, Kiattawee; Carlin, Cathleen R.; Sonnichsen, Frank D.


    The epidermal growth factor receptor (EGFR) is a member of the receptor tyrosine kinase family involved in the regulation of cellular proliferation and differentiation. Its juxtamembrane domain (JX), the region located between the transmembrane and kinase domains, plays important roles in receptor trafficking. Two sorting signals, a PXXP motif and a 658LL659 motif, are responsible for basolateral sorting in polarized epithelial cells, and a 679LL680 motif targets the ligand-activated receptor for lysosomal degradation. To understand the regulation of these signals, we characterized the structural properties of recombinant JX domain in aqueous solution and in dodecylphosphocholine (DPC) detergent. JX is inherently unstructured in aqueous solution, albeit a nascent helix encompasses the lysosomal sorting signal. In DPC micelles, structures derived from NMR data showed three amphipathic, helical segments. A large, internally inconsistent group of long range nuclear Overhauser effects suggest a close proximity of the helices, and the presence of significant conformational averaging. Models were determined for the average JX conformation using restraints representing the translational restriction due to micelle-surface adsorption, and the helix orientations were determined from residual dipolar couplings. Two equivalent average structural models were obtained that differ only in the relative orientation between first and second helices. In these models, the 658LL659 and 679LL680 motifs are located in the first and second helices and face the micelle surface, whereas the PXXP motif is located in a flexible helix-connecting region. The data suggest that the activity of these signals may be regulated by their membrane association and restricted accessibility in the intact receptor.

  8. Multivalent structure of galectin-1-nanogold complex serves as potential therapeutics for rheumatoid arthritis by enhancing receptor clustering

    Directory of Open Access Journals (Sweden)

    Y-J Huang


    Full Text Available Cellular behaviour is controlled by numerous processes, including intracellular signalling pathways that are triggered by the binding of ligands with cell surface receptors. Multivalent ligands have multiple copies of a recognition element that binds to receptors and influences downstream signals. Nanoparticle-ligand complexes may form multivalent structures to crosslink receptors with high avidity and specificity. After conjugation onto gold nanoparticles, galectin-1 (Au-Gal1 bound with higher affinity to Jurkat cells to promote CD45 clustering and inhibition of its phosphatase activity, resulting in enhancement of apoptosis via caspase-dependent pathways. Au-Gal1 injected intra-articularly into rats with collagen-induced arthritis (CIA promoted apoptosis of CD4+ T cells and reduced pro-inflammatory cytokine levels in the ankle joints as well as ameliorated clinical symptoms of arthritis. These observed therapeutic effects indicate that the multivalent structure of nanoparticle-ligands can regulate the distribution of cell surface receptors and subsequent intracellular signalling, and this may provide new insights into nanoparticle applications.

  9. Structure, Receptor Binding, and Antigenicity of Influenza Virus Hemagglutinins from the 1957 H2N2 Pandemic

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Rui; McBride, Ryan; Paulson, James C.; Basler, Christopher F.; Wilson, Ian A. (Sinai); (Scripps)


    The hemagglutinin (HA) envelope protein of influenza viruses mediates essential viral functions, including receptor binding and membrane fusion, and is the major viral antigen for antibody neutralization. The 1957 H2N2 subtype (Asian flu) was one of the three great influenza pandemics of the last century and caused 1 million deaths globally from 1957 to 1968. Three crystal structures of 1957 H2 HAs have been determined at 1.60 to 1.75 {angstrom} resolutions to investigate the structural basis for their antigenicity and evolution from avian to human binding specificity that contributed to its introduction into the human population. These structures, which represent the highest resolutions yet recorded for a complete ectodomain of a glycosylated viral surface antigen, along with the results of glycan microarray binding analysis, suggest that a hydrophobicity switch at residue 226 and elongation of receptor-binding sites were both critical for avian H2 HA to acquire human receptor specificity. H2 influenza viruses continue to circulate in birds and pigs and, therefore, remain a substantial threat for transmission to humans. The H2 HA structure also reveals a highly conserved epitope that could be harnessed in the design of a broader and more universal influenza A virus vaccine.

  10. Receptor binding profiles and quantitative structure-affinity relationships of some 5-substituted-N,N-diallyltryptamines. (United States)

    Cozzi, Nicholas V; Daley, Paul F


    N,N-Diallyltryptamine (DALT) and 5-methoxy-N,N-diallyltryptamine (5-MeO-DALT) are two tryptamines synthesized and tested by Alexander Shulgin. In self-experiments, 5-MeO-DALT was reported to be psychoactive in the 12-20mg range, while the unsubstituted compound DALT had few discernible effects in the 42-80 mg range. Recently, 5-MeO-DALT has been used in nonmedical settings for its psychoactive effects, but these effects have been poorly characterized and little is known of its pharmacological properties. We extended the work of Shulgin by synthesizing additional 5-substituted-DALTs. We then compared them to DALT and 5-MeO-DALT for their binding affinities at 45 cloned receptors and transporter proteins. Based on in vitro binding affinity, we identified 27 potential receptor targets for the 5-substituted-DALT compounds. Five of the DALT compounds had affinity in the 10-80 nM range for serotonin 5-HT1A and 5-HT2B receptors, while the affinity of DALT itself at 5-HT1A receptors was slightly lower at 100 nM. Among the 5-HT2 subtypes, the weakest affinity was at 5-HT2A receptors, spanning 250-730 nM. Five of the DALT compounds had affinity in the 50-400 nM range for serotonin 5-HT1D, 5-HT6, and 5-HT7 receptors; again, it was the unsubstituted DALT that had the weakest affinity at all three subtypes. The test drugs had even weaker affinity for 5-HT1B, 5-HT1E, and 5-HT5A subtypes and little or no affinity for the 5-HT3 subtype. These compounds also had generally nanomolar affinities for adrenergic α2A, α2B, and α2C receptors, sigma receptors σ1 and σ2, histamine H1 receptors, and norepinephrine and serotonin uptake transporters. They also bound to other targets in the nanomolar-to-low micromolar range. Based on these binding results, it is likely that multiple serotonin receptors, as well as several nonserotonergic sites are important for the psychoactive effects of DALT drugs. To learn whether any quantitative structure-affinity relationships existed, we evaluated

  11. Structural Diversity in Conserved Regions Like the DRY-Motif among Viral 7TM Receptors-A Consequence of Evolutionary Pressure?

    DEFF Research Database (Denmark)

    Mølleskov-Jensen, Ann-Sofie; Sparre-Ulrich, Alexander Hovard; Davis-Poynter, Nicholas


    and the C-terminal tail. In the DRY-motif, the arginine residue serves important purposes by being directly involved in G protein coupling. Interestingly, among the viral receptors there is a greater diversity in the DRY-motif compared to their endogenous receptor homologous. The C-terminal receptor tail......Several herpes- and poxviruses have captured chemokine receptors from their hosts and modified these to their own benefit. The human and viral chemokine receptors belong to class A 7 transmembrane (TM) receptors which are characterized by several structural motifs like the DRY-motif in TM3...... motifs and discuss their role in viral 7TM receptor signaling compared to their endogenous counterparts....

  12. Structural basis of RNA recognition and activation by innate immune receptor RIG-I

    Energy Technology Data Exchange (ETDEWEB)

    Jiang, Fuguo; Ramanathan, Anand; Miller, Matthew T.; Tang, Guo-Qing; Gale, Jr., Michael; Patel, Smita S.; Marcotrigiano, Joseph (Rutgers); (RWJ-Med); (UW-MED)


    Retinoic-acid-inducible gene-I (RIG-I; also known as DDX58) is a cytoplasmic pathogen recognition receptor that recognizes pathogen-associated molecular pattern (PAMP) motifs to differentiate between viral and cellular RNAs. RIG-I is activated by blunt-ended double-stranded (ds)RNA with or without a 5'-triphosphate (ppp), by single-stranded RNA marked by a 5'-ppp and by polyuridine sequences. Upon binding to such PAMP motifs, RIG-I initiates a signalling cascade that induces innate immune defences and inflammatory cytokines to establish an antiviral state. The RIG-I pathway is highly regulated and aberrant signalling leads to apoptosis, altered cell differentiation, inflammation, autoimmune diseases and cancer. The helicase and repressor domains (RD) of RIG-I recognize dsRNA and 5'-ppp RNA to activate the two amino-terminal caspase recruitment domains (CARDs) for signalling. Here, to understand the synergy between the helicase and the RD for RNA binding, and the contribution of ATP hydrolysis to RIG-I activation, we determined the structure of human RIG-I helicase-RD in complex with dsRNA and an ATP analogue. The helicase-RD organizes into a ring around dsRNA, capping one end, while contacting both strands using previously uncharacterized motifs to recognize dsRNA. Small-angle X-ray scattering, limited proteolysis and differential scanning fluorimetry indicate that RIG-I is in an extended and flexible conformation that compacts upon binding RNA. These results provide a detailed view of the role of helicase in dsRNA recognition, the synergy between the RD and the helicase for RNA binding and the organization of full-length RIG-I bound to dsRNA, and provide evidence of a conformational change upon RNA binding. The RIG-I helicase-RD structure is consistent with dsRNA translocation without unwinding and cooperative binding to RNA. The structure yields unprecedented insight into innate immunity and has a broader impact on other areas of biology, including

  13. Anion complexation with cyanobenzoyl substituted first and second generation tripodal amide receptors: crystal structure and solution studies. (United States)

    Hoque, Md Najbul; Gogoi, Abhijit; Das, Gopal


    Anion complexation properties of two new tripodal amide receptors have been extensively studied here. Two tripodal receptors have been synthesized from the reaction of cyanobenzoyl acid chloride with two tri-amine building blocks such as (i) tris(2-aminoethyl)amine and (ii) tris(2-(4-aminophenoxy)ethyl)amine, which resulted in the first (L1) and second (L2) generation tripodal amides respectively. A detailed comparison of their coordination behavior with anions is also described by crystallographic and solution state experiments. The crystal structure demonstrates various types of spatial orientations of tripodal arms in two receptors and concomitantly interacts with anions distinctively. Intramolecular H-bonding between amide N–H and CO prevents opening of the receptor cavity in the crystal, which leads to a locked conformation of L1 having C(3v) symmetry and makes amide hydrogen unavailable for the anion which results in side cleft anion binding. However, in L2 we conveniently shift the anion binding sites to a distant position which increases cavity size as well as rules out any intramolecular H-bonding between amide N–H and CO. The crystal structure shows a different orientation of the arms in L2; it adopts a quasi-planar arrangement with C(2v) symmetry. In the crystal structure two arms are pointed in the same direction and while extending the contact the third arm is H-bonded with the apical N-atom through a –CN group, making a pseudo capsular cavity where the anion interacts. Most importantly spatial reorientation of the receptor L2 from a C(2v) symmetry to a folded conformation with a C(3v) symmetry was observed only in the presence of an octahedral SiF6(2-) anion and forms a sandwich type complex. Receptors L1 and L2 are explored for their solution state anion binding abilities. The substantial changes in chemical shifts were observed for the amide (-NH) and aromatic hydrogen (-CH) (especially for F(-)), indicating the role of these hydrogens in

  14. Structure and Function of CC-Chemokine Receptor 5 Homologues Derived from Representative Primate Species and Subspecies of the Taxonomic Suborders Prosimii and Anthropoidea



    A chemokine receptor from the seven-transmembrane-domain G-protein-coupled receptor superfamily is an essential coreceptor for the cellular entry of human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) strains. To investigate nonhuman primate CC-chemokine receptor 5 (CCR5) homologue structure and function, we amplified CCR5 DNA sequences from peripheral blood cells obtained from 24 representative species and subspecies of the primate suborders Prosimii (family L...

  15. Novel histamine H3-receptor antagonists and partial agonists with a non-aminergic structure


    Nickel, Tobias; Bauer, Ulrich; Schlicker, Eberhard; Kathmann, Markus; Göthert, Manfred; Sasse, Astrid; Stark, Holger; Schunack, Walter


    We determined the affinities of eight novel histamine H3-receptor ligands (ethers and carbamates) for H3-receptor binding sites and their agonistic/antagonistic effects in two functional H3-receptor models. The compounds differ from histamine in that the ethylamine chain is replaced by a propyloxy chain; in the three ethers mentioned below (FUB 335, 373 and 407), R is n-pentyl, 3-methylbutyl and 3,3-dimethylbutyl, respectively.The compounds monophasically inhibited [3H]-Nα-methylhistamine bin...


    Directory of Open Access Journals (Sweden)

    Jonathan P. Carrillo-Vázquez


    Full Text Available Leptin Receptor (LEPR is a component of a signaling pathway related to appetite and energy expenditure. Single Nucleotide Polymorphisms (SNP of Leptin receptor gene (lepr have been proposed as possible modulator of adipose tissue and body weight. The main phenomenological consequence reported of these SNPs is the modulation of the LEP-LEPR interaction promoting the weight gain. Particularly, Q223R polymorphism has been associated with human obesity in some populations. In this work, we analyze the structural effects of Q223R substitution in a model of the extracellular region of LEPR comparing the stability between LEPR-Q and its Q223R variant (rs1137101 by Molecular Dynamics (MD simulations. These results showed different behavior between both molecules after one nanosecond (ns of simulation and significant differences in the secondary structure content were evidenced.

  17. Structure of Human G Protein-Coupled Receptor Kinase 2 in Complex with the Kinase Inhibitor Balanol

    Energy Technology Data Exchange (ETDEWEB)

    Tesmer, John J.G.; Tesmer, Valerie M.; Lodowski, David T.; Steinhagen, Henning; Huber, Jochen (Sanofi); (Michigan); (Texas)


    G protein-coupled receptor kinase 2 (GRK2) is a pharmaceutical target for the treatment of cardiovascular diseases such as congestive heart failure, myocardial infarction, and hypertension. To better understand how nanomolar inhibition and selectivity for GRK2 might be achieved, we have determined crystal structures of human GRK2 in complex with G{beta}{gamma} in the presence and absence of the AGC kinase inhibitor balanol. The selectivity of balanol among human GRKs is assessed.

  18. Genomic structure, characterization, and identification of the promotor of the human IL-8 receptor A gene

    Energy Technology Data Exchange (ETDEWEB)

    Sprenger, H.; Lloyd, A.R.; Meyer, R.G.; Johnston, J.A.; Kelvin, D.J. [National Cancer Institute, Frederick, MA (United States)


    Two unique but homologous receptors for the neutrophil chemoattractant IL-8 have been cloned (designated IL-8RA and IL-8RB), each of which binds IL-8 with high affinity. IL-8RA mRNA expression was found to be regulated by granulocyte-CSF and LPS. In an attempt to understand the tissue-specific expression and to identify transcriptional regulatory elements, the authors have cloned, sequenced, and characterized the human IL-8RA gene. A {lambda}-DASH clone encoding the entire human IL-8RA gene was isolated by screening a genomic library with a PCR-generated cDNA. After mapping, subcloning, and sequencing several restriction fragments, a 9.2-kb continuous DNA sequence was obtained. As the sizes of the published cDNA (1.9 kb) and the mRNA determined by Northern blot analysis (2.1 kb) were not in agreement, a full-length cDNA was cloned by using a modified rapid amplification of cDNA ends technique. They identified a 5{prime}-untranslated region of 119 bp. After comparison with the genomic sequence, they found the gene consisted of two exons interrupted by an intron of 1.7 kb. A 1050-bp ORF was encoded entirely in the second exon together with a 834-bp 3{prime}-untranslated region. The immediate GC-rich 5{prime}-flanking region upstream of exon 1 could serve as a constitutively active promoter in chloramphenicolacetyl-transferase-expression assays. Expression analysis of additional upstream regions suggested the presence of silencer elements between positions -841 and -280. In conclusion, cloning a full-length cDNA permitted cloning of the human IL-8RA gene, identification of the genomic structure, and characterization of the promoter region. 45 refs., 6 figs.

  19. 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: [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)


    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.

  20. Structure of human cytomegalovirus UL141 binding to TRAIL-R2 reveals novel, non-canonical death receptor interactions.

    Directory of Open Access Journals (Sweden)

    Ivana Nemčovičová


    Full Text Available The TRAIL (TNF-related apoptosis inducing ligand death receptors (DRs of the tumor necrosis factor receptor superfamily (TNFRSF can promote apoptosis and regulate antiviral immunity by maintaining immune homeostasis during infection. In turn, human cytomegalovirus (HCMV expresses immunomodulatory proteins that down-regulate cell surface expression of TNFRSF members as well as poliovirus receptor-related proteins in an effort to inhibit host immune effector pathways that would lead to viral clearance. The UL141 glycoprotein of human cytomegalovirus inhibits host defenses by blocking cell surface expression of TRAIL DRs (by retention in ER and poliovirus receptor CD155, a nectin-like Ig-fold molecule. Here we show that the immunomodulatory function of HCMV UL141 is associated with its ability to bind diverse proteins, while utilizing at least two distinct binding sites to selectively engage TRAIL DRs or CD155. Binding studies revealed high affinity interaction of UL141 with both TRAIL-R2 and CD155 and low affinity binding to TRAIL-R1. We determined the crystal structure of UL141 bound to TRAIL-R2 at 2.1 Å resolution, which revealed that UL141 forms a homodimer that engages two TRAIL-R2 monomers 90° apart to form a heterotetrameric complex. Our structural and biochemical data reveal that UL141 utilizes its Ig-domain to facilitate non-canonical death receptor interactions while UL141 partially mimics the binding site of TRAIL on TRAIL-R2, which we found to be distinct from that of CD155. Moreover, UL141 also binds to an additional surface patch on TRAIL-R2 that is distinct from the TRAIL binding site. Therefore, the breadth of UL141-mediated effects indicates that HCMV has evolved sophisticated strategies to evade the immune system by modulating multiple effector pathways.

