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首页> 外文学位 >The structural basis of Gbeta5-RGS7 interaction with the muscarinic M3 receptor and implications for its role in functional pathway selectivity.
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The structural basis of Gbeta5-RGS7 interaction with the muscarinic M3 receptor and implications for its role in functional pathway selectivity.

机译：Gbeta5-RGS7与毒蕈碱M3受体相互作用的结构基础及其对功能途径选择性的影响。

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摘要

The neurotransmitter acetylcholine (Ach) is arguably the most important in the CNS. Two classes of receptors are activated by Ach: nicotinic (ionotropic) and muscarinic (metabotropic). Muscarinic receptor pharmacology is well-defined, but still lacks receptor sub-type specificity seen in other GPCR families like adrenergic receptors. We have found that Gbeta5-RGS7, a unique G-protein obligate heterodimer complex, selectively attenuates Ca2+ signaling through the muscarinic M3 receptor (M3R).;The G protein beta subunit Gbeta-RGS7 uniquely forms heterodimers with R7 family regulators of G protein signaling (RGS) proteins (RGS6, RGS7, RGS9, and RGS11) instead of Ggamma. While the Gbeta5-RGS7 complex attenuates Ca2+ signaling mediated by M3R, the route of Ca2+ entry (i.e., release from intracellular stores and/or influx across the plasma membrane) is unknown. Here I show that in addition to suppressing carbachol-stimulated Ca2+ release, Gbeta5-RGS7 enhanced Ca2+ influx. This novel effect of Gbeta5-RGS7 was blocked by nifedipine and 2-APB. Experiments with pertussis toxin, RGS domain-deficient mutant of RGS7 and a novel inhibitor of Gq, showed that Gbeta5-RGS7 modulated a Gq-mediated pathway. These studies indicate that Gbeta5-RGS7, independent of RGS7 GAP activity, couples M3R to a nifedipine-sensitive Ca2+ channel.;In neurons and glands, muscarinic signaling plays a major role in secretion. The novel finding that GGbeta5-RGS7 enhances M3R-stimulated insulin secretion can explain why loss of Gbeta5 results in impaired insulin secretion in mice. In insulin secreting cells, I found that the mechanism of GGbeta5-RGS7-enhanced Ca2+ signaling is similar to the one identified in CHO-K1 cells as it is sensitive to nifedipine.;I also compared the action of Gbeta5-RGS7 on M3R-induced Ca 2+ influx and release elicited by different muscarinic agonists. Responses to oxotremorine-m were insensitive to Gbeta5-RGS7. Pilocarpine responses consisted of a large release and modest influx components, of which the former was strongly inhibited whereas the latter was insensitive to Gbeta5-RGS7. McN-A-343 was the only compound whose total Ca2+ response was enhanced by Gbeta5-RGS7, attributed to, in part, by the relatively small Ca2+ release this partial agonist stimulated. Together these results show that distinct agonists not only have differential M3R functional selectivity, but also confer specific sensitivity to the GGbeta5-RGS7 complex.;A deeper understanding of the structural basis of this ligand bias towards sensitivity to Gbeta5-RGS7 may lead to new strategies for selective therapeutics. My biophysical studies provide additional insights into the structural basis of Gbeta5-RGS7 regulation of M3R signaling.;Altogether, this dissertation work led to the novel finding that Gbeta5-RGS7 has a dual effect on M3R-stimulated Ca2+ signaling, and the newly discovered positive effect on Ca2+ influx plays an important role in hormone and/or neurotransmitter secretion stimulated by the M3R pathway.
机译：神经递质乙酰胆碱（Ach）可以说是中枢神经系统中最重要的。 Ach激活两类受体：烟碱（离子型）和毒蕈碱（代谢型）。毒蕈碱受体的药理学已明确定义，但仍缺乏其他GPCR家族（如肾上腺素能受体）所见的受体亚型特异性。我们发现，Gbeta5-RGS7，一种独特的G蛋白专性异二聚体复合物，通过毒蕈碱M3受体（M3R）选择性地减弱Ca2 +信号传导； （RGS）蛋白（RGS6，RGS7，RGS9和RGS11）代替Ggamma。虽然Gbeta5-RGS7复合物减弱了由M3R介导的Ca2 +信号传导，但Ca2 +进入的途径（即从细胞内存储释放和/或跨质膜流入）尚不清楚。在这里我表明，除了抑制卡巴胆碱刺激的Ca2 +释放外，Gbeta5-RGS7还增强了Ca2 +的内流。 Gbeta5-RGS7的这种新作用被硝苯地平和2-APB阻断。百日咳毒素，RGS7的RGS域缺陷型突变体和新型Gq抑制剂的实验表明，Gbeta5-RGS7调节了Gq介导的途径。这些研究表明，独立于RGS7 GAP活性的Gbeta5-RGS7将M3R偶联到硝苯地平敏感的Ca2 +通道上。在神经元和腺体中，毒蕈碱信号在分泌中起主要作用。 GGbeta5-RGS7增强M3R刺激的胰岛素分泌的新发现可以解释为什么Gbeta5的丢失会导致小鼠胰岛素分泌受损。在胰岛素分泌细胞中，我发现GGbeta5-RGS7增强的Ca2 +信号转导机制与CHO-K1细胞中确定的机制相似，因为它对硝苯地平敏感。我还比较了Gbeta5-RGS7对M3R诱导的作用。 Ca 2+的流入和释放是由不同的毒蕈碱激动剂引起的。对oxotremorine-m的反应对Gbeta5-RGS7不敏感。毛果芸香碱反应由大量释放和适度的内流成分组成，其中前者受到强烈抑制，而后者对Gbeta5-RGS7不敏感。 McN-A-343是唯一的化合物，其总Ca2 +响应可通过Gbeta5-RGS7增强，部分归因于该部分激动剂所刺激的相对较小的Ca2 +释放。这些结果共同表明，不同的激动剂不仅具有不同的M3R功能选择性，而且还赋予GGbeta5-RGS7复合物特定的敏感性。；对该配体偏向于对Gbeta5-RGS7敏感性的偏向的结构基础的更深入了解可能会导致新的策略用于选择性治疗。我的生物物理研究为Gbeta5-RGS7调控M3R信号传导的结构基础提供了更多见解。总而言之，本论文的工作导致了一个新发现，即Gbeta5-RGS7对M3R刺激的Ca2 +信号传导具有双重作用，并且新发现的阳性结果对Ca2 +内流的影响在M3R途径刺激的激素和/或神经递质分泌中起重要作用。

著录项

  • 作者

    Karpinsky-Semper, Darla.;

  • 作者单位

    University of Miami.;

  • 授予单位 University of Miami.;
  • 学科 Pharmacology.;Cellular biology.;Molecular biology.
  • 学位 Ph.D.
  • 年度 2014
  • 页码 129 p.
  • 总页数 129
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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