  1. Structure of human cytomegalovirus UL141 binding to TRAIL-R2 reveals novel, non-canonical death receptor interactions. (United States)

    Nemčovičová, Ivana; Benedict, Chris A; Zajonc, Dirk M


    The TRAIL (TNF-related apoptosis inducing ligand) death receptors (DRs) of the tumor necrosis factor receptor superfamily (TNFRSF) can promote apoptosis and regulate antiviral immunity by maintaining immune homeostasis during infection. In turn, human cytomegalovirus (HCMV) expresses immunomodulatory proteins that down-regulate cell surface expression of TNFRSF members as well as poliovirus receptor-related proteins in an effort to inhibit host immune effector pathways that would lead to viral clearance. The UL141 glycoprotein of human cytomegalovirus inhibits host defenses by blocking cell surface expression of TRAIL DRs (by retention in ER) and poliovirus receptor CD155, a nectin-like Ig-fold molecule. Here we show that the immunomodulatory function of HCMV UL141 is associated with its ability to bind diverse proteins, while utilizing at least two distinct binding sites to selectively engage TRAIL DRs or CD155. Binding studies revealed high affinity interaction of UL141 with both TRAIL-R2 and CD155 and low affinity binding to TRAIL-R1. We determined the crystal structure of UL141 bound to TRAIL-R2 at 2.1 Å resolution, which revealed that UL141 forms a homodimer that engages two TRAIL-R2 monomers 90° apart to form a heterotetrameric complex. Our structural and biochemical data reveal that UL141 utilizes its Ig-domain to facilitate non-canonical death receptor interactions while UL141 partially mimics the binding site of TRAIL on TRAIL-R2, which we found to be distinct from that of CD155. Moreover, UL141 also binds to an additional surface patch on TRAIL-R2 that is distinct from the TRAIL binding site. Therefore, the breadth of UL141-mediated effects indicates that HCMV has evolved sophisticated strategies to evade the immune system by modulating multiple effector pathways.

  2. Structural proof of a dimeric positive modulator bridging two identical AMPA receptor-binding sites

    DEFF Research Database (Denmark)

    Kaae, Birgitte Høiriis; Harpsøe, Kasper; Kastrup, Jette Sandholm Jensen;


    Dimeric positive allosteric modulators of ionotropic glutamate receptors were designed, synthesized, and characterized pharmacologically in electrophysiological experiments. The designed compounds are dimers of arylpropylsulfonamides and have been constructed without a linker. The monomeric...

  3. Virus-resembling nano-structures for near infrared fluorescence imaging of ovarian cancer HER2 receptors (United States)

    Guerrero, Yadir A.; Bahmani, Baharak; Singh, Sheela P.; Vullev, Valentine I.; Kundra, Vikas; Anvari, Bahman


    Ovarian cancer remains the dominant cause of death due to malignancies of the female reproductive system. The capability to identify and remove all tumors during intraoperative procedures may ultimately reduce cancer recurrence, and lead to increased patient survival. The objective of this study is to investigate the effectiveness of an optical nano-structured system for targeted near infrared (NIR) imaging of ovarian cancer cells that over-express the human epidermal growth factor receptor 2 (HER2), an important biomarker associated with ovarian cancer. The nano-structured system is comprised of genome-depleted plant-infecting brome mosaic virus doped with NIR chromophore, indocyanine green, and functionalized at the surface by covalent attachment of monoclonal antibodies against the HER2 receptor. We use absorption and fluorescence spectroscopy, and dynamic light scattering to characterize the physical properties of the constructs. Using fluorescence imaging and flow cytometry, we demonstrate the effectiveness of these nano-structures for targeted NIR imaging of HER2 receptors in vitro. These functionalized nano-materials may provide a platform for NIR imaging of ovarian cancer.

  4. The structure of haemoglobin bound to the haemoglobin receptor IsdH from Staphylococcus aureus shows disruption of the native α-globin haem pocket. (United States)

    Dickson, Claire F; Jacques, David A; Clubb, Robert T; Guss, J Mitchell; Gell, David A


    Staphylococcus aureus is a common and serious cause of infection in humans. The bacterium expresses a cell-surface receptor that binds to, and strips haem from, human haemoglobin (Hb). The binding interface has previously been identified; however, the structural changes that promote haem release from haemoglobin were unknown. Here, the structure of the receptor-Hb complex is reported at 2.6 Å resolution, which reveals a conformational change in the α-globin F helix that disrupts the haem-pocket structure and alters the Hb quaternary interactions. These features suggest potential mechanisms by which the S. aureus Hb receptor induces haem release from Hb.

  5. Efficient cell-free production of olfactory receptors: detergent optimization, structure, and ligand binding analyses. (United States)

    Kaiser, Liselotte; Graveland-Bikker, Johanna; Steuerwald, Dirk; Vanberghem, Mélanie; Herlihy, Kara; Zhang, Shuguang


    High-level production of membrane proteins, particularly of G protein-coupled receptors (GPCRs) in heterologous cell systems encounters a number of difficulties from their inherent hydrophobicity in their transmembrane domains, which frequently cause protein aggregation and cytotoxicity and thus reduce the protein yield. Recent advances in cell-free protein synthesis circumvent those problems to produce membrane proteins with a yield sometimes exceeding the cell-based approach. Here, we report cell-free production of a human olfactory receptor 17-4 (hOR17-4) using the wheat germ extract. Using the simple method, we also successful produced two additional olfactory receptors. To obtain soluble olfactory receptors and to increase yield, we directly added different detergents in varying concentrations to the cell-free reaction. To identify a purification buffer system that maintained the receptor in a nonaggregated form, we developed a method that uses small-volume size-exclusion column chromatography combined with rapid and sensitive dot-blot detection. Different buffer components including salt concentration, various detergents and detergent concentration, and reducing agent and its concentrations were evaluated for their ability to maintain the cell-free produced protein stable and nonaggregated. The purified olfactory receptor displays a typical a alpha-helical CD spectrum. Surface plasmon resonance measurements were used to show binding of a known ligand undecanal to hOR17-4. Our approach to produce a high yield of purified olfactory receptor is a milestone toward obtaining a large quantity of olfactory receptors for designing bionic sensors. Furthermore, this simple approach may be broadly useful not only for other classes of GPCRs but also for other membrane proteins.

  6. Immunological studies on the structure and function of the nicotinic acetylcholine receptor in mammalian muscle

    Energy Technology Data Exchange (ETDEWEB)

    Gu, Y.


    The specificity of the antibodies in the serum of a patient with myasthenia gravis for a the {alpha}-bungarotoxin binding sites of the acetylcholine receptor (AChR) was examined using AChRs in the C2 mouse muscle cell line as a model. The antibodies were shown to be specific for one of the two toxin-binding sites. The effect of the antibodies in this myasthenic serum on the functional response of the receptor to cholinergic agonists was also examined using carbamylcholine-induced {sup 22}Na uptake into C2 myotubes as a measured of the receptor function. Antibodies specific for the {gamma}, {delta}, and {epsilon} subunit, respectively, of mammalian muscle AChRs were developed using subunit-specific synthetic peptides as antigens. Using these antibodies and monoclonal antibodies for other subunits as probes, I have identified four ({alpha}, {beta}, {gamma}, and {delta}) subunits of mammalian muscle AChRs on immunoblots. When AChRs from embryonic, neonatal, normal and denervated adult muscles were compared on immunoblots, the {alpha}, {beta}, and {delta} subunits were identical in all four receptor preparations, with or without endoglycosidase digestion. The spatial and temporal distribution of the {gamma}- and {epsilon}- AChRs in developing and in denervated muscles corresponds to the distribution of AChRs with slow and fast channels, respectively, and that the development changes in the channel properties of the receptor arise from a change in the subunit composition of the receptor, in which the {gamma} is replaced by {epsilon}.

  7. Selective Allosteric Antagonists for the G Protein-Coupled Receptor GPRC6A Based on the 2-Phenylindole Privileged Structure Scaffold

    DEFF Research Database (Denmark)

    Johansson, Henrik; Boesgaard, Michael Worch; Nørskov-Lauritsen, Lenea


    G protein-coupled receptors (GPCRs) represent a biological target class of fundamental importance in drug therapy. The GPRC6A receptor is a newly deorphanized class C GPCR that we recently reported for the first allosteric antagonists based on the 2-arylindole privileged structure scaffold (e.g., 1...

  8. X-ray structure of a minor group human rhinovirus bound to a fragment of its cellular receptor protein. (United States)

    Verdaguer, Nuria; Fita, Ignacio; Reithmayer, Manuela; Moser, Rosita; Blaas, Dieter


    Although many viral receptors have been identified, the ways in which they interact with their cognate viruses are not understood at the molecular level. We have determined the X-ray structure of a complex between calcium-containing modules of the very low-density lipoprotein receptor and the minor group human rhinovirus HRV2. The receptor binds close to the icosahedral five-fold vertex, with only one module per virus protomer. The binding face of this module is defined by acidic calcium-chelating residues and, in particular, by an exposed tryptophan that is highly conserved. The attachment site on the virus involves only residues from VP1, particularly a lysine strictly conserved in all minor group HRVs. The disposition of the attached ligand-binding repeats around the five-fold axis, together with the proximity of the N- and C-terminal ends of adjacent modules, suggests that more than one repeat in a single receptor molecule might attach simultaneously.

  9. Structural and functional divergence of growth hormone-releasing hormone receptors in early sarcopterygians: lungfish and Xenopus.

    Directory of Open Access Journals (Sweden)

    Janice K V Tam

    Full Text Available The evolutionary trajectories of growth hormone-releasing hormone (GHRH receptor remain enigmatic since the discovery of physiologically functional GHRH-GHRH receptor (GHRHR in non-mammalian vertebrates in 2007. Interestingly, subsequent studies have described the identification of a GHRHR(2 in chicken in addition to the GHRHR and the closely related paralogous receptor, PACAP-related peptide (PRP receptor (PRPR. In this article, we provide information, for the first time, on the GHRHR in sarcopterygian fish and amphibians by the cloning and characterization of GHRHRs from lungfish (P. dolloi and X. laevis. Sequence alignment and phylogenetic analyses demonstrated structural resemblance of lungfish GHRHR to their mammalian orthologs, while the X. laevis GHRHR showed the highest homology to GHRHR(2 in zebrafish and chicken. Functionally, lungfish GHRHR displayed high affinity towards GHRH in triggering intracellular cAMP and calcium accumulation, while X. laevis GHRHR(2 was able to react with both endogenous GHRH and PRP. Tissue distribution analyses showed that both lungfish GHRHR and X. laevis GHRHR(2 had the highest expression in brain, and interestingly, X. laevis(GHRHR2 also had high abundance in the reproductive organs. These findings, together with previous reports, suggest that early in the Sarcopterygii lineage, GHRHR and PRPR have already established diverged and specific affinities towards their cognate ligands. GHRHR(2, which has only been found in xenopus, zebrafish and chicken hitherto, accommodates both GHRH and PRP.

  10. Structure-guided optimization of estrogen receptor binding affinity and antagonist potency of pyrazolopyrimidines with basic side chains.

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, H.; Sheng, S.; Compton, D.; Kim, Y.; Joachimiak, A.; Sharma, S.; Carlson, K.; Katzenellenbogen, B.; Nettles, K.; Greene, G.; Katzenellenbogen, J.; Biosciences Division; Univ. of Illinois; Univ. of Chicago; The Scripps Research Inst.


    2,3-Diarylpyrazolo[1,5-a]pyrimidines are estrogen receptor (ER) antagonists of modest potency that we have described previously. Guided by the crystal structure of an ER-ligand complex that we have obtained with one of these compounds, we prepared analogs that contain a basic side chain at the 2- or 3-aryl group and quickly found one that, according to the structure-based prediction, shows an increase in binding affinity and antagonist potency and a loss of residual agonist activity.

  11. The structural basis of androgen receptor activation: Intramolecular and intermolecular amino–carboxy interactions (United States)

    Schaufele, Fred; Carbonell, Xavier; Guerbadot, Martin; Borngraeber, Sabine; Chapman, Mark S.; Ma, Aye Aye K.; Miner, Jeffrey N.; Diamond, Marc I.


    Nuclear receptors (NRs) are ligand-regulated transcription factors important in human physiology and disease. In certain NRs, including the androgen receptor (AR), ligand binding to the carboxy-terminal domain (LBD) regulates transcriptional activation functions in the LBD and amino-terminal domain (NTD). The basis for NTD–LBD communication is unknown but may involve NTD–LBD interactions either within a single receptor or between different members of an AR dimer. Here, measurement of FRET between fluorophores attached to the NTD and LBD of the AR established that agonist binding initiated an intramolecular NTD–LBD interaction in the nucleus and cytoplasm. This intramolecular folding was followed by AR self-association, which occurred preferentially in the nucleus. Rapid, ligand-induced intramolecular folding and delayed association also were observed for estrogen receptor-α but not for peroxisome proliferator activated receptor-γ2. An antagonist ligand, hydroxyflutamide, blocked the NTD–LBD association within AR. NTD–LBD association also closely correlated with the transcriptional activation by heterologous ligands of AR mutants isolated from hormone-refractory prostate tumors. Intramolecular folding, but not AR–AR affinity, was disrupted by mutation of an α-helical (23FQNLF27) motif in the AR NTD previously described to interact with the AR LBD in vitro. This work establishes an intramolecular NTD–LBD conformational change as an initial component of ligand-regulated NR function. PMID:15994236

  12. Molecular Cloning, Genomic Organization and Developmental Regulation of a Novel Receptor from Drosophila melanogaster Structurally Related to Gonadotropin-Releasing Hormone Receptors from Vertebrates

    DEFF Research Database (Denmark)

    Hauser, Frank; Søndergaard, Leif; Grimmelikhuijzen, Cornelis J.P.


    RH) receptors from vertebrates. Using the polymerase chain reaction, withDrosophilacDNA as a template, and oligonucleotide probes coding for the presumed exons of this gene, we were able to clone the cDNA coding for this receptor. The transmembrane region of the receptor shows 36% amino acid residue identity...

  13. Crystal Structure of the Ligand Binding Suppressor Domain of Type 1 Inositol 1,4,5-Trisphosphate Receptor

    Energy Technology Data Exchange (ETDEWEB)

    Bosanac, Ivan; Yamazaki, Haruka; Matsu-ura, Toru; Michikawa, Takayuki; Mikoshiba, Katsuhiko; Ikura, Mitsuhiko (U. of Texas-SMED)


    Binding of inositol 1,4,5-trisphosphate (IP{sub 3}) to the amino-terminal region of IP{sub 3} receptor promotes Ca{sup 2+} release from the endoplasmic reticulum. Within the amino terminus, the first 220 residues directly preceding the IP{sub 3} binding core domain play a key role in IP{sub 3} binding suppression and regulatory protein interaction. Here we present a crystal structure of the suppressor domain of the mouse type 1 IP{sub 3} receptor at 1.8 {angstrom}. Displaying a shape akin to a hammer, the suppressor region contains a Head subdomain forming the {beta}-trefoil fold and an Arm subdomain possessing a helix-turn-helix structure. The conserved region on the Head subdomain appeared to interact with the IP{sub 3} binding core domain and is in close proximity to the previously proposed binding sites of Homer, RACK1, calmodulin, and CaBP1. The present study sheds light onto the mechanism underlying the receptor's sensitivity to the ligand and its communication with cellular signaling proteins.

  14. cDNA heterogeneity suggests structural variants related to the high-affinity IgE receptor. (United States)

    Liu, F T; Albrandt, K; Robertson, M W


    The high-affinity IgE receptor present on mast cells and basophils is responsible for the IgE-mediated activation of these cells. The current model for this receptor depicts a four-subunit structure, alpha beta gamma 2. A cDNA for the alpha subunit was recently cloned and predicts a structure consisting of two homologous extracellular domains, a transmembrane segment, and a cytoplasmic tail. Using a synthetic oligonucleotide corresponding to the amino-terminal sequence of the alpha subunit, we identified a number of cDNA clones from a rat basophilic leukemia cell cDNA library. Nucleotide sequencing established four different forms of cDNA: one is nearly identical to the published cDNA; the second differs from the first in the 5' untranslated sequence; the other two forms use either one or the other of the 5'-end sequences as above and lack 163 base pairs in the region coding for the second extracellular domain. RNase protection analysis with radioactive RNA probes established the heterogeneity of rat basophilic leukemia cell mRNA with regard to both the 5' and the internal sequences. Our results suggest the existence of at least four different protein forms related to the alpha subunit of the high-affinity IgE receptor.

  15. Structure-function analysis of STRUBBELIG, an Arabidopsis atypical receptor-like kinase involved in tissue morphogenesis.

    Directory of Open Access Journals (Sweden)

    Prasad Vaddepalli

    Full Text Available Tissue morphogenesis in plants requires the coordination of cellular behavior across clonally distinct histogenic layers. The underlying signaling mechanisms are presently being unraveled and are known to include the cell surface leucine-rich repeat receptor-like kinase STRUBBELIG in Arabidopsis. To understand better its mode of action an extensive structure-function analysis of STRUBBELIG was performed. The phenotypes of 20 EMS and T-DNA-induced strubbelig alleles were assessed and homology modeling was applied to rationalize their possible effects on STRUBBELIG protein structure. The analysis was complemented by phenotypic, cell biological, and pharmacological investigations of a strubbelig null allele carrying genomic rescue constructs encoding fusions between various mutated STRUBBELIG proteins and GFP. The results indicate that STRUBBELIG accepts quite some sequence variation, reveal the biological importance for the STRUBBELIG N-capping domain, and reinforce the notion that kinase activity is not essential for its function in vivo. Furthermore, individual protein domains of STRUBBELIG cannot be related to specific STRUBBELIG-dependent biological processes suggesting that process specificity is mediated by factors acting together with or downstream of STRUBBELIG. In addition, the evidence indicates that biogenesis of a functional STRUBBELIG receptor is subject to endoplasmic reticulum-mediated quality control, and that an MG132-sensitive process regulates its stability. Finally, STRUBBELIG and the receptor-like kinase gene ERECTA interact synergistically in the control of internode length. The data provide genetic and molecular insight into how STRUBBELIG regulates intercellular communication in tissue morphogenesis.

  16. Crystal structure of human TWEAK in complex with the Fab fragment of a neutralizing antibody reveals insights into receptor binding.

    Directory of Open Access Journals (Sweden)

    Alfred Lammens

    Full Text Available The tumor necrosis factor-like weak inducer of apoptosis (TWEAK is a multifunctional cytokine playing a key role in tissue regeneration and remodeling. Dysregulation of TWEAK signaling is involved in various pathological processes like autoimmune diseases and cancer. The unique interaction with its cognate receptor Fn14 makes both ligand and receptor promising targets for novel therapeutics. To gain insights into this important signaling pathway, we determined the structure of soluble human TWEAK in complex with the Fab fragment of an antibody selected for inhibition of receptor binding. In the crystallized complex TWEAK is bound by three Fab fragments of the neutralizing antibody. Homology modeling shows that Fab binding overlaps with the putative Fn14 binding site of TWEAK. Docking of the Fn14 cysteine rich domain (CRD to that site generates a highly complementary interface with perfectly opposing charged and hydrophobic residues. Taken together the presented structure provides new insights into the biology of TWEAK and the TWEAK/Fn14 pathway, which will help to optimize the therapeutic strategy for treatment of related cancer types and autoimmune diseases.

  17. Structural insights of homotypic interaction domains in the ligand-receptor signal transduction of tumor necrosis factor (TNF) (United States)

    Park, Young-Hoon; Jeong, Mi Suk; Jang, Se Bok


    Several members of tumor necrosis factor receptor (TNFR) superfamily that these members activate caspase-8 from death-inducing signaling complex (DISC) in TNF ligand-receptor signal transduction have been identified. In the extrinsic pathway, apoptotic signal transduction is induced in death domain (DD) superfamily; it consists of a hexahelical bundle that contains 80 amino acids. The DD superfamily includes about 100 members that belong to four subfamilies: death domain (DD), caspase recruitment domain (CARD), pyrin domain (PYD), and death effector domain (DED). This superfamily contains key building blocks: with these blocks, multimeric complexes are formed through homotypic interactions. Furthermore, each DD-binding event occurs exclusively. The DD superfamily regulates the balance between death and survival of cells. In this study, the structures, functions, and unique features of DD superfamily members are compared with their complexes. By elucidating structural insights of DD superfamily members, we investigate the interaction mechanisms of DD domains; these domains are involved in TNF ligand-receptor signaling. These DD superfamily members play a pivotal role in the development of more specific treatments of cancer. [BMB Reports 2016; 49(3): 159-166] PMID:26615973

  18. Structure activity relationship of uridine 5′-diphosphate analogues at the human P2Y6 receptor (United States)

    Besada, Pedro; Shin, Dae Hong; Costanzi, Stefano; Ko, Hyojin; Mathé, Christophe; Gagneron, Julien; Gosselin, Gilles; Maddileti, Savitri; Harden, T. Kendall; Jacobsona, Kenneth A.


    The structure activity relationships and molecular modeling of the uracil nucleotide-activated P2Y6 receptor have been studied. A series of UDP analogues bearing substitutions of the ribose moiety, the uracil ring, and the diphosphate group was synthesized and assayed for activity at the human P2Y6 receptor. The uracil ring was modified at the 4-position, with the synthesis of 4-substituted-thiouridine-5′-diphosphate analogues, as well as at positions 3 and 5. The effect of modifications at the level of the phosphate chain was studied by preparing a cyclic 3′,5′-diphosphate analogue, a 3′-diphosphate analogue and several dinucleotide diphosphates. 5-Iodo-UDP 32 (EC50 0.15 μM) was equipotent to UDP, while substitutions of the 2′-hydroxyl (amino, azido) greatly reduce potency. 2- and 4-Thio analogues, 20 and 21, respectively, were also relatively potent in comparison to UDP. However, most other modifications greatly reduced potency. Molecular modeling indicates that the β-phosphate of 5′-UDP and analogs is essential for the establishment of electrostatic interactions with two of the three conserved cationic residues of the receptor. Among 4-thioether derivatives, a 4-ethylthio analogue 23 displayed an EC50 of 0.28 μM, indicative of favorable interactions predicted for a small 4-alkylthio moiety with the aromatic ring of Y33 in TM1. The activity of analogue 19 in which the ribose was substituted with a 2-oxabicyclohexane ring in a rigid (S) conformation (P= 126°, 1′-exo) was consistent with molecular modeling. These results provide a better understanding of molecular recognition at the P2Y6 receptor and will be helpful in designing selective and potent P2Y6 receptor ligands PMID:16942026

  19. Structural requirements for inducible shedding of the p55 tumor necrosis factor receptor

    DEFF Research Database (Denmark)

    Brakebusch, C; Varfolomeev, E E; Batkin, M


    Induced shedding of the p55 tumor necrosis factor receptor (p55-R) was previously shown to be independent of the amino acid sequence properties of the intracellular domain of this receptor. We now find it also independent of the sequence properties of the transmembrane domain and of the cysteine......-rich region that constitutes most of the extracellular domain of the receptor. The shedding is shown to depend solely on the sequence properties of a small region within the spacer that links the cysteine-rich region in the extracellular domain to the transmembrane domain. Detailed tests of effects......, however, by some mutations that seem to change the conformation of the spacer region. These findings suggest that a short amino acid sequence in the p55-R is essential and sufficient for its shedding and that the shedding is mediated either by a protease with limited sequence specificity or by several...

  20. Synthesis and structure-activity studies on acidic amino acids and related diacids as NMDA receptor ligands

    DEFF Research Database (Denmark)

    Johansen, T N; Frydenvang, Karla Andrea; Ebert, B;


    The 3-isoxazolol amino acids (S)-2-amino-3-(3-hydroxy-5-methyl-4- isoxazolyl)propionic acid [(S)-AMPA, 2] and (R,S)-2-amino-2-(3-hydroxy-5-methyl-4-isoxazolyl)acetic acid (AMAA, 5a) (Figure 1) are potent and specific agonists at the AMPA and N-methyl-D-aspartic acid (NMDA) subtypes, respectively......, of (S)-glutamic acid (1) receptors. A number of amino acids and diacids structurally related to AMAA were synthesized and tested electrophysiologically and in receptor-binding assays. The hydroxymethyl analogue 7c of AMAA was an NMDA agonist approximately equipotent with AMAA in the [3H...... by molecular mechanics calculations. Compound 7a possesses extra steric bulk and shows significant restriction of conformational flexibility compared to AMAA and 7c, which may be determining factors for the observed differences in biological activity. Although the nitrogen atom of quinolinic acid (6) has very...

  1. Structure-Activity Relationships and Identification of Optmized CC-Chemokine Receptor CCR1, 5, and 8 Metal-Ion Chelators

    DEFF Research Database (Denmark)

    Chalikiopoulos, Alexander; Thiele, Stefanie; Malmgaard-Clausen, Mikkel;


    Chemokine receptors are involved in trafficking of leukocytes and represent targets for autoimmune conditions, inflammatory diseases, viral infections, and cancer. We recently published CCR1, CCR8, and CCR5 agonists and positive modulators based on a three metal-ion chelator series: 2,2'-bipyridine...... bipyridine (23). The structure-activity relationships contribute to small-molecule drug development, and the novel chelators constitute valuable tools for studies of structural mechanisms for chemokine receptor activation....

  2. Assessment of structurally diverse philanthotoxin analogues for inhibitory activity on ionotropic glutamate receptor subtypes

    DEFF Research Database (Denmark)

    Frølund, Sidsel; Bella, Angelo; Kristensen, Anders Skov;


    -electrode voltage-clamp electrophysiology employing Xenopus laevis oocytes expressing GluA1(i) AMPA or GluN1/2A NMDA receptors. Several of the analogues showed significantly increased inhibition of the GluN1/2A NMDA receptor. Thus, an analogue containing N-(1-naphtyl)acetyl group showed an IC(50) value of 47 n......M. For the diamino acid-based analogues, the optimal spacer length between two N-acyl groups was determined, resulting in an analogue with an IC(50) value of 106 nM....

  3. Synthesis, structure-activity relationships, and characterization of novel nonsteroidal and selective androgen receptor modulators. (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


    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.

  4. The foot structure from the type 1 ryanodine receptor is required for functional coupling to store-operated channels. (United States)

    Sampieri, Alicia; Diaz-Muñoz, Mauricio; Antaramian, Anaid; Vaca, Luis


    In the present study we have explored structural determinants of the functional interaction between skeletal muscle ryanodine receptor (RyR1) and transient receptor potential channel 1 (TRPC1) channels expressed in Chinese hamster ovary cells. We have illustrated a functional interaction between TRPC1 channels and RyR1 for the regulation of store-operated calcium entry (SOCE) initiated after releasing calcium from a caffeine-sensitive intracellular calcium pool. RNA interference experiments directed to reduce the amount of TRPC1 protein indicate that RyR1 associates to at least two different types of store-operated channels (SOCs), one dependent and one independent of TRPC1. In contrast, bradykinin-induced SOCE is completely dependent on the presence of TRPC1 protein, as we have previously illustrated. Removing the foot structure from RyR1 results in normal caffeine-induced release of calcium from internal stores but abolishes the activation of SOCE, indicating that this structure is require for functional coupling to SOCs. The footless RyR1 protein shows a different cellular localization when compared with wild type RyR1. The later protein shows a higher percentage of colocalization with FM-464, a marker of plasma membrane. The implications of the foot structure for the functional and physical coupling to TRPC and SOCs is discussed.

  5. Endoglin structure and function - Determinants of endoglin phosphorylation by transforming growth factor-beta receptors

    NARCIS (Netherlands)

    Koleva, Rositsa I.; Conley, Barbara A.; Romero, Diana; Riley, Kristin S.; Marto, Jarrod A.; Lux, Andreas; Vary, Calvin P. H.


    Determination of the functional relationship between the transforming growth factor-beta(TGF beta) receptor proteins endoglin and ALK1 is essential to the understanding of the human vascular disease, hereditary hemorrhagic telangiectasia. TGF beta 1 caused recruitment of ALK1 into a complex with end

  6. Crystal structure of the β2 adrenergic receptor-Gs protein complex

    DEFF Research Database (Denmark)

    Rasmussen, Søren Gøgsig Faarup; DeVree, Brian T; Zou, Yaozhong;


    G protein-coupled receptors (GPCRs) are responsible for the majority of cellular responses to hormones and neurotransmitters as well as the senses of sight, olfaction and taste. The paradigm of GPCR signalling is the activation of a heterotrimeric GTP binding protein (G protein) by an agonist...

  7. Structural basis for receptor recognition of vitamin-B(12)-intrinsic factor complexes

    DEFF Research Database (Denmark)

    Andersen, Christian Brix Folsted; Madsen, Mette; Storm, Tina;


    Cobalamin (Cbl, vitamin B(12)) is a bacterial organic compound and an essential coenzyme in mammals, which take it up from the diet. This occurs by the combined action of the gastric intrinsic factor (IF) and the ileal endocytic cubam receptor formed by the 460-kilodalton (kDa) protein cubilin...

  8. Structural analysis of the receptors for granulocyte colony-stimulating factor on neutrophils

    Energy Technology Data Exchange (ETDEWEB)

    Hanazono, Y.; Hosoi, T.; Kuwaki, T.; Matsuki, S.; Miyazono, K.; Miyagawa, K.; Takaku, F. (Univ. of Tokyo (Japan))


    We investigated granulocyte colony-stimulating factor (G-CSF) receptors on neutrophils from three patients with chronic myelogenous leukemia (CML) in the chronic phase, in comparison with four normal volunteers. Because we experienced some difficulties in radioiodinating intact recombinant human G-CSF, we developed a new derivative of human G-CSF termed YPY-G-CSF. It was easy to iodinate this protein using the lactoperoxidase method because of two additional tyrosine residues, and its radioactivity was higher than that previously reported. The biological activity of YPY-G-CSF as G-CSF was fully retained. Scatchard analysis demonstrated that CML neutrophils had a single class of binding sites (1400 +/- 685/cell) with a dissociation constant (Kd) of 245 +/- 66 pM. The number of sites and Kd value of CML neutrophils were not significantly different from those of normal neutrophils (p greater than 0.9). Cross-linking studies revealed two specifically labeled bands of (125I)YPY-G-CSF-receptor complexes with apparent molecular masses of 160 and 110 kd on both normal and CML neutrophils. This is the first report describing two receptor proteins on neutrophils. According to the analyses of the proteolytic process of these cross-linked complexes and proteolytic mapping, we assume that alternative splicing or processing from a single gene may generate two distinct receptor proteins that bind specifically to G-CSF but have different fates in intracellular metabolism.

  9. A common allele in the oxytocin receptor gene (OXTR) impacts prosocial temperament and human hypothalamic-limbic structure and function. (United States)

    Tost, Heike; Kolachana, Bhaskar; Hakimi, Shabnam; Lemaitre, Herve; Verchinski, Beth A; Mattay, Venkata S; Weinberger, Daniel R; Meyer-Lindenberg, Andreas


    The evolutionarily highly conserved neuropeptide oxytocin is a key mediator of social and emotional behavior in mammals, including humans. A common variant (rs53576) in the oxytocin receptor gene (OXTR) has been implicated in social-behavioral phenotypes, such as maternal sensitivity and empathy, and with neuropsychiatric disorders associated with social impairment, but the intermediate neural mechanisms are unknown. Here, we used multimodal neuroimaging in a large sample of healthy human subjects to identify structural and functional alterations in OXTR risk allele carriers and their link to temperament. Activation and interregional coupling of the amygdala during the processing of emotionally salient social cues was significantly affected by genotype. In addition, evidence for structural alterations in key oxytocinergic regions emerged, particularly in the hypothalamus. These neural characteristics predicted lower levels of reward dependence, specifically in male risk allele carriers. Our findings identify sex-dependent mechanisms impacting the structure and function of hypothalamic-limbic circuits that are of potential clinical and translational significance.

  10. A structural and mutagenic blueprint for molecular recognition of strychnine and d-tubocurarine by different cys-loop receptors.

    Directory of Open Access Journals (Sweden)

    Marijke Brams


    Full Text Available Cys-loop receptors (CLR are pentameric ligand-gated ion channels that mediate fast excitatory or inhibitory transmission in the nervous system. Strychnine and d-tubocurarine (d-TC are neurotoxins that have been highly instrumental in decades of research on glycine receptors (GlyR and nicotinic acetylcholine receptors (nAChR, respectively. In this study we addressed the question how the molecular recognition of strychnine and d-TC occurs with high affinity and yet low specificity towards diverse CLR family members. X-ray crystal structures of the complexes with AChBP, a well-described structural homolog of the extracellular domain of the nAChRs, revealed that strychnine and d-TC adopt multiple occupancies and different ligand orientations, stabilizing the homopentameric protein in an asymmetric state. This introduces a new level of structural diversity in CLRs. Unlike protein and peptide neurotoxins, strychnine and d-TC form a limited number of contacts in the binding pocket of AChBP, offering an explanation for their low selectivity. Based on the ligand interactions observed in strychnine- and d-TC-AChBP complexes we performed alanine-scanning mutagenesis in the binding pocket of the human α1 GlyR and α7 nAChR and showed the functional relevance of these residues in conferring high potency of strychnine and d-TC, respectively. Our results demonstrate that a limited number of ligand interactions in the binding pocket together with an energetic stabilization of the extracellular domain are key to the poor selective recognition of strychnine and d-TC by CLRs as diverse as the GlyR, nAChR, and 5-HT(3R.

  11. Genetic and structural basis for selection of a ubiquitous T cell receptor deployed in Epstein-Barr virus infection.

    Directory of Open Access Journals (Sweden)

    John J Miles

    Full Text Available Despite the ∼10(18 αβ T cell receptor (TCR structures that can be randomly manufactured by the human thymus, some surface more frequently than others. The pinnacles of this distortion are public TCRs, which exhibit amino acid-identical structures across different individuals. Public TCRs are thought to result from both recombinatorial bias and antigen-driven selection, but the mechanisms that underlie inter-individual TCR sharing are still largely theoretical. To examine this phenomenon at the atomic level, we solved the co-complex structure of one of the most widespread and numerically frequent public TCRs in the human population. The archetypal AS01 public TCR recognizes an immunodominant BMLF1 peptide, derived from the ubiquitous Epstein-Barr virus, bound to HLA-A*0201. The AS01 TCR was observed to dock in a diagonal fashion, grasping the solvent exposed peptide crest with two sets of complementarity-determining region (CDR loops, and was fastened to the peptide and HLA-A*0201 platform with residue sets found only within TCR genes biased in the public response. Computer simulations of a random V(DJ recombination process demonstrated that both TCRα and TCRβ amino acid sequences could be manufactured easily, thereby explaining the prevalence of this receptor across different individuals. Interestingly, the AS01 TCR was encoded largely by germline DNA, indicating that the TCR loci already comprise gene segments that specifically recognize this ancient pathogen. Such pattern recognition receptor-like traits within the αβ TCR system further blur the boundaries between the adaptive and innate immune systems.

  12. Crystal structure of R-spondin 2 in complex with the ectodomains of its receptors LGR5 and ZNRF3. (United States)

    Zebisch, Matthias; Jones, E Yvonne


    The four secreted R-spondin (Rspo1-4) proteins of vertebrates function as stem cell growth factors and potentiate canonical Wnt signalling. Rspo proteins act by cross-linking members of two cell surface receptor families, complexing the stem cell markers LGR4-6 with the Frizzled-specific E3 ubiquitin ligases ZNRF3/RNF43. The consequent internalisation of the ternary LGR-Rspo-E3 complex removes the E3 ligase activity, which otherwise targets the Wnt receptor Frizzled for degradation, and thus enhances Wnt signalling. Multiple combinations of LGR4-6, Rspo1-4 and ZNRF3/RNF43 are possible, implying the existence of generic interaction determinants, but also of specific differences in complex architecture and activity. We present here a high resolution crystal structure of an ectodomain variant of human LGR5 (hLGR5ecto) complexed with a signalling competent fragment of mouse Rspo2 (mRspo2Fu1-Fu2). The structure shows that the particularly potent Rspo2 ligand engages LGR5 in a fashion almost identical to that reported for hRSPO1. Comparison of our hLGR5ecto structure with previously published structures highlights a surprising plasticity of the LGR ectodomains, characterised by a nearly 9° or larger rotation of the N-terminal half of the horseshoe-like fold relative to the C-terminal half. We also report a low resolution hLGR5-mRspo2Fu1-Fu2-mZNRF3ecto ternary complex structure. This crystal structure confirms our previously suggested hypothesis, showing that Rspo proteins cross-link LGRs and ZNRF3 into a 2:2:2 complex, whereas a 1:1:1 complex is formed with RNF43.

  13. Advances in the Development and Application of Computational Methodologies for Structural Modeling of G-Protein Coupled Receptors (United States)

    Mobarec, Juan Carlos


    Background Despite the large amount of experimental data accumulated in the past decade on G-protein coupled receptor (GPCR) structure and function, understanding of the molecular mechanisms underlying GPCR signaling is still far from being complete, thus impairing the design of effective and selective pharmaceuticals. Objective Understanding of GPCR function has been challenged even further by more recent experimental evidence that several of these receptors are organized in the cell membrane as homo- or hetero-oligomers, and that they may exhibit unique pharmacological properties. Given the complexity of these new signaling systems, researcher’s efforts are turning increasingly to molecular modeling, bioinformatics and computational simulations for mechanistic insights of GPCR functional plasticity. Methods We review here current advances in the development and application of computational approaches to improve prediction of GPCR structure and dynamics, thus enhancing current understanding of GPCR signaling. Results/Conclusions Models resulting from use of these computational approaches further supported by experiments are expected to help elucidate the complex allosterism that propagates through GPCR complexes, ultimately aiming at successful structure-based rational drug design. PMID:19672320

  14. Using Molecular Initiating Events to Develop a Structural Alert Based Screening Workflow for Nuclear Receptor Ligands Associated with Hepatic Steatosis. (United States)

    Mellor, Claire L; Steinmetz, Fabian P; Cronin, Mark T D


    In silico models are essential for the development of integrated alternative methods to identify organ level toxicity and lead toward the replacement of animal testing. These models include (quantitative) structure-activity relationships ((Q)SARs) and, importantly, the identification of structural alerts associated with defined toxicological end points. Structural alerts are able both to predict toxicity directly and assist in the formation of categories to facilitate read-across. They are particularly important to decipher the myriad mechanisms of action that result in organ level toxicity. The aim of this study was to develop novel structural alerts for nuclear receptor (NR) ligands that are associated with inducing hepatic steatosis and to show the vast number of existing data that are available. Current knowledge on NR agonists was extended with data from the ChEMBL database (12,713 chemicals in total) of bioactive molecules and from studying NR ligand-binding interactions within the protein database (PDB, 624 human NR structure files). A computational structural alert based workflow was developed using KNIME from these data using molecular fragments and other relevant chemical features. In total, 214 structural features were recorded computationally as SMARTS strings, and therefore, they can be used for grouping and screening during drug development and hazard assessment and provide knowledge to anchor adverse outcome pathways (AOPs) via their molecular initiating events (MIEs).

  15. Molecular mechanism of agonism and inverse agonism in the melanocortin receptors: Zn(2+) as a structural and functional probe

    DEFF Research Database (Denmark)

    Holst, Birgitte; Schwartz, Thue W


    -ion Zn(2+) increases the signaling activity of at least the MC1 and MC4 receptors in three distinct ways: (1). by directly functioning as an agonist; (2). by potentiating the action of the endogenous agonist; and (3). by inhibiting the binding of the endogenous inverse agonist. Structurally the MC...... extracellular loop 2 is ultrashort because TM-IV basically connects directly into TM-V, whereas extracellular loop 3 appears to be held in a particular, constrained conformation by a putative, internal disulfide bridge. The interaction mode for the small and well-defined zinc-ion between a third, free Cys...

  16. Structure-Based Prediction of Subtype Selectivity of Histamine H3 Receptor Selective Antagonists in Clinical Trials

    DEFF Research Database (Denmark)

    Kim, Soo-Kyung; Fristrup, Peter; Abrol, Ravinder


    applications, including treatment of Alzheimer’s disease, attention deficit hyperactivity disorder (ADHD), epilepsy, and obesity.(1) However, many of these drug candidates cause undesired side effects through the cross-reactivity with other histamine receptor subtypes. In order to develop improved selectivity......–5–6 and the aliphatic ring located in TMs 2–3–7. These 3D structures for all four HRs should help guide the rational design of novel drugs for the subtype selective antagonists and agonists with reduced side effects....

  17. Interaction of structure-specific and promiscuous G-protein-coupled receptors mediates small-molecule signaling in Caenorhabditis elegans. (United States)

    Park, Donha; O'Doherty, Inish; Somvanshi, Rishi K; Bethke, Axel; Schroeder, Frank C; Kumar, Ujendra; Riddle, Donald L


    A chemically diverse family of small-molecule signals, the ascarosides, control developmental diapause (dauer), olfactory learning, and social behaviors of the nematode model organism, Caenorhabditis elegans. The ascarosides act upstream of conserved signaling pathways, including the insulin, TGF-β, serotonin, and guanylyl cyclase pathways; however, the sensory processes underlying ascaroside function are poorly understood. Because ascarosides often are multifunctional and show strongly synergistic effects, characterization of their receptors will be essential for understanding ascaroside biology and may provide insight into molecular mechanisms that produce synergistic outcomes in small-molecule sensing. Based on DAF-8 immunoprecipitation, we here identify two G-protein-coupled receptors, DAF-37 and DAF-38, which cooperatively mediate ascaroside perception. daf-37 mutants are defective in all responses to ascr#2, one of the most potent dauer-inducing ascarosides, although this mutant responds normally to other ascarosides. In contrast, daf-38 mutants are partially defective in responses to several different ascarosides. Through cell-specific overexpression, we show that DAF-37 regulates dauer when expressed in ASI neurons and adult behavior when expressed in ASK neurons. Using a photoaffinity-labeled ascr#2 probe and amplified luminescence assays (AlphaScreen), we demonstrate that ascr#2 binds to DAF-37. Photobleaching fluorescent energy transfer assays revealed that DAF-37 and DAF-38 form heterodimers, and we show that heterodimerization strongly increases cAMP inhibition in response to ascr#2. These results suggest that that the ascarosides' intricate signaling properties result in part from the interaction of highly structure-specific G-protein-coupled receptors such as DAF-37 with more promiscuous G-protein-coupled receptors such as DAF-38.

  18. Interaction of structure-specific and promiscuous G-protein–coupled receptors mediates small-molecule signaling in Caenorhabditis elegans (United States)

    Park, Donha; O'Doherty, Inish; Somvanshi, Rishi K.; Bethke, Axel; Schroeder, Frank C.; Kumar, Ujendra; Riddle, Donald L.


    A chemically diverse family of small-molecule signals, the ascarosides, control developmental diapause (dauer), olfactory learning, and social behaviors of the nematode model organism, Caenorhabditis elegans. The ascarosides act upstream of conserved signaling pathways, including the insulin, TGF-β, serotonin, and guanylyl cyclase pathways; however, the sensory processes underlying ascaroside function are poorly understood. Because ascarosides often are multifunctional and show strongly synergistic effects, characterization of their receptors will be essential for understanding ascaroside biology and may provide insight into molecular mechanisms that produce synergistic outcomes in small-molecule sensing. Based on DAF-8 immunoprecipitation, we here identify two G-protein–coupled receptors, DAF-37 and DAF-38, which cooperatively mediate ascaroside perception. daf-37 mutants are defective in all responses to ascr#2, one of the most potent dauer-inducing ascarosides, although this mutant responds normally to other ascarosides. In contrast, daf-38 mutants are partially defective in responses to several different ascarosides. Through cell-specific overexpression, we show that DAF-37 regulates dauer when expressed in ASI neurons and adult behavior when expressed in ASK neurons. Using a photoaffinity-labeled ascr#2 probe and amplified luminescence assays (AlphaScreen), we demonstrate that ascr#2 binds to DAF-37. Photobleaching fluorescent energy transfer assays revealed that DAF-37 and DAF-38 form heterodimers, and we show that heterodimerization strongly increases cAMP inhibition in response to ascr#2. These results suggest that that the ascarosides' intricate signaling properties result in part from the interaction of highly structure-specific G-protein–coupled receptors such as DAF-37 with more promiscuous G-protein–coupled receptors such as DAF-38. PMID:22665789

  19. Structural Stereochemistry of Androstene Hormones Determines Interactions with Human Androgen, Estrogen, and Glucocorticoid Receptors

    Directory of Open Access Journals (Sweden)

    Thomas L. Shaak


    Full Text Available DHEA, 17α-AED, 17β-AED, and 17β-AET exhibit strong biological activity that has been attributed to androgenic, estrogenic, or antiglucocorticoid activity in vivo and in vitro. This study compared DHEA, 17α-AED, 17β-AED, and 17β-AET for their ability to activate the human AR, ER, and GR and determine the relative androgenicity, estrogenicity, and glucocorticoid activity. The results show that, at the receptor level, these androstene hormones are weak AR and even weaker ER activators. Direct androstene hormone activation of the human AR, ERα, and ERβ may not be essential for their biological function. Similarly, these hormones indirectly activated the human GR, only in the presence of high dexamethasone concentrations. These results underscore the major difference between androstene hormone interactions with these nuclear receptors and their biological effects.

  20. Structure of metabotropic glutamate receptor C-terminal domains in contact with interacting proteins


    Enz, Ralf


    Metabotropic glutamate receptors (mGluRs) regulate intracellular signal pathways that control several physiological tasks, including neuronal excitability, learning, and memory. This is achieved by the formation of synaptic signal complexes, in which mGluRs assemble with functionally related proteins such as enzymes, scaffolds, and cytoskeletal anchor proteins. Thus, mGluR associated proteins actively participate in the regulation of glutamatergic neurotransmission. Importantly, dysfunction o...

  1. Discovery of non-LBD inhibitor for androgen receptor by structure-guide design. (United States)

    Ryu, Byung Jun; Kim, Nakjeong; Kim, Jun Tae; Koo, Tae-Sung; Yoo, Sung-Eun; Jeong, Seo Hee; Kim, Seong Hwan; Kang, Nam Sook


    In this study, we synthesized the BF-3 binding small molecules, a series of pyridazinone-based compounds, as a novel class of non-LBP antiandrogens for treating prostate cancer by inhibiting androgen receptor. The new class compound was discovered to inhibitor the viability of AR-dependent human prostate LNCap cells and AR activity combining with the computational method. It showed a good physicochemical and PK property.

  2. Structural aspects of molecular recognition in the immune system. Part II: Pattern recognition receptors



    The vertebrate immune system uses pattern recognition receptors (PRRs) to detect a large variety of molecular signatures (pathogen-associated molecular patterns, PAMPs) from a broad range of different invading pathogens. The PAMPs range in size from relatively small molecules, to others of intermediate size such as bacterial lipopolysaccharide, lipopeptides, and oligosaccharides, to macromolecules such as viral DNA, RNA, and pathogen-derived proteins such as flagellin. Underlying this functio...

  3. Structural Determinants for the Binding of Morphinan Agonists to the μ-Opioid Receptor.

    Directory of Open Access Journals (Sweden)

    Xiaojing Cong

    Full Text Available Atomistic descriptions of the μ-opioid receptor (μOR noncovalently binding with two of its prototypical morphinan agonists, morphine (MOP and hydromorphone (HMP, are investigated using molecular dynamics (MD simulations. Subtle differences between the binding modes and hydration properties of MOP and HMP emerge from the calculations. Alchemical free energy perturbation calculations show qualitative agreement with in vitro experiments performed in this work: indeed, the binding free energy difference between MOP and HMP computed by forward and backward alchemical transformation is 1.2±1.1 and 0.8±0.8 kcal/mol, respectively, to be compared with 0.4±0.3 kcal/mol from experiment. Comparison with an MD simulation of μOR covalently bound with the antagonist β-funaltrexamine hints to agonist-induced conformational changes associated with an early event of the receptor's activation: a shift of the transmembrane helix 6 relative to the transmembrane helix 3 and a consequent loss of the key R165-T279 interhelical hydrogen bond. This finding is consistent with a previous proposal suggesting that the R165-T279 hydrogen bond between these two helices indicates an inactive receptor conformation.

  4. Interleukin 9 and its receptor: an overview of structure and function. (United States)

    Demoulin, J B; Renauld, J C


    Interleukin-9 (IL-9) is a multifunctional cytokine produced by activated TH2 clones in vitro and during TH2-like T cell responses in vivo. Although IL-9 was initially described as a T cell growth factor, its role in T cell responses is still unclear. While freshly isolated normal T cells do not respond to IL-9, this cytokine induces the proliferation of murine T cell lymphomas in vitro, and in vivo overexpression of IL-9 results in the development of thymic lymphomas. In the human, the existence of an IL-9 mediated autocrine loop has been suggested for some malignancies such as Hodgkin's disease. Various observations indicate that IL-9 is actively involved in mast cells responses by inducing the proliferation and differentiation of these cells. Other potential biological targets for IL-9 include B lymphocytes, and hematopoietic progenitors, for which higher responses were observed with foetal or transformed cells as compared to normal adult progenitors. The IL-9 receptor is a member of the hemopoietin receptor superfamily and interacts with the gamma chain of the IL-2 receptor for signaling. Signal transduction studies have stressed the role of the Jak-STAT pathway in various IL-9 bioactivities, whereas the 4PS/IRS2 adaptor protein might also play a significant role in IL-9 signaling.

  5. The serotonin receptor 7 and the structural plasticity of brain circuits

    Directory of Open Access Journals (Sweden)

    Floriana eVolpicelli


    Full Text Available Serotonin (5-hydroxytryptamine, 5-HT modulates numerous physiological processes in the nervous system. Together with its function as neurotrasmitter, 5-HT regulates neurite outgrowth, dendritic spine shape and density, growth cone motility and synapse formation during development. In the mammalian brain 5-HT innervation is virtually ubiquitous and the diversity and specificity of its signaling and function arise from at least 20 different receptors, grouped in 7 classes. Here we will focus on the role 5-HT7 receptor (5-HT7R in the correct establishment of neuronal cytoarchitecture during development, as also suggested by its involvement in several neurodevelopmental disorders. The emerging picture shows that this receptor is a key player contributing not only to shape brain networks during development but also to remodel neuronal wiring in the mature brain, thus controlling cognitive and emotional responses. The activation of 5-HT7R might be one of the mechanisms underlying the ability of the CNS to respond to different stimuli by modulation of its circuit configuration.

  6. Structure of the human angiotensin II type 1 (AT1) receptor bound to angiotensin II from multiple chemoselective photoprobe contacts reveals a unique peptide binding mode. (United States)

    Fillion, Dany; Cabana, Jérôme; Guillemette, Gaétan; Leduc, Richard; Lavigne, Pierre; Escher, Emanuel


    Breakthroughs in G protein-coupled receptor structure determination based on crystallography have been mainly obtained from receptors occupied in their transmembrane domain core by low molecular weight ligands, and we have only recently begun to elucidate how the extracellular surface of G protein-coupled receptors (GPCRs) allows for the binding of larger peptide molecules. In the present study, we used a unique chemoselective photoaffinity labeling strategy, the methionine proximity assay, to directly identify at physiological conditions a total of 38 discrete ligand/receptor contact residues that form the extracellular peptide-binding site of an activated GPCR, the angiotensin II type 1 receptor. This experimental data set was used in homology modeling to guide the positioning of the angiotensin II (AngII) peptide within several GPCR crystal structure templates. We found that the CXC chemokine receptor type 4 accommodated the results better than the other templates evaluated; ligand/receptor contact residues were spatially grouped into defined interaction clusters with AngII. In the resulting receptor structure, a β-hairpin fold in extracellular loop 2 in conjunction with two extracellular disulfide bridges appeared to open and shape the entrance of the ligand-binding site. The bound AngII adopted a somewhat vertical binding mode, allowing concomitant contacts across the extracellular surface and deep within the transmembrane domain core of the receptor. We propose that such a dualistic nature of GPCR interaction could be well suited for diffusible linear peptide ligands and a common feature of other peptidergic class A GPCRs.

  7. Gene structure and expression of serotonin receptor HTR2C in hypothalamic samples from infanticidal and control sows

    Directory of Open Access Journals (Sweden)

    Quilter Claire R


    Full Text Available Abstract Background The serotonin pathways have been implicated in behavioural phenotypes in a number of species, including human, rat, mouse, dog and chicken. Components of the pathways, including the receptors, are major targets for drugs used to treat a variety of physiological and psychiatric conditions in humans. In our previous studies we have identified genetic loci potentially contributing to maternal infanticide in pigs, which includes a locus on the porcine X chromosome long arm. The serotonin receptor HTR2C maps to this region, and is therefore an attractive candidate for further study based on its function and its position in the genome. Results In this paper we describe the structure of the major transcripts produced from the porcine HTR2C locus using cDNA prepared from porcine hypothalamic and pooled total brain samples. We have confirmed conservation of sites altered by RNA editing in other mammalian species, and identified polymorphisms in the gene sequence. Finally, we have analysed expression and editing of HTR2C in hypothalamus samples from infanticidal and control animals. Conclusions The results confirm that although the expression of the long transcriptional variant of HTR2C is raised in infanticidal animals, the overall patterns of editing in the hypothalamus are similar between the two states. Sequences associated with the cDNA and genomic structures of HTR2C reported in this paper are deposited in GenBank under accession numbers FR720593, FR720594 and FR744452.

  8. Structure-activity relationships of substituted 1H-indole-2-carboxamides as CB1 receptor allosteric modulators. (United States)

    Nguyen, Thuy; German, Nadezhda; Decker, Ann M; Li, Jun-Xu; Wiley, Jenny L; Thomas, Brian F; Kenakin, Terry P; Zhang, Yanan


    A series of substituted 1H-indole-2-carboxamides structurally related to compounds Org27569 (1), Org29647 (2) and Org27759 (3) were synthesized and evaluated for CB1 allosteric modulating activity in calcium mobilization assays. Structure-activity relationship studies showed that the modulation potency of this series at the CB1 receptor was enhanced by the presence of a diethylamino group at the 4-position of the phenyl ring, a chloro or fluoro group at the C5 position and short alkyl groups at the C3 position on the indole ring. The most potent compound (45) had an IC₅₀ value of 79 nM which is ∼2.5 and 10 fold more potent than the parent compounds 3 and 1, respectively. These compounds appeared to be negative allosteric modulators at the CB1 receptor and dose-dependently reduced the Emax of agonist CP55,940. These analogs may provide the basis for further optimization and use of CB1 allosteric modulators.

  9. Biophysical and structural investigation of bacterially expressed and engineered CCR5, a G protein-coupled receptor. (United States)

    Wiktor, Maciej; Morin, Sébastien; Sass, Hans-Jürgen; Kebbel, Fabian; Grzesiek, Stephan


    The chemokine receptor CCR5 belongs to the class of G protein-coupled receptors. Besides its role in leukocyte trafficking, it is also the major HIV-1 coreceptor and hence a target for HIV-1 entry inhibitors. Here, we report Escherichia coli expression and a broad range of biophysical studies on E. coli-produced CCR5. After systematic screening and optimization, we obtained 10 mg of purified, detergent-solubilized, folded CCR5 from 1L culture in a triply isotope-labeled ((2)H/(15)N/(13)C) minimal medium. Thus the material is suitable for NMR spectroscopic studies. The expected α-helical secondary structure content is confirmed by circular dichroism spectroscopy. The solubilized CCR5 is monodisperse and homogeneous as judged by transmission electron microscopy. Interactions of CCR5 with its ligands, RANTES and MIP-1β were assessed by surface plasmon resonance yielding K(D) values in the nanomolar range. Using size exclusion chromatography, stable monomeric CCR5 could be isolated. We show that cysteine residues affect both the yield and oligomer distribution of CCR5. HSQC spectra suggest that the transmembrane domains of CCR5 are in equilibrium between several conformations. In addition we present a model of CCR5 based on the crystal structure of CXCR4 as a starting point for protein engineering.

  10. Biophysical and structural investigation of bacterially expressed and engineered CCR5, a G protein-coupled receptor

    Energy Technology Data Exchange (ETDEWEB)

    Wiktor, Maciej; Morin, Sebastien; Sass, Hans-Juergen [University of Basel, Focal Area Structural Biology and Biophysics, Biozentrum (Switzerland); Kebbel, Fabian [University of Basel, Center for Cellular Imaging and NanoAnalytics (C-CINA), Biozentrum (Switzerland); Grzesiek, Stephan, E-mail: [University of Basel, Focal Area Structural Biology and Biophysics, Biozentrum (Switzerland)


    The chemokine receptor CCR5 belongs to the class of G protein-coupled receptors. Besides its role in leukocyte trafficking, it is also the major HIV-1 coreceptor and hence a target for HIV-1 entry inhibitors. Here, we report Escherichia coli expression and a broad range of biophysical studies on E. coli-produced CCR5. After systematic screening and optimization, we obtained 10 mg of purified, detergent-solubilized, folded CCR5 from 1L culture in a triply isotope-labeled ({sup 2}H/{sup 15}N/{sup 13}C) minimal medium. Thus the material is suitable for NMR spectroscopic studies. The expected {alpha}-helical secondary structure content is confirmed by circular dichroism spectroscopy. The solubilized CCR5 is monodisperse and homogeneous as judged by transmission electron microscopy. Interactions of CCR5 with its ligands, RANTES and MIP-1{beta} were assessed by surface plasmon resonance yielding K{sub D} values in the nanomolar range. Using size exclusion chromatography, stable monomeric CCR5 could be isolated. We show that cysteine residues affect both the yield and oligomer distribution of CCR5. HSQC spectra suggest that the transmembrane domains of CCR5 are in equilibrium between several conformations. In addition we present a model of CCR5 based on the crystal structure of CXCR4 as a starting point for protein engineering.

  11. CXC chemokine CXCL12 and its receptor CXCR4 in tree shrews (Tupaia belangeri): structure, expression and function. (United States)

    Chen, Guiyuan; Wang, Wei; Meng, Shengke; Zhang, Lichao; Wang, Wenxue; Jiang, Zongmin; Yu, Min; Cui, Qinghua; Li, Meizhang


    Chemokines are small secreted proteins functionally involved in the immune system's regulation of lymphocyte migration across numerous mammalian species. Given its growing popularity in immunological models, we investigated the structure and function of chemokine CXCL12 protein in tree shrews. We found that CXCL12 and its receptor CXCR4 in tree shrew had structural similarities to their homologous human proteins. Phylogenetic analysis supports the view that tree shrew is evolutionarily-close to the primates. Our results also showed that the human recombinant CXCL12 protein directly enhanced the migration of tree shrew's lymphocytes in vitro, while AMD3100 enhanced the mobilization of hematopoietic progenitor cells (HPCs) from bone marrow into peripheral blood in tree shrew in vivo. Collectively, these findings suggested that chemokines in tree shrews may play the same or similar roles as those in humans, and that the tree shrew is a viable animal model for studying human immunological diseases.

  12. CXC chemokine CXCL12 and its receptor CXCR4 in tree shrews (Tupaia belangeri: structure, expression and function.

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    Guiyuan Chen

    Full Text Available Chemokines are small secreted proteins functionally involved in the immune system's regulation of lymphocyte migration across numerous mammalian species. Given its growing popularity in immunological models, we investigated the structure and function of chemokine CXCL12 protein in tree shrews. We found that CXCL12 and its receptor CXCR4 in tree shrew had structural similarities to their homologous human proteins. Phylogenetic analysis supports the view that tree shrew is evolutionarily-close to the primates. Our results also showed that the human recombinant CXCL12 protein directly enhanced the migration of tree shrew's lymphocytes in vitro, while AMD3100 enhanced the mobilization of hematopoietic progenitor cells (HPCs from bone marrow into peripheral blood in tree shrew in vivo. Collectively, these findings suggested that chemokines in tree shrews may play the same or similar roles as those in humans, and that the tree shrew is a viable animal model for studying human immunological diseases.

  13. Cloning and localization of two multigene receptor families in goldfish olfactory epithelium (United States)

    Cao, Yanxiang; Oh, Bryan C.; Stryer, Lubert


    Goldfish reproduction is coordinated by pheromones that are released by ovulating females and detected by males. Two highly potent pheromones, a dihydroxyprogesterone and a prostaglandin, previously have been identified, and their effects on goldfish behavior have been studied in depth. We have cloned goldfish olfactory epithelium cDNAs belonging to two multigene G-protein coupled receptor families as a step toward elucidating the molecular basis of pheromone recognition. One gene family (GFA) consists of homologs of putative odorant receptors (≈320 residues) found in the olfactory epithelium of other fish and mammals. The other family (GFB) consists of homologs of putative pheromone receptors found in the vomeronasal organ (VNO) of mammals and also in the nose of pufferfish. GFB receptors (≈840 residues) are akin to the V2R family of VNO receptors, which possess a large extracellular N-terminal domain and are homologs of calcium-sensing and metabotropic glutamate receptors. In situ hybridization showed that the two families of goldfish receptors are differentially expressed in the olfactory epithelium. GFB mRNA is abundant in rather compact cells whose nuclei are near the apical surface. In contrast, GFA mRNA is found in elongated cells whose nuclei are positioned deeper in the epithelium. Our findings support the hypothesis that the separate olfactory organ and VNO of terrestrial vertebrates arose in evolution by the segregation of distinct classes of neurons that were differentially positioned in the olfactory epithelium of a precursor aquatic vertebrate. PMID:9751777

  14. Structure-based stabilization of HIV-1 gp120 enhances humoral immune responses to the induced co-receptor binding site.

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    Barna Dey


    Full Text Available The human immunodeficiency virus type 1 (HIV-1 exterior envelope glycoprotein, gp120, possesses conserved binding sites for interaction with the primary virus receptor, CD4, and also for the co-receptor, generally CCR5. Although gp120 is a major target for virus-specific neutralizing antibodies, the gp120 variable elements and its malleable nature contribute to evasion of effective host-neutralizing antibodies. To understand the conformational character and immunogenicity of the gp120 receptor binding sites as potential vaccine targets, we introduced structure-based modifications to stabilize gp120 core proteins (deleted of the gp120 major variable regions into the conformation recognized by both receptors. Thermodynamic analysis of the re-engineered core with selected ligands revealed significant stabilization of the receptor-binding regions. Stabilization of the co-receptor-binding region was associated with a marked increase in on-rate of ligand binding to this site as determined by surface plasmon resonance. Rabbit immunization studies showed that the conformational stabilization of core proteins, along with increased ligand affinity, was associated with strikingly enhanced humoral immune responses against the co-receptor-binding site. These results demonstrate that structure-based approaches can be exploited to stabilize a conformational site in a large functional protein to enhance immunogenic responses specific for that region.

  15. Crystal Structure of the Receptor-Binding Domain of Botulinum Neurotoxin Type HA, Also Known as Type FA or H

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    Guorui Yao


    Full Text Available Botulinum neurotoxins (BoNTs, which have been exploited as cosmetics and muscle-disorder treatment medicines for decades, are well known for their extreme neurotoxicity to humans. They pose a potential bioterrorism threat because they cause botulism, a flaccid muscular paralysis-associated disease that requires immediate antitoxin treatment and intensive care over a long period of time. In addition to the existing seven established BoNT serotypes (BoNT/A–G, a new mosaic toxin type termed BoNT/HA (aka type FA or H was reported recently. Sequence analyses indicate that the receptor-binding domain (HC of BoNT/HA is ~84% identical to that of BoNT/A1. However, BoNT/HA responds differently to some potent BoNT/A-neutralizing antibodies (e.g., CR2 that target the HC. Therefore, it raises a serious concern as to whether BoNT/HA poses a new threat to our biosecurity. In this study, we report the first high-resolution crystal structure of BoNT/HA-HC at 1.8 Å. Sequence and structure analyses reveal that BoNT/HA and BoNT/A1 are different regarding their binding to cell-surface receptors including both polysialoganglioside (PSG and synaptic vesicle glycoprotein 2 (SV2. Furthermore, the new structure also provides explanations for the ~540-fold decreased affinity of antibody CR2 towards BoNT/HA compared to BoNT/A1. Taken together, these new findings advance our understanding of the structure and function of this newly identified toxin at the molecular level, and pave the way for the future development of more effective countermeasures.

  16. Evolutionary diversification of retinoic acid receptor ligand-binding pocket structure by molecular tinkering (United States)

    Gutierrez-Mazariegos, Juliana; Nadendla, Eswar Kumar; Studer, Romain A.; Alvarez, Susana; de Lera, Angel R.; Kuraku, Shigehiro; Bourguet, William; Laudet, Vincent


    Whole genome duplications (WGDs) have been classically associated with the origin of evolutionary novelties and the so-called duplication–degeneration–complementation model describes the possible fates of genes after duplication. However, how sequence divergence effectively allows functional changes between gene duplicates is still unclear. In the vertebrate lineage, two rounds of WGDs took place, giving rise to paralogous gene copies observed for many gene families. For the retinoic acid receptors (RARs), for example, which are members of the nuclear hormone receptor (NR) superfamily, a unique ancestral gene has been duplicated resulting in three vertebrate paralogues: RARα, RARβ and RARγ. It has previously been shown that this single ancestral RAR was neofunctionalized to give rise to a larger substrate specificity range in the RARs of extant jawed vertebrates (also called gnathostomes). To understand RAR diversification, the members of the cyclostomes (lamprey and hagfish), jawless vertebrates representing the extant sister group of gnathostomes, provide an intermediate situation and thus allow the characterization of the evolutionary steps that shaped RAR ligand-binding properties following the WGDs. In this study, we assessed the ligand-binding specificity of cyclostome RARs and found that their ligand-binding pockets resemble those of gnathostome RARα and RARβ. In contrast, none of the cyclostome receptors studied showed any RARγ-like specificity. Together, our results suggest that cyclostome RARs cover only a portion of the specificity repertoire of the ancestral gnathostome RARs and indicate that the establishment of ligand-binding specificity was a stepwise event. This iterative process thus provides a rare example for the diversification of receptor–ligand interactions of NRs following WGDs. PMID:27069642

  17. The structure and function of vertebrate fibroblast growth factor receptor 1. (United States)

    Groth, Casper; Lardelli, Michael


    The vertebrate fibroblast growth factor receptor 1 (FGFR1) is alternatively spliced generating multiple splice variants that are differentially expressed during embryo development and in the adult body. The restricted expression patterns of FGFR1 isoforms, together with differential expression and binding of specific ligands, leads to activation of common FGFR1 signal transduction pathways, but may result in distinctively different biological responses as a result of differences in cellular context. FGFR1 isoforms are also present in the nucleus in complex with various fibroblast growth factors where they function to regulate transcription of target genes.

  18. Glucocorticoid receptor gene haplotype structure and steroid therapy outcome in IBD patients

    Institute of Scientific and Technical Information of China (English)

    Jessica; Mwinyi; Christa; Wenger; Jyrki; J; Eloranta; Gerd; A; Kullak-Ublick


    AIM: To study whether the glucocorticoid receptor (GR/ NR3C1) gene haplotypes influence the steroid therapy outcome in inflammatory bowel disease (IBD). METHODS: We sequenced all coding exons and flanking intronic sequences of the NR3C1 gene in 181 IBD patients, determined the single nucleotide polymorphisms, and predicted the NR3C1 haplotypes. Furthermore, we investigated whether certain NR3C1 haplotypes are significantly associated with steroid therapy outcomes. RESULTS: We detected 13 NR3C1 variants, whi...

  19. Support for 5-HT2C receptor functional selectivity in vivo utilizing structurally diverse, selective 5-HT2C receptor ligands and the 2,5-dimethoxy-4-iodoamphetamine elicited head-twitch response model. (United States)

    Canal, Clinton E; Booth, Raymond G; Morgan, Drake


    There are seemingly conflicting data in the literature regarding the role of serotonin (5-HT) 5-HT2C receptors in the mouse head-twitch response (HTR) elicited by the hallucinogenic 5-HT2A/2B/2C receptor agonist 2,5-dimethoxy-4-iodoamphetamine (DOI). Namely, both 5-HT2C receptor agonists and antagonists, regarding 5-HT2C receptor-mediated Gq-phospholipase C (PLC) signaling, reportedly attenuate the HTR response. The present experiments tested the hypothesis that both classes of 5-HT2C receptor compounds could attenuate the DOI-elicited-HTR in a single strain of mice, C57Bl/6J. The expected results were considered in accordance with ligand functional selectivity. Commercially-available 5-HT2C agonists (CP 809101, Ro 60-0175, WAY 161503, mCPP, and 1-methylpsilocin), novel 4-phenyl-2-N,N-dimethyl-aminotetralin (PAT)-type 5-HT2C agonists (with 5-HT2A/2B antagonist activity), and antagonists selective for 5-HT2A (M100907), 5-HT2C (SB-242084), and 5-HT2B/2C (SB-206553) receptors attenuated the DOI-elicited-HTR. In contrast, there were differential effects on locomotion across classes of compounds. The 5-HT2C agonists and M100907 decreased locomotion, SB-242084 increased locomotion, SB-206553 resulted in dose-dependent biphasic effects on locomotion, and the PATs did not alter locomotion. In vitro molecular pharmacology studies showed that 5-HT2C agonists potent for attenuating the DOI-elicited-HTR also reduced the efficacy of DOI to activate mouse 5-HT2C receptor-mediated PLC signaling in HEK cells. Although there were differences in affinities of a few compounds at mouse compared to human 5-HT2A or 5-HT2C receptors, all compounds tested retained their selectivity for either receptor, regardless of receptor species. Results indicate that 5-HT2C receptor agonists and antagonists attenuate the DOI-elicited-HTR in C57Bl/6J mice, and suggest that structurally diverse 5-HT2C ligands result in different 5-HT2C receptor signaling outcomes compared to DOI.

  20. All-atom structural models of insulin binding to the insulin receptor in the presence of a tandem hormone-binding element. (United States)

    Vashisth, Harish; Abrams, Cameron F


    Insulin regulates blood glucose levels in higher organisms by binding to and activating insulin receptor (IR), a constitutively homodimeric glycoprotein of the receptor tyrosine kinase (RTK) superfamily. Therapeutic efforts in treating diabetes have been significantly impeded by the absence of structural information on the activated form of the insulin/IR complex. Mutagenesis and photo-crosslinking experiments and structural information on insulin and apo-IR strongly suggest that the dual-chain insulin molecule, unlike the related single-chain insulin-like growth factors, binds to IR in a very different conformation than what is displayed in storage forms of the hormone. In particular, hydrophobic residues buried in the core of the folded insulin molecule engage the receptor. There is also the possibility of plasticity in the receptor structure based on these data, which may in part be due to rearrangement of the so-called CT-peptide, a tandem hormone-binding element of IR. These possibilities provide opportunity for large-scale molecular modeling to contribute to our understanding of this system. Using various atomistic simulation approaches, we have constructed all-atom structural models of hormone/receptor complexes in the presence of CT in its crystallographic position and a thermodynamically favorable displaced position. In the "displaced-CT" complex, many more insulin-receptor contacts suggested by experiments are satisfied, and our simulations also suggest that R-insulin potentially represents the receptor-bound form of hormone. The results presented in this work have further implications for the design of receptor-specific agonists/antagonists.

  1. Structure of the activation domain of the GM-CSF/IL-3/IL-5 receptor common beta-chain bound to an antagonist. (United States)

    Rossjohn, J; McKinstry, W J; Woodcock, J M; McClure, B J; Hercus, T R; Parker, M W; Lopez, A F; Bagley, C J


    Heterodimeric cytokine receptors generally consist of a major cytokine-binding subunit and a signaling subunit. The latter can transduce signals by more than 1 cytokine, as exemplified by the granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-2 (IL-2), and IL-6 receptor systems. However, often the signaling subunits in isolation are unable to bind cytokines, a fact that has made it more difficult to obtain structural definition of their ligand-binding sites. This report details the crystal structure of the ligand-binding domain of the GM-CSF/IL-3/IL-5 receptor beta-chain (beta(c)) signaling subunit in complex with the Fab fragment of the antagonistic monoclonal antibody, BION-1. This is the first single antagonist of all 3 known eosinophil-producing cytokines, and it is therefore capable of regulating eosinophil-related diseases such as asthma. The structure reveals a fibronectin type III domain, and the antagonist-binding site involves major contributions from the loop between the B and C strands and overlaps the cytokine-binding site. Furthermore, tyrosine(421) (Tyr(421)), a key residue involved in receptor activation, lies in the neighboring loop between the F and G strands, although it is not immediately adjacent to the cytokine-binding residues in the B-C loop. Interestingly, functional experiments using receptors mutated across these loops demonstrate that they are cooperatively involved in full receptor activation. The experiments, however, reveal subtle differences between the B-C loop and Tyr(421), which is suggestive of distinct functional roles. The elucidation of the structure of the ligand-binding domain of beta(c) also suggests how different cytokines recognize a single receptor subunit, which may have implications for homologous receptor systems. (Blood. 2000;95:2491-2498)

  2. Crystal Structure of the Complex of Human FasL and Its Decoy Receptor DcR3. (United States)

    Liu, Weifeng; Ramagopal, Udupi; Cheng, Huiyong; Bonanno, Jeffrey B; Toro, Rafael; Bhosle, Rahul; Zhan, Chenyang; Almo, Steven C


    The apoptotic effect of FasL:Fas signaling is disrupted by DcR3, a unique secreted member of the tumor necrosis factor receptor superfamily, which also binds and neutralizes TL1A and LIGHT. DcR3 is highly elevated in patients with various tumors and contributes to mechanisms by which tumor cells to evade host immune surveillance. Here we report the crystal structure of FasL in complex with DcR3. Comparison of FasL:DcR3 structure with our earlier TL1A:DcR3 and LIGHT:DcR3 structures supports a paradigm involving the recognition of invariant main-chain and conserved side-chain functionalities, which is responsible for the recognition of multiple TNF ligands exhibited by DcR3. The FasL:DcR3 structure also provides insight into the FasL:Fas recognition surface. We demonstrate that the ability of recombinant FasL to induce Jurkat cell apoptosis is significantly enhanced by native glycosylation or by structure-inspired mutations, both of which result in reduced tendency to aggregate. All of these activities are efficiently inhibited by recombinant DcR3.

  3. Structural model for the interaction of a designed Ankyrin Repeat Protein with the human epidermal growth factor receptor 2.

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    V Chandana Epa

    Full Text Available Designed Ankyrin Repeat Proteins are a class of novel binding proteins that can be selected and evolved to bind to targets with high affinity and specificity. We are interested in the DARPin H10-2-G3, which has been evolved to bind with very high affinity to the human epidermal growth factor receptor 2 (HER2. HER2 is found to be over-expressed in 30% of breast cancers, and is the target for the FDA-approved therapeutic monoclonal antibodies trastuzumab and pertuzumab and small molecule tyrosine kinase inhibitors. Here, we use computational macromolecular docking, coupled with several interface metrics such as shape complementarity, interaction energy, and electrostatic complementarity, to model the structure of the complex between the DARPin H10-2-G3 and HER2. We analyzed the interface between the two proteins and then validated the structural model by showing that selected HER2 point mutations at the putative interface with H10-2-G3 reduce the affinity of binding up to 100-fold without affecting the binding of trastuzumab. Comparisons made with a subsequently solved X-ray crystal structure of the complex yielded a backbone atom root mean square deviation of 0.84-1.14 Ångstroms. The study presented here demonstrates the capability of the computational techniques of structural bioinformatics in generating useful structural models of protein-protein interactions.

  4. Structural dynamics of V3 loop with different electrostatics: implications on co-receptor recognition: a molecular dynamics study of HIV gp120. (United States)

    Chandramouli, Balasubramanian; Chillemi, Giovanni; Giombini, Emanuela; Capobianchi, Maria R; Rozera, Gabriella; Desideri, Alessandro


    The HIV's envelope glycoprotein gp120 plays a major role in the entry of the virus into the host cell, through its successive interactions with the cell surface CD4 receptor and a co-receptor (CCR5 or CXCR4). The choice of a specific co-receptor by gp120 has an important consequence on HIV infection and pathogenesis. The third variable region within gp120, the V3 loop, is the principal determinant of the co-receptor usage by gp120. Here, we report the long time molecular dynamics simulations of four gp120 structures, having a V3 loop charge of +3 and +5, from both R5 and X4 specific strains of HIV. The results of the study highlight the properties of the V3 loop that can be critical for dictating the co-receptor recognition and selection in structural context. In detail, we observe that the structural orientation of the V3 loop in the 3D space is modulated by its net charge, whilst its co-receptor choice is likely dictated by a combined effect of both the electrostatics of the loop and its conformational variability at the level of its central crown region.

  5. Crystal Structure of Botulinum Neurotoxin Type a in Complex With the Cell Surface Co-Receptor GT1b-Insight Into the Toxin-Neuron Interaction

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    Stenmark, P.; Dupuy, J.; Inamura, A.; Kiso, M.; Stevens, R.C.


    Botulinum neurotoxins have a very high affinity and specificity for their target cells requiring two different co-receptors located on the neuronal cell surface. Different toxin serotypes have different protein receptors; yet, most share a common ganglioside co-receptor, GT1b. We determined the crystal structure of the botulinum neurotoxin serotype A binding domain (residues 873-1297) alone and in complex with a GT1b analog at 1.7 A and 1.6 A, respectively. The ganglioside GT1b forms several key hydrogen bonds to conserved residues and binds in a shallow groove lined by Tryptophan 1266. GT1b binding does not induce any large structural changes in the toxin; therefore, it is unlikely that allosteric effects play a major role in the dual receptor recognition. Together with the previously published structures of botulinum neurotoxin serotype B in complex with its protein co-receptor, we can now generate a detailed model of botulinum neurotoxin's interaction with the neuronal cell surface. The two branches of the GT1b polysaccharide, together with the protein receptor site, impose strict geometric constraints on the mode of interaction with the membrane surface and strongly support a model where one end of the 100 A long translocation domain helix bundle swing into contact with the membrane, initiating the membrane anchoring event.

  6. Structure of the gene for human. beta. /sub 2/-adrenergic receptor: expression and promoter characterization

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    Emorine, L.J.; Marullo, S.; Delavier-Klutchko, C.; Kaveri, S.V.; Durieu-Trautmann, O.; Strosberg, A.D.


    The genomic gene coding for the human ..beta../sub 2/-adrenergic receptor (..beta../sub 2/AR) from A431 epidermoid cells has been isolated. Transfection of the gene into eukaryotic cells restores a fully active receptor/GTP-binding protein/adenylate cyclase complex with ..beta../sub 2/AR properties. Southern blot analyses with ..beta../sub 2/AR-specific probes show that a single ..beta../sub 2/AR gene is common to various human tissues and that its flanking sequences are highly conserved among humans and between man and rabbit, mouse, and hamster. Functional significance of these regions is supported by the presence of a promoter region (including mRNA cap sites, two TATA boxes, a CAAT box, and three G + C-rich regions that resemble binding sites for transcription factor Sp1) 200-300 base pairs 5' to the translation initiation codon. In the 3' flanking region, sequences homologous to glucocorticoid-response elements might be responsible for the increased expression of the ..beta../sub 2/AR gene observed after treatment of the transfected cells with hydrocortisone. In addition, 5' to the promoter region, an open reading frame encodes a 251-residue polypeptide that displays striking homologies with protein kinases and other nucleotide-binding proteins.

  7. The meaning and relevance of B-cell receptor structure and function in chronic lymphocytic leukemia. (United States)

    Stevenson, Freda K; Forconi, Francesco; Packham, Graham


    The B-cell receptor (BCR) is of critical importance for normal B cells and for the majority of B-cell malignancies, especially chronic lymphocytic leukemia (CLL). The two major subsets of CLL are biologically distinct, being derived from B cells at different stages of differentiation and carrying unmutated (U-CLL) or mutated (M-CLL) IGHV genes. U-CLL, which has a poorer prognosis, often has relatively conserved (stereotypic) IGHV-HD-HJ sequences, indicative of interaction with large (super)antigens and similar to those in normal naive innate B cells. Conserved sequences are less evident in M-CLL, in keeping with its postfollicular origin. However, both subsets exhibit features of chronic antigen exposure in tissue sites, with local proliferative events, but also downregulation of surface immunoglobulin M but not surface immunoglobulin D, a characteristic of normal anergic B cells. BCR-mediated anergy can spread to other receptors such as CXCR4. Circulating CLL cells retain a shadow of tissue-based events that can reverse over time, but the overall extent of anergy is greater in M-CLL. Despite this stereotypic variety and more genomic complexity, BCR-mediated responses in vitro appear relatively homogeneous in U-CLL, but M-CLL is more heterogeneous. The differential balance between antigen-induced proliferation or anergy is the likely determinant of clinical behavior and possibly of response to kinase inhibitors.

  8. Structural Requirements of N-Substituted Spiropiperidine Analogues as Agonists of Nociceptin/Orphanin FQ Receptor

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    Ling Yang


    Full Text Available The nociceptin/orphanin FQ (NOP receptor is involved in a wide range of biological functions, including pain, anxiety, depression and drug abuse. Especially, its agonists have great potential to be developed into anxiolytics. In this work, both the ligand- and receptor-based three-dimensional quantitative structure–activity relationship (3D-QSAR studies were carried out using comparative molecular field analysis (CoMFA and comparative molecular similarity indices analysis (CoMSIA techniques on 103 N-substituted spiropiperidine analogues as NOP agonists. The resultant optimal ligand-based CoMSIA model exhibited Q2 of 0.501, R2ncv of 0.912 and its predictive ability was validated by using an independent test set of 26 compounds which gave R2pred value of 0.818. In addition, docking analysis and molecular dynamics simulation (MD were also applied to elucidate the probable binding modes of these agonists. Interpretation of the 3D contour maps, in the context of the topology of the active site of NOP, provided insight into the NOP-agonist interactions. The information obtained from this work can be used to accurately predict the binding affinity of related agonists and also facilitate the future rational design of novel agonists with improved activity.

  9. Controlling the taste receptor accessible structure of rebaudioside A via binding to bovine serum albumin. (United States)

    Mudgal, Samriddh; Keresztes, Ivan; Feigenson, Gerald W; Rizvi, S S H


    We illustrate a method that uses bovine serum albumin (BSA) to control the receptor-accessible part of rebaudioside A (Reb A). The critical micelle concentration (CMC) of Reb A was found to be 4.5 mM and 5 mM at pH 3 and 6.7 respectively. NMR studies show that below its CMC, Reb A binds weakly to BSA to generate a Reb A-protein complex ("RPC"), which is only modestly stable under varying conditions of pH (3.0-6.7) and temperature (4-40°C) with its binding affinities determined to be in the range of 5-280 mM. Furthermore, saturation transfer difference (STD) NMR experiments confirm that the RPC has fast exchange of the bitterness-instigating diterpene of Reb A into the binding sites of BSA. Our method can be used to alter the strength of Reb A-receptor interaction, as a result of binding of Reb A to BSA, which may ultimately lead to moderation of its taste.

  10. Pressure-selective modulation of NMDA receptor subtypes may reflect 3D structural differences. (United States)

    Mor, Amir; Kuttner, Yosef Y; Levy, Shiri; Mor, Merav; Hollmann, Michael; Grossman, Yoram


    Professional deep-water divers exposed to high pressure (HP) above 1.1 MPa suffer from High Pressure Neurological Syndrome (HPNS), which is associated with CNS hyperexcitability. We have previously reported that HP augments N-methyl-D-aspartate receptor (NMDAR) synaptic responses, increases neuronal excitability, and potentially causes irreversible neuronal damage. We now report that HP (10.1 MPa) differentially affects eight specific NMDAR subtypes. GluN1(1a or 1b) was co-expressed with one of the four GluN2(A-D) subunits in Xenopus laevis oocytes. HP increased ionic currents (measured by two electrode voltage clamps) of one subtype, reduced the current in four others, and did not affect the current in the remaining three. 3D theoretical modeling was aimed at revealing specific receptor domains involved with HP selectivity. In light of the information on the CNS spatial distribution of the different NMDAR subtypes, we conclude that the NMDAR's diverse responses to HP may lead to selective HP effects on different brain regions. These discoveries call for further and more specific investigation of deleterious HP effects and suggest the need for a re-evaluation of deep-diving safety guidelines.

  11. The structural basis of gas-responsive transcription by the human nuclear hormone receptor REV-ERBbeta.

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    Keith I Pardee


    Full Text Available Heme is a ligand for the human nuclear receptors (NR REV-ERBalpha and REV-ERBbeta, which are transcriptional repressors that play important roles in circadian rhythm, lipid and glucose metabolism, and diseases such as diabetes, atherosclerosis, inflammation, and cancer. Here we show that transcription repression mediated by heme-bound REV-ERBs is reversed by the addition of nitric oxide (NO, and that the heme and NO effects are mediated by the C-terminal ligand-binding domain (LBD. A 1.9 A crystal structure of the REV-ERBbeta LBD, in complex with the oxidized Fe(III form of heme, shows that heme binds in a prototypical NR ligand-binding pocket, where the heme iron is coordinately bound by histidine 568 and cysteine 384. Under reducing conditions, spectroscopic studies of the heme-REV-ERBbeta complex reveal that the Fe(II form of the LBD transitions between penta-coordinated and hexa-coordinated structural states, neither of which possess the Cys384 bond observed in the oxidized state. In addition, the Fe(II LBD is also able to bind either NO or CO, revealing a total of at least six structural states of the protein. The binding of known co-repressors is shown to be highly dependent upon these various liganded states. REV-ERBs are thus highly dynamic receptors that are responsive not only to heme, but also to redox and gas. Taken together, these findings suggest new mechanisms for the systemic coordination of molecular clocks and metabolism. They also raise the possibility for gas-based therapies for the many disorders associated with REV-ERB biological functions.

  12. Toxic and non-toxic aggregates from the SBMA and normal forms of androgen receptor have distinct oligomeric structures. (United States)

    Jochum, Tobias; Ritz, Manuela E; Schuster, Christoph; Funderburk, Sarah F; Jehle, Katja; Schmitz, Katja; Brinkmann, Falko; Hirtz, Michael; Moss, David; Cato, Andrew C B


    Hormone-dependent aggregation of the androgen receptor (AR) with a polyglutamine (polyQ) stretch amplification (>38) is considered to be the causative agent of the neurodegenerative disorder spinal and bulbar muscular atrophy (SBMA), consistent with related neurodegenerative diseases involving polyQ-extended proteins. In spite of the widespread acceptance of this common causal hypothesis, little attention has been paid to its apparent incompatibility with the observation of AR aggregation in healthy individuals with no polyQ stretch amplification. Here we used atomic force microscopy (AFM) to characterize sub-micrometer scale aggregates of the wild-type (22 glutamines) and the SBMA form (65 glutamines), as well as a polyQ deletion mutant (1 glutamine) and a variant with a normal length polyQ stretch but with a serine to alanine double mutation elsewhere in the protein. We used a baculovirus-insect cell expression system to produce full-length proteins for these structural analyses. We related the AFM findings to cytotoxicity as measured by expression of the receptors in Drosophila motoneurons or in neuronal cells in culture. We found that the pathogenic AR mutants formed oligomeric fibrils up to 300-600nm in length. These were clearly different from annular oligomers 120-180nm in diameter formed by the nonpathogenic receptors. We could also show that melatonin, which is known to ameliorate the pathological phenotype in the fly model, caused polyQ-extended AR to form annular oligomers. Further comparative investigation of these reproducibly distinct toxic and non-toxic oligomers could advance our understanding of the molecular basis of the polyQ pathologies.

  13. Novel information on the epitope of an inverse agonist monoclonal antibody provides insight into the structure of the TSH receptor.

    Directory of Open Access Journals (Sweden)

    Chun-Rong Chen

    Full Text Available The TSH receptor (TSHR comprises an extracellular leucine-rich domain (LRD linked by a hinge region to the transmembrane domain (TMD. Insight into the orientation of these components to each other is required for understanding how ligands activate the receptor. We previously identified residue E251 at the LRD-hinge junction as contributing to coupling TSH binding with receptor activation. However, a single residue cannot stabilize the LRD-hinge unit. Therefore, based on the LRD crystal structure we selected for study four other potential LRD-hinge interface charged residues. Alanine substitutions of individual residues K244, E247, K250 and R255 (as well as previously known E251A did not affect TSH binding or function. However, the cumulative mutation of these residues in varying permutations, primarily K250A and R255A when associated with E251A, partially uncoupled TSH binding and function. These data suggest that these three residues, spatially very close to each other at the LRD base, interact with the hinge region. Unexpectedly and most important, monoclonal antibody CS-17, a TSHR inverse agonist whose epitope straddles the LRD-hinge, was found to interact with residues K244 and E247 at the base of the convex LRD surface. These observations, together with the functional data, exclude residues K244 and E247 from the TSHR LRD-hinge interface. Further, for CS-17 accessibility to K244 and E247, the concave surface of the TSHR LRD must be tilted forwards towards the hinge region and plasma membrane. Overall, these data provide insight into the mechanism by which ligands either activate the TSHR or suppress its constitutive activity.

  14. Polymeric structure and host Toll-like receptor 4 dictate immunogenicity of NY-ESO-1 antigen in vivo. (United States)

    Liu, Yanan; Tian, Xiaoli; Leitner, Wolfgang W; Aldridge, Michael E; Zheng, Junying; Yu, Zhiya; Restifo, Nicholas P; Weiss, Richard; Scheiblhofer, Sandra; Xie, Chong; Sun, Ren; Cheng, Genhong; Zeng, Gang


    In search of intrinsic factors that contribute to the distinctively strong immunogenicity of a non-mutated cancer/testis antigen, we found that NY-ESO-1 forms polymeric structures through disulfide bonds. NY-ESO-1 binding to immature dendritic cells was dependent on its polymeric structure and involved Toll-like receptor-4 (TLR4) on the surface of immature dendritic cells in mouse and human. Gene gun-delivered plasmid encoding the wild-type NY-ESO-1 readily induced T cell-dependent antibody (Ab) responses in wild-type C57BL/10 mice but not TLR4-knock-out C57BL/10ScNJ mice. Disrupting polymeric structures of NY-ESO-1 by cysteine-to-serine (Cys-to-Ser) substitutions lead to diminished immunogenicity and altered TLR4-dependence in the induced Ab response. To demonstrate its adjuvant effect, NY-ESO-1 was fused with a major mugwort pollen allergen Art v 1 and a tumor-associated antigen, carbonic anhydrase 9. Plasmid DNA vaccines encoding the fusion genes generated robust immune responses against otherwise non-immunogenic targets in mice. Polymeric structure and TLR4 may play important roles in rendering NY-ESO-1 immunogenic and thus serve as a potent molecular adjuvant. NY-ESO-1 thus represents the first example of a cancer/testis antigen that is a also damage-associated molecular pattern.

  15. Evaluation of Phytocannabinoids from High Potency Cannabis sativa using In Vitro Bioassays to Determine Structure-Activity Relationships for Cannabinoid Receptor 1 and Cannabinoid Receptor 2



    Cannabis has been around for thousands of years and has been used recreationally, medicinally, and for fiber. Over 500 compounds have been isolated from Cannabis sativa with approximately 105 being cannabinoids. Of those 105 compounds, Δ9-tetrahydrocannabinol has been determined as the primary constituent, which is also responsible for the psychoactivity associated with Cannabis. Cannabinoid receptors belong to the large superfamily of G protein-coupled receptors. Targeting the cannabinoid re...

  16. Structure-activity relationship of miltirone, an active central benzodiazepine receptor ligand isolated from Salvia miltiorrhiza Bunge (Danshen)

    Energy Technology Data Exchange (ETDEWEB)

    Chang, H.M.; Chui, K.Y.; Tan, F.W.; Yang, Y.; Zhong, Z.P.; Lee, C.M.; Sham, H.L.; Wong, H.N. (Chinese Univ. of Hong Kong, Shatin (Hong Kong))


    Twenty one o-quinonoid-type compounds and one coumarin-type compound related to miltirone (1) have been synthesized with the aim to identify the key structural elements involved in miltirone's interaction with the central benzodiazepine receptor. On the basis of their inhibition of ({sup 3}H)flunitrazepam binding to bovine cerebral cortex membranes, it is apparent that ring A of miltirone is essential for affinity. Although increasing the size of ring A from six-membered to seven- and eight-membered is well-tolerated, the introduction of polar hydroxyl groups greatly reduces binding affinity. The presence of 1,1-dimethyl groups on ring A is, however, not essential. On the other hand, the isopropyl group on ring C appears to be critical for binding as its removal decreases affinity by more than 30-fold. It can, however, be replaced with a methyl group with minimal reduction in affinity. Finally, linking ring A and B with a -CH{sub 2}CH{sub 2}- bridge results in analogue 89, which is 6 times more potent than miltirone at the central benzodiazepine receptor (IC50 = 0.05 microM).

  17. Deriving structural and functional insights from a ligand-based hierarchical classification of G protein-coupled receptors. (United States)

    Attwood, T K; Croning, M D R; Gaulton, A


    G protein-coupled receptors (GPCRs) constitute the largest known family of cell-surface receptors. With hundreds of members populating the rhodopsin-like GPCR superfamily and many more awaiting discovery in the human genome, they are of interest to the pharmaceutical industry because of the opportunities they afford for yielding potentially lucrative drug targets. Typical sequence analysis strategies for identifying novel GPCRs tend to involve similarity searches using standard primary database search tools. This will reveal the most similar sequence, generally without offering any insight into its family or superfamily relationships. Conversely, searches of most 'pattern' or family databases are likely to identify the superfamily, but not the closest matching subtype. Here we describe a diagnostic resource that allows identification of GPCRs in a hierarchical fashion, based principally upon their ligand preference. This resource forms part of the PRINTS database, which now houses approximately 250 GPCR-specific fingerprints ( This collection of fingerprints is able to provide more sensitive diagnostic opportunities than have been realized by related approaches and is currently the only diagnostic tool for assigning GPCR subtypes. Mapping such fingerprints on to three-dimensional GPCR models offers powerful insights into the structural and functional determinants of subtype specificity.

  18. Cloning, structural characterization, and chromosomal localization of the gene encoding the human prostaglandin E(2) receptor EP2 subtype. (United States)

    Smock, S L; Pan, L C; Castleberry, T A; Lu, B; Mather, R J; Owen, T A


    Northern blot analysis of human placental RNA using a probe to the 5' end of the human prostaglandin E(2) (PGE(2)) EP2 receptor subtype coding region revealed the existence of a high abundance, low molecular weight transcript. To investigate the origin of this transcript, and its possible relationship to the human EP2 mRNA, we have cloned and characterized the gene encoding the human PGE(2) EP2 receptor subtype, identified transcriptional initiation and termination sites in two tissues (spleen and thymus), and determined its chromosomal localization. The human EP2 gene consists of two exons separated by a large intron, utilizes a common initiation site in both spleen and thymus at 1113 bp upstream of the translation initiation site, and has 3' transcript termini at 1140 bp and 1149 bp downstream of the translation stop site in spleen and thymus respectively. Southern and fluorescence in situ hybridization analysis demonstrated the human EP2 gene to be a single copy gene located in band 22 of the long arm of chromosome 14 (14q22). Though our initial interest in this gene was to investigate potential differential splicing of the human EP2 gene in placenta, this work demonstrates that the atypical transcript observed in placenta probably arises from a distinct, yet related, gene. Knowledge of the sequence, structure, and transcription events associated with the human EP2 gene will enable a broader understanding of its regulation and potential role in normal physiology and disease.

  19. GABA receptor antagonists and insecticides: common structural features of 4-alkyl-1-phenylpyrazoles and 4-alkyl-1-phenyltrioxabicyclooctanes. (United States)

    Sammelson, Robert E; Caboni, Pierluigi; Durkin, Kathleen A; Casida, John E


    Fipronil [5-amino-3-cyano-1-(2,6-dichloro-4-trifluoromethylphenyl)-4-trifluoromethylsulfinylpyrazole] is one of the most important insecticides. Structure-activity studies described here reveal that fipronil retains its very high binding potency at the human beta3 and house fly gamma-aminobutyric acid (GABA) receptors and toxicity to house flies on replacing the pyrazole trifluoromethylsulfinyl moiety with tert-butyl or isopropyl and the phenyl trifluoromethyl substituent with ethynyl, trifluoromethoxy, bromo or chloro. Among the compounds studied, those with other alkyl groups at the 4-position of the pyrazole, as well as phenyl substitution without one or both of the 2,6-dichloro groups, are less effective. 5-Amino-4-tert-butyl-3-cyano-1-(2,6-dichloro-4-ethynylphenyl)pyrazole is highly effective and almost isosteric with 4-tert-butyl-3-cyano-1-(4-ethynylphenyl)-2,6,7-trioxabicyclo[2.2.2]octane (the most potent 4-alkyl-1-phenyltrioxabicyclooctane) as a noncompetitive GABA antagonist and insecticide. These findings are interpreted as three binding subsites in the GABA receptor: a hydrophobic site undergoing steric interaction with the tert-butyl or equivalent group; a hydrogen bonding site to pyrazole N-2; a pi bonding site to the face of the phenyl moiety; with supplemental enhancement by the 3-cyano and 4-ethynyl substituents.

  20. Design, synthesis, and structure-affinity relationships of regioisomeric N-benzyl alkyl ether piperazine derivatives as sigma-1 receptor ligands. (United States)

    Moussa, Iman A; Banister, Samuel D; Beinat, Corinne; Giboureau, Nicolas; Reynolds, Aaron J; Kassiou, Michael


    A series of N-(benzofuran-2-ylmethyl)-N'-benzylpiperazines bearing alkyl or fluoroalkyl aryl ethers were synthesized and evaluated at various central nervous system receptors. Examination of in vitro sigma1 {[3H]+-pentazocine} and sigma2 ([3H]DTG) receptor binding profiles of piperazines 11-13 and 25-36 revealed several highly potent and sigma1 selective ligands, notably, N-(benzofuran-2-ylmethyl)-N'-(4'-methoxybenzyl)piperazine (13, Ki=2.7 nM, sigma2/sigma1=38) and N-(benzofuran-2-ylmethyl)-N'-(4'-(2''-fluoroethoxy)benzyl)piperazine (30, Ki=2.6 nM, sigma2/sigma1=187). Structural features for optimal sigma1 receptor affinity and selectivity over the sigma2 receptor were identified. On the basis of its favorable log D value, 13 was selected as a candidate for the development of a sigma1 receptor positron emission tomography radiotracer. [11C]13 showed high uptake in the brain and other sigma receptor-rich organs of a Papio hamadryas baboon. The in vivo evaluation of [11C]13 indicates that this radiotracer is a suitable candidate for imaging the sigma1 receptor in neurodegenerative processes.

  1. Characterisation of SNP haplotype structure in chemokine and chemokine receptor genes using CEPH pedigrees and statistical estimation

    Directory of Open Access Journals (Sweden)

    Clark Vanessa J


    Full Text Available Abstract Chemokine signals and their cell-surface receptors are important modulators of HIV-1 disease and cancer. To aid future case/control association studies, aim to further characterise the haplotype structure of variation in chemokine and chemokine receptor genes. To perform haplotype analysis in a population-based association study, haplotypes must be determined by estimation, in the absence of family information or laboratory methods to establish phase. Here, test the accuracy of estimates of haplotype frequency and linkage disequilibrium by comparing estimated haplotypes generated with the expectation maximisation (EM algorithm to haplotypes determined from Centre d'Etude Polymorphisme Humain (CEPH pedigree data. To do this, they have characterised haplotypes comprising alleles at 11 biallelic loci in four chemokine receptor genes (CCR3, CCR2, CCR5 and CCRL2, which span 150 kb on chromosome 3p21, and haplotyes of nine biallelic loci in six chemokine genes [MCP-1(CCL2, Eotaxin(CCL11, RANTES(CCL5, MPIF-1(CCL23, PARC(CCL18 and MIP-1α(CCL3 ] on chromosome 17q11-12. Forty multi-generation CEPH families, totalling 489 individuals, were genotyped by the TaqMan 5'-nuclease assay. Phased haplotypes and haplotypes estimated from unphased genotypes were compared in 103 grandparents who were assumed to have mated at random. For the 3p21 single nucleotide polymorphism (SNP data, haplotypes determined by pedigree analysis and haplotypes generated by the EM algorithm were nearly identical. Linkage disequilibrium, measured by the D' statistic, was nearly maximal across the 150 kb region, with complete disequilibrium maintained at the extremes between CCR3-Y17Y and CCRL2-1243V. D'-values calculated from estimated haplotypes on 3p21 had high concordance with pairwise comparisons between pedigree-phased chromosomes. Conversely, there was less agreement between analyses of haplotype frequencies and linkage disequilibrium using estimated haplotypes when

  2. The predicted 3D structure of the human D2 dopamine receptor and the binding site and binding affinities for agonists and antagonists (United States)

    Kalani, M. Yashar S.; Vaidehi, Nagarajan; Hall, Spencer E.; Trabanino, Rene J.; Freddolino, Peter L.; Kalani, Maziyar A.; Floriano, Wely B.; Tak Kam, Victor Wai; Goddard, William A., III


    Dopamine neurotransmitter and its receptors play a critical role in the cell signaling process responsible for information transfer in neurons functioning in the nervous system. Development of improved therapeutics for such disorders as Parkinson's disease and schizophrenia would be significantly enhanced with the availability of the 3D structure for the dopamine receptors and of the binding site for dopamine and other agonists and antagonists. We report here the 3D structure of the long isoform of the human D2 dopamine receptor, predicted from primary sequence using first-principles theoretical and computational techniques (i.e., we did not use bioinformatic or experimental 3D structural information in predicting structures). The predicted 3D structure is validated by comparison of the predicted binding site and the relative binding affinities of dopamine, three known dopamine agonists (antiparkinsonian), and seven known antagonists (antipsychotic) in the D2 receptor to experimentally determined values. These structures correctly predict the critical residues for binding dopamine and several antagonists, identified by mutation studies, and give relative binding affinities that correlate well with experiments. The predicted binding site for dopamine and agonists is located between transmembrane (TM) helices 3, 4, 5, and 6, whereas the best antagonists bind to a site involving TM helices 2, 3, 4, 6, and 7 with minimal contacts to TM helix 5. We identify characteristic differences between the binding sites of agonists and antagonists.

  3. Intracellular Delivery of a Planar DNA Origami Structure by the Transferrin-Receptor Internalization Pathway

    DEFF Research Database (Denmark)

    Schaffert, David Henning; Okholm, Anders Hauge; Sørensen, Rasmus Schøler;


    DNA origami provides rapid access to easily functionalized, nanometer-sized structures making it an intriguing platform for the development of defined drug delivery and sensor systems. Low cellular uptake of DNA nanostructures is a major obstacle in the development of DNA-based delivery platforms....... Herein, significant strong increase in cellular uptake in an established cancer cell line by modifying a planar DNA origami structure with the iron transport protein transferrin (Tf) is demonstrated. A variable number of Tf molecules are coupled to the origami structure using a DNA-directed, site...... on the origami surface....

  4. Structural and thermodynamic bases for the design of pure prolactin receptor antagonists: X-ray structure of Del1-9-G129R-hPRL

    DEFF Research Database (Denmark)

    Jomain, Jean-Baptiste; Tallet, Estelle; Broutin, Isabelle;


    Competitive antagonists of the human prolactin (hPRL) receptor are a novel class of molecules of potential therapeutic interest in the context of cancer. We recently developed the pure antagonist Del1-9-G129R-hPRL by deleting the nine N-terminal residues of G129R-hPRL, a first generation partial...... antagonist. We determined the crystallographic structure of Del1-9-G129R-hPRL, which revealed no major change compared with wild type hPRL, indicating that its pure antagonistic properties are intrinsically due to the mutations. To decipher the molecular bases of pure antagonism, we compared the biological...... agonism can be abolished either by further disrupting hormone site 2-receptor contacts by N-terminal deletion, as in Del1-9-G129R-hPRL, or by stabilizing hPRL and constraining its intrinsic flexibility, as in G129V-hPRL. Udgivelsesdato: 2007-Nov-9...

  5. Structural mapping of the voltage-dependent sodium channel. Distance between the tetrodotoxin and Centruroides suffusus suffusus II beta-scorpion toxin receptors. (United States)

    Darbon, H; Angelides, K J


    A 7- dimethylaminocoumarin -4-acetate fluorescent derivative of toxin II from the venom of the scorpion Centruroides suffusus suffusus (Css II) has been prepared to study the structural, conformational, and cellular properties of the beta-neurotoxin receptor site on the voltage-dependent sodium channel. The derivative retains high affinity for its receptor site on the synaptosomal sodium channel with a KD of 7 nM and site capacity of 1.5 pmol/mg of synaptosomal protein. The fluorescent toxin is very environmentally sensitive and the fluorescence emission upon binding indicates that the Css II receptor is largely hydrophobic. Binding of tetrodotoxin or batrachotoxin does not alter the spectroscopic properties of bound Css II, whereas toxin V from Leiurus quinquestriatus effects a 10-nm blue shift to a more hydrophobic environment. This is the first direct indication of conformational coupling between these separate neurotoxin receptor sites. The distance between the tetrodotoxin and Css II scorpion toxin receptors on the sodium channel was measured by fluorescence resonance energy transfer. Efficiencies were measured by both donor quenching and acceptor-sensitized emission. The distance between these two neurotoxin sites is about 34 A. The implications of these receptor locations together with other known molecular distances are discussed in terms of a molecular structure of the voltage-dependent sodium channel.

  6. Use of Monoclonal Antibodies to Study the Structural Basis of the Function of Nicotinic Acetylcholine Receptors on Electric Organ and Muscle and to Determine the Structure of Nicotinic Acetylcholine Receptors on Neurons (United States)


    of chicken neurona .4receptor subunits. Sequences of al and a2 are from Net .Ot al. -l Sequences of a3 and a4 were determintl from clones described...Sucrose gradient analysis of neurona & nicotinic receptors was conducted as follows. Chicken ind rat brain receptors were extracted from crude

  7. NCBI nr-aa BLAST: CBRC-OCUN-01-1504 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-OCUN-01-1504 sp|Q9QY96|CASR_MOUSE Extracellular calcium-sensing receptor precu...rsor (CaSR) (Parathyroid Cell calcium-sensing receptor) gb|AAD28371.1|AF110178_1 calcium-sensing receptor [Mus musculus] Q9QY96 0.0 95% ...

  8. NCBI nr-aa BLAST: CBRC-CINT-01-0064 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-CINT-01-0064 sp|Q9QY96|CASR_MOUSE Extracellular calcium-sensing receptor precu...rsor (CaSR) (Parathyroid Cell calcium-sensing receptor) gb|AAD28371.1|AF110178_1 calcium-sensing receptor [Mus musculus] Q9QY96 0.0 41% ...

  9. NCBI nr-aa BLAST: CBRC-RNOR-11-0116 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-RNOR-11-0116 sp|Q9QY96|CASR_MOUSE Extracellular calcium-sensing receptor precu...rsor (CaSR) (Parathyroid Cell calcium-sensing receptor) gb|AAD28371.1|AF110178_1 calcium-sensing receptor [Mus musculus] Q9QY96 0.0 97% ...

  10. Discovery and Structure-Activity Relationships of the Neoseptins: A New Class of Toll-like Receptor-4 (TLR4) Agonists. (United States)

    Morin, Matthew D; Wang, Ying; Jones, Brian T; Su, Lijing; Surakattula, Murali M R P; Berger, Michael; Huang, Hua; Beutler, Elliot K; Zhang, Hong; Beutler, Bruce; Boger, Dale L


    Herein, we report studies leading to the discovery of the neoseptins and a comprehensive examination of the structure-activity relationships (SARs) of this new class of small-molecule mouse Toll-like receptor 4 (mTLR4) agonists. The compounds in this class, which emerged from screening an α-helix mimetic library, stimulate the immune response, act by a well-defined mechanism (mouse TLR4 agonist), are easy to produce and structurally manipulate, exhibit exquisite SARs, are nontoxic, and elicit improved and qualitatively different responses compared to lipopolysaccharide, even though they share the same receptor.

  11. Intracellular Delivery of a Planar DNA Origami Structure by the Transferrin-Receptor Internalization Pathway. (United States)

    Schaffert, David H; Okholm, Anders H; Sørensen, Rasmus S; Nielsen, Jesper S; Tørring, Thomas; Rosen, Christian B; Kodal, Anne Louise B; Mortensen, Michael R; Gothelf, Kurt V; Kjems, Jørgen


    DNA origami provides rapid access to easily functionalized, nanometer-sized structures making it an intriguing platform for the development of defined drug delivery and sensor systems. Low cellular uptake of DNA nanostructures is a major obstacle in the development of DNA-based delivery platforms. Herein, significant strong increase in cellular uptake in an established cancer cell line by modifying a planar DNA origami structure with the iron transport protein transferrin (Tf) is demonstrated. A variable number of Tf molecules are coupled to the origami structure using a DNA-directed, site-selective labeling technique to retain ligand functionality. A combination of confocal fluorescence microscopy and quantitative (qPCR) techniques shows up to 22-fold increased cytoplasmic uptake compared to unmodified structures and with an efficiency that correlates to the number of transferrin molecules on the origami surface.

  12. Screening of 397 chemicals and development of a quantitative structure-activity relationship model for androgen receptor antagonism

    DEFF Research Database (Denmark)

    Vinggaard, Annemarie; Niemelä, Jay Russell; Wedebye, Eva Bay;


    We have screened 397 chemicals for human androgen receptor (AR) antagonism by a sensitive reporter gene assay to generate data for the development of a quantitative structure-activity relationship (QSAR) model. A total of 523 chemicals comprising data on 292 chemicals from our laboratory and data...... by the synthetic androgen R1881. The MultiCASE expert system was used to construct a QSAR model for AR antagonizing potential. A "5 Times, 2-Fold 50% Cross Validation" of the model showed a sensitivity of 64%, a specificity of 84%, and a concordance of 76%. Data for 102 chemicals were generated for an external...... validation of the model resulting in a sensitivity of 57%, a specificity of 98%, and a concordance of 92% of the model. The model was run on a set of 176103 chemicals, and 47% were within the domain of the model. Approximately 8% of chemicals was predicted active for AR antagonism. We conclude...

  13. The structure and function of the urokinase receptor, a membrane protein governing plasminogen activation on the cell surface

    DEFF Research Database (Denmark)

    Behrendt, N; Rønne, E; Danø, K


    PA receptor, uPAR, is a cell-surface protein which plays an important role in the localization and regulation of these processes. In the present article a number of established conclusions concerning the structure and function of uPAR are presented, and in addition various models are discussed which might...... explain additional observations for which the mechanisms involved have not yet been clarified experimentally. uPAR is a highly glycosylated, 3-domain protein, anchored in the plasma membrane by a glycolipid moiety. The domain organization is important for efficient ligand-binding, and the NH2-terminal...... to an interplay between uPAR and other, unidentified components. In addition to the function in the regulation of proteolysis, uPAR seems to play a role in internalization processes and in cellular signal transduction and adhesion. A few reagents have been identified which are capable to inhibit the interaction...

  14. Structural complexes of the agonist, inverse agonist and antagonist bound C5a receptor: insights into pharmacology and signaling. (United States)

    Rana, Soumendra; Sahoo, Amita Rani; Majhi, Bharat Kumar


    The C5a receptor (C5aR) is a pharmacologically important G-protein coupled receptor (GPCR) that interacts with (h)C5a, by recruiting both the "orthosteric" sites (site1 at the N-terminus and site2 at the ECS, extra cellular surface) on C5aR in a two site-binding model. However, the complex pharmacological landscape and the distinguishing chemistry operating either at the "orthosteric" site1 or at the functionally important "orthosteric" site2 of C5aR are still not clear, which greatly limits the understanding of C5aR pharmacology. One of the major bottlenecks is the lack of an experimental structure or a refined model structure of C5aR with appropriately defined active sites. The study attempts to understand the pharmacology at the "orthosteric" site2 of C5aR rationally by generating a highly refined full-blown model structure of C5aR through advanced molecular modeling techniques, and further subjecting it to automated docking and molecular dynamics (MD) studies in the POPC bilayer. The first series of structural complexes of C5aR respectively bound to a linear native peptide agonist ((h)C5a-CT), a small molecule inverse agonist (NDT) and a cyclic peptide antagonist (PMX53) are reported, apparently establishing the unique pharmacological landscape of the "orthosteric" site2, which also illustrates an energetically distinct but coherent competitive chemistry ("cation-π" vs. "π-π" interactions) involved in distinguishing the established ligands known for targeting the "orthosteric" site2 of C5aR. Over a total of 1 μs molecular dynamics (MD) simulation in the POPC bilayer, it is evidenced that while the agonist prefers a "cation-π" interaction, the inverse agonist prefers a "cogwheel/L-shaped" interaction in contrast to the "edge-to-face/T-shaped" type π-π interactions demonstrated by the antagonist by engaging the F275(7.28) of the C5aR. In the absence of a NMR or crystallographically guided model structure of C5aR, the computational model complexes not only

  15. Crystal Structure of the Urokinase Receptor in a Ligand-Free Form

    DEFF Research Database (Denmark)

    Xu, Xiang; Gårdsvoll, Henrik; Yuan, Cai;


    . The crystal structure of human uPAR in its ligand-free state would clarify this issue, but such information remains unfortunately elusive. We now report the crystal structures of a stabilized, human uPAR (H47C/N259C) in its ligand-free form to 2.4 Å and in complex with amino-terminal fragment (ATF) to 3.2 Å...

  16. Secreted Isoform of Human Lynx1 (SLURP-2): Spatial Structure and Pharmacology of Interactions with Different Types of Acetylcholine Receptors (United States)

    Lyukmanova, E. N.; Shulepko, M. A.; Shenkarev, Z. O.; Bychkov, M. L.; Paramonov, A. S.; Chugunov, A. O.; Kulbatskii, D. S.; Arvaniti, M.; Dolejsi, Eva; Schaer, T.; Arseniev, A. S.; Efremov, R. G.; Thomsen, M. S.; Dolezal, V.; Bertrand, D.; Dolgikh, D. A.; Kirpichnikov, M. P.


    Human-secreted Ly-6/uPAR-related protein-2 (SLURP-2) regulates the growth and differentiation of epithelial cells. Previously, the auto/paracrine activity of SLURP-2 was considered to be mediated via its interaction with the α3β2 subtype of the nicotinic acetylcholine receptors (nAChRs). Here, we describe the structure and pharmacology of a recombinant analogue of SLURP-2. Nuclear magnetic resonance spectroscopy revealed a ‘three-finger’ fold of SLURP-2 with a conserved β-structural core and three protruding loops. Affinity purification using cortical extracts revealed that SLURP-2 could interact with the α3, α4, α5, α7, β2, and β4 nAChR subunits, revealing its broader pharmacological profile. SLURP-2 inhibits acetylcholine-evoked currents at α4β2 and α3β2-nAChRs (IC50 ~0.17 and >3 μM, respectively) expressed in Xenopus oocytes. In contrast, at α7-nAChRs, SLURP-2 significantly enhances acetylcholine-evoked currents at concentrations <1 μM but induces inhibition at higher concentrations. SLURP-2 allosterically interacts with human M1 and M3 muscarinic acetylcholine receptors (mAChRs) that are overexpressed in CHO cells. SLURP-2 was found to promote the proliferation of human oral keratinocytes via interactions with α3β2-nAChRs, while it inhibited cell growth via α7-nAChRs. SLURP-2/mAChRs interactions are also probably involved in the control of keratinocyte growth. Computer modeling revealed possible SLURP-2 binding to the ‘classical’ orthosteric agonist/antagonist binding sites at α7 and α3β2-nAChRs.

  17. Synthesis and Structure-Activity Relationship Analysis of 5-HT7 Receptor Antagonists: Piperazin-1-yl Substituted Unfused Heterobiaryls

    Directory of Open Access Journals (Sweden)

    Lucjan Strekowski


    Full Text Available A series of piperazin-1-yl substituted unfused heterobiaryls was synthesized as ligands of the 5-HT7 receptors. The goal of this project was to elucidate the structural features that affect the 5-HT7 binding affinity of this class of compounds represented by the model ligand 4-(3-furyl-2-(4-methylpiperazin-1-ylpyrimidine (2. The SAR studies included systematical structural changes of the pyrimidine core moiety in 2 to quinazoline, pyridine and benzene, changes of the 3-furyl group to other heteroaryl substituents, the presence of various analogs of the 4-methylpiperazin-1-yl group, as well as additional substitutions at positions 5 and 6 of the pyrimidine. Substitution of position 6 of the pyrimidine in the model ligand with an alkyl group results in a substantial increase of the binding affinity (note a change in position numbers due to the nomenclature rules. It was also demonstrated that 4-(3-furyl moiety is crucial for the 5-HT7 binding affinity of the substituted pyrimidines, although, the pyrimidine core can be replaced with a pyridine ring without a dramatic loss of the binding affinity. The selected ethylpyrimidine (12 and butylpyrimidine (13 analogs of high 5-HT7 binding affinity showed antagonistic properties in cAMP functional test and varied selectivity profile—compound 12 can be regarded as a dual 5-HT7/5-HT2AR ligand, and 13 as a multi-receptor (5-HT7, 5-HT2A, 5-HT6 and D2 agent.

  18. Structure of the Receptor-Binding Protein of Bacteriophage Det7: a Podoviral Tail Spike in a Myovirus▿ (United States)

    Walter, Monika; Fiedler, Christian; Grassl, Renate; Biebl, Manfred; Rachel, Reinhard; Hermo-Parrado, X. Lois; Llamas-Saiz, Antonio L.; Seckler, Robert; Miller, Stefan; van Raaij, Mark J.


    A new Salmonella enterica phage, Det7, was isolated from sewage and shown by electron microscopy to belong to the Myoviridae morphogroup of bacteriophages. Det7 contains a 75-kDa protein with 50% overall sequence identity to the tail spike endorhamnosidase of podovirus P22. Adsorption of myoviruses to their bacterial hosts is normally mediated by long and short tail fibers attached to a contractile tail, whereas podoviruses do not contain fibers but attach to host cells through stubby tail spikes attached to a very short, noncontractile tail. The amino-terminal 150 residues of the Det7 protein lack homology to the P22 tail spike and are probably responsible for binding to the base plate of the myoviral tail. Det7 tail spike lacking this putative particle-binding domain was purified from Escherichia coli, and well-diffracting crystals of the protein were obtained. The structure, determined by molecular replacement and refined at a 1.6-Å resolution, is very similar to that of bacteriophage P22 tail spike. Fluorescence titrations with an octasaccharide suggest Det7 tail spike to bind its receptor lipopolysaccharide somewhat less tightly than the P22 tail spike. The Det7 tail spike is even more resistant to thermal unfolding than the already exceptionally stable homologue from P22. Folding and assembly of both trimeric proteins are equally temperature sensitive and equally slow. Despite the close structural, biochemical, and sequence similarities between both proteins, the Det7 tail spike lacks both carboxy-terminal cysteines previously proposed to form a transient disulfide during P22 tail spike assembly. Our data suggest receptor-binding module exchange between podoviruses and myoviruses in the course of bacteriophage evolution. PMID:18077713

  19. The role of the class A scavenger receptors, SR-A and MARCO, in the immune system. Part 1. The structure of receptors, their ligand binding repertoires and ability to initiate intracellular signaling

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    Szczepan Józefowski


    Full Text Available  Recognition of pathogens by innate immune cells is mediated by pattern recognition receptors (PRR, which include scavenger receptors (SR. The class A SR, SR-A/CD204 and MARCO, are characterized by the presence of collagenous and SR cysteine-rich domains in their extracellular portions. Both receptors are expressed mainly on macrophages and dendritic cells. Thanks to their ability to bind to a wide range of polyanionic ligands, the class A SR may participate in numerous functions of these cells, such as endocytosis, and adhesion to extracellular matrix and to other cells. Among SR-A ligands are oxidized lipoproteins and β-amyloid fibrils, which link SR-A to the pathogenesis of arteriosclerosis and Alzheimer’s disease. Despite the demonstration of class A SR involvement in so many processes, the lack of selective ligands precluded reaching definite conclusions concerning their signaling abilities. Using specific receptor ligation with antibodies, we showed that SR-A and MARCO trigger intracellular signaling, modulating pro-inflammatory and microbicidal activities of macrophages. Surprisingly, despite similarities in structure and ligand binding repertoires, SR-A and MARCO exert opposite effects on interleukin-12 (IL-12 production in macrophages. SR-A ligation also stimulated H2O2 and IL-10 production, but had no effect on the release of several other cytokines. These limited effects of specific SR-A ligation contrast with generalized enhancement of immune responses observed in SR-A-deficient mice. Recent studies have revealed that many of these effects of SR-A deficiency may be caused by compensatory changes in the expression of other receptors and/or disinhibition of signal transduction from receptors belonging to the Toll/IL-1R family, rather than by the loss of the receptor function of SR-A.

  20. Structure of the hepatitis E virus-like particle suggests mechanisms for virus assembly and receptor binding

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    Guu, Tom S.Y.; Liu, Zheng; Ye, Qiaozhen; Mata, Douglas A.; Li, Kunpeng; Yin, Changcheng; Zhang, Jingqiang; Tao, Yizhi Jane; (Sun Yat-Sen); (Rice); (Peking)


    Hepatitis E virus (HEV), a small, non-enveloped RNA virus in the family Hepeviridae, is associated with endemic and epidemic acute viral hepatitis in developing countries. Our 3.5-{angstrom} structure of a HEV-like particle (VLP) shows that each capsid protein contains 3 linear domains that form distinct structural elements: S, the continuous capsid; P1, 3-fold protrusions; and P2, 2-fold spikes. The S domain adopts a jelly-roll fold commonly observed in small RNA viruses. The P1 and P2 domains both adopt {beta}-barrel folds. Each domain possesses a potential polysaccharide-binding site that may function in cell-receptor binding. Sugar binding to P1 at the capsid protein interface may lead to capsid disassembly and cell entry. Structural modeling indicates that native T = 3 capsid contains flat dimers, with less curvature than those of T = 1 VLP. Our findings significantly advance the understanding of HEV molecular biology and have application to the development of vaccines and antiviral medications.

  1. Design, synthesis, biochemical studies, cellular characterization, and structure-based computational studies of small molecules targeting the urokinase receptor. (United States)

    Wang, Fang; Eric Knabe, W; Li, Liwei; Jo, Inha; Mani, Timmy; Roehm, Hartmut; Oh, Kyungsoo; Li, Jing; Khanna, May; Meroueh, Samy O


    The urokinase receptor (uPAR) serves as a docking site to the serine protease urokinase-type plasminogen activator (uPA) to promote extracellular matrix (ECM) degradation and tumor invasion and metastasis. Previously, we had reported a small molecule inhibitor of the uPAR·uPA interaction that emerged from structure-based virtual screening. Here, we measure the affinity of a large number of derivatives from commercial sources. Synthesis of additional compounds was carried out to probe the role of various groups on the parent compound. Extensive structure-based computational studies suggested a binding mode for these compounds that led to a structure-activity relationship study. Cellular studies in non-small cell lung cancer (NSCLC) cell lines that include A549, H460 and H1299 showed that compounds blocked invasion, migration and adhesion. The effects on invasion of active compounds were consistent with their inhibition of uPA and MMP proteolytic activity. These compounds showed weak cytotoxicity consistent with the confined role of uPAR to metastasis.

  2. Crystal structure of a complete ternary complex of T-cell receptor, peptide-MHC, and CD4

    Energy Technology Data Exchange (ETDEWEB)

    Yin, Yiyuan; Wang, Xin Xiang; Mariuzza, Roy A [Maryland


    Adaptive immunity depends on specific recognition by a T-cell receptor (TCR) of an antigenic peptide bound to a major histocompatibility complex (pMHC) molecule on an antigen-presenting cell (APC). In addition, T-cell activation generally requires binding of this same pMHC to a CD4 or CD8 coreceptor. Here, we report the structure of a complete TCR-pMHC-CD4 ternary complex involving a human autoimmune TCR, a myelin-derived self-peptide bound to HLA-DR4, and CD4. The complex resembles a pointed arch in which TCR and CD4 are each tilted ~65° relative to the T-cell membrane. By precluding direct contacts between TCR and CD4, the structure explains how TCR and CD4 on the T cell can simultaneously, yet independently, engage the same pMHC on the APC. The structure, in conjunction with previous mutagenesis data, places TCR-associated CD3εγ and CD3εδ subunits, which transmit activation signals to the T cell, inside the TCR-pMHC-CD4 arch, facing CD4. By establishing anchor points for TCR and CD4 on the T-cell membrane, the complex provides a basis for understanding how the CD4 coreceptor focuses TCR on MHC to guide TCR docking on pMHC during thymic T-cell selection.

  3. Structural correlates of affinity in fetal versus adult endplate nicotinic receptors (United States)

    Nayak, Tapan Kumar; Chakraborty, Srirupa; Zheng, Wenjun; Auerbach, Anthony


    Adult-type nicotinic acetylcholine receptors (AChRs) mediate signalling at mature neuromuscular junctions and fetal-type AChRs are necessary for proper synapse development. Each AChR has two neurotransmitter binding sites located at the interface of a principal and a complementary subunit. Although all agonist binding sites have the same core of five aromatic amino acids, the fetal site has ~30-fold higher affinity for the neurotransmitter ACh. Here we use molecular dynamics simulations of adult versus fetal homology models to identify complementary-subunit residues near the core that influence affinity, and use single-channel electrophysiology to corroborate the results. Four residues in combination determine adult versus fetal affinity. Simulations suggest that at lower-affinity sites, one of these unsettles the core directly and the others (in loop E) increase backbone flexibility to unlock a key, complementary tryptophan from the core. Swapping only four amino acids is necessary and sufficient to exchange function between adult and fetal AChRs.

  4. Crystal Structure of the PAC1R Extracellular Domain Unifies a Consensus Fold for Hormone Recognition by Class B G-Protein Coupled Receptors

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    Kumar, Shiva; Pioszak, Augen; Zhang, Chenghai; Swaminathan, Kunchithapadam; Xu, H. Eric (Van Andel); (NU Singapore)


    Pituitary adenylate cyclase activating polypeptide (PACAP) is a member of the PACAP/glucagon family of peptide hormones, which controls many physiological functions in the immune, nervous, endocrine, and muscular systems. It activates adenylate cyclase by binding to its receptor, PAC1R, a member of class B G-protein coupled receptors (GPCR). Crystal structures of a number of Class B GPCR extracellular domains (ECD) bound to their respective peptide hormones have revealed a consensus mechanism of hormone binding. However, the mechanism of how PACAP binds to its receptor remains controversial as an NMR structure of the PAC1R ECD/PACAP complex reveals a different topology of the ECD and a distinct mode of ligand recognition. Here we report a 1.9 {angstrom} crystal structure of the PAC1R ECD, which adopts the same fold as commonly observed for other members of Class B GPCR. Binding studies and cell-based assays with alanine-scanned peptides and mutated receptor support a model that PAC1R uses the same conserved fold of Class B GPCR ECD for PACAP binding, thus unifying the consensus mechanism of hormone binding for this family of receptors.

  5. Crystal structure of the PAC1R extracellular domain unifies a consensus fold for hormone recognition by class B G-protein coupled receptors.

    Directory of Open Access Journals (Sweden)

    Shiva Kumar

    Full Text Available Pituitary adenylate cyclase activating polypeptide (PACAP is a member of the PACAP/glucagon family of peptide hormones, which controls many physiological functions in the immune, nervous, endocrine, and muscular systems. It activates adenylate cyclase by binding to its receptor, PAC1R, a member of class B G-protein coupled receptors (GPCR. Crystal structures of a number of Class B GPCR extracellular domains (ECD bound to their respective peptide hormones have revealed a consensus mechanism of hormone binding. However, the mechanism of how PACAP binds to its receptor remains controversial as an NMR structure of the PAC1R ECD/PACAP complex reveals a different topology of the ECD and a distinct mode of ligand recognition. Here we report a 1.9 Å crystal structure of the PAC1R ECD, which adopts the same fold as commonly observed for other members of Class B GPCR. Binding studies and cell-based assays with alanine-scanned peptides and mutated receptor support a model that PAC1R uses the same conserved fold of Class B GPCR ECD for PACAP binding, thus unifying the consensus mechanism of hormone binding for this family of receptors.

  6. Differential effects of CSF-1R D802V and KIT D816V homologous mutations on receptor tertiary structure and allosteric communication.

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    Priscila Da Silva Figueiredo Celestino Gomes

    Full Text Available The colony stimulating factor-1 receptor (CSF-1R and the stem cell factor receptor KIT, type III receptor tyrosine kinases (RTKs, are important mediators of signal transduction. The normal functions of these receptors can be compromised by gain-of-function mutations associated with different physiopatological impacts. Whereas KIT D816V/H mutation is a well-characterized oncogenic event and principal cause of systemic mastocytosis, the homologous CSF-1R D802V has not been identified in human cancers. The KIT D816V oncogenic mutation triggers resistance to the RTK inhibitor Imatinib used as first line treatment against chronic myeloid leukemia and gastrointestinal tumors. CSF-1R is also sensitive to Imatinib and this sensitivity is altered by mutation D802V. Previous in silico characterization of the D816V mutation in KIT evidenced that the mutation caused a structure reorganization of the juxtamembrane region (JMR and facilitated its departure from the kinase domain (KD. In this study, we showed that the equivalent CSF-1R D802V mutation does not promote such structural effects on the JMR despite of a reduction on some key H-bonds interactions controlling the JMR binding to the KD. In addition, this mutation disrupts the allosteric communication between two essential regulatory fragments of the receptors, the JMR and the A-loop. Nevertheless, the mutation-induced shift towards an active conformation observed in KIT D816V is not observed in CSF-1R D802V. The distinct impact of equivalent mutation in two homologous RTKs could be associated with the sequence difference between both receptors in the native form, particularly in the JMR region. A local mutation-induced perturbation on the A-loop structure observed in both receptors indicates the stabilization of an inactive non-inhibited form, which Imatinib cannot bind.

  7. Muscarinic M3 receptors on structural cells regulate cigarette smoke-indu