Structure and Functions of Signal-Transducing G-Proteins

信号转导 G 蛋白的结构和功能

• 批准号: 6104237
• 负责人: JOHN K NORTHUP
• 金额: --
• 依托单位: NATIONAL INSTITUTE ON DEAFNESS AND OTHER COMMUNICATION DISORDERS
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6104237
• 关键词: 3T3 cells; G protein; Sf9 cell line; allosteric site; biological signal transduction; bombesin; conformation; cyclic GMP; ketanserin; neuropeptide receptor; phosphodiesterases; piperazines; protein reconstitution; protein structure function; receptor coupling; receptor sensitivity; serotonin inhibitor; serotonin receptor; structural biology; visual perception; 3T3 cells; G protein; Sf9 cell line; allosteric site; biological signal transduction; bombesin; conformation; cyclic GMP; ketanserin; neuropeptide receptor; phosphodiesterases; piperazines; protein reconstitution; protein structure function; receptor coupling; receptor sensitivity; serotonin inhibitor; serotonin receptor; structural biology; visual perception

In the past year we have characterized and extended our new in situ receptor-G-protein assay systems to increase their utility for a diverse family of receptor structures. We have improved the in situ reconstitution procedures to facilitate the investigation of differences in G-protein interactions for receptors expressed in mammalian cells as well as baculoviral expression in Sf9 cells. Using these techniques we have examined the bombesin receptor family (Gastrin releasing peptide receptor -- GRPr, Neuromedin B receptor -- NMBr, and bombesin receptor subtype 3, BRS3) stably expressed in mouse 3T3 fibroblasts. The GRPr and NMBr both couple exclusively to G-alpha-q and display similar selectivity for G-beta-gamma dimers. However, the two receptors differ quantitatively in Km for G-alpha-q, K1/2 for G- beta-gamma, and Vmax for catalyzed GTP exchange. The BRS3 receptor failed to couple to any of the tested G-proteins. To identify the G-protein coupling partner for BRS3, we have co- infected Sf9 cells with this receptor or GRPr together with G- alpha and G-beta-gamma viruses. In these experiments we find that the BRS3 receptor can activate, albeit partially, the mammalian G-alpha-q. Thus, we conclude that the closely related bombesin receptor structures have distinct G-protein selectivity. These data show the utility of our in situ approach for the investigation of receptor-selectivity of G-proteins. We have also extended our investigations of 5HT2c receptors by expressing the two human allelic variants of this receptor (cys and ser 23). Both of these receptors display a high basal activity, which is inhibited by classical antagonists of the 5HT2 receptor. This basal activity of the receptor is increased by high G-protein concentrations. Increasing G-protein increases the efficacy of the partial agonist mCPP to be identical to the full agonist 5HT, and at full 5HT2c-G-protein saturations the receptors are fully activated in the absence of agonists. The inverse agonist efficacy of mianserin, ketanserin and mesulergine is also increased by high G-protein saturation, so that at full saturations these antagonists inhibit receptor activity 30-fold. These studies provide a molecular insight into the activation mechanism of G-protein coupled receptors, revealing that the activated conformation of the receptor is stabilized by G-protein interaction. In situ reconstitution thus has provided a powerful approach for molecular analysis of important CNS receptor function. Lastly, we have identified the biological function of the helical domain (HD) portion of the G-protein alpha subunit revealed by the recently solved crystal structures for the retinal G-protein. We succeeded in the autonomous expression of the HD proteins from representatives of all G-protein families, and we examined the biochemical functions for the purified HD proteins using the vertebrate visual model. The HD functions as an allosteric modulator of the target enzyme for visual transduction, cyclic GMP phosphodiesterase (PDE). This HD regulation is biologically selective, only the HD proteins from G-proteins previously known to regulate the PDE displayed this activity. Further, we have characterized the molecular basis for the HD function as an entirely novel allosteric mechanism, which we suggest, may apply to all G-protein regulated enzymes.

在过去的一年中，我们进行了描述和扩展 我们的新原位受体-G蛋白测定系统，以增加其 多元化受体结构家族的实用程序。我们有所改善 现场重建程序，以促进调查 G蛋白相互作用的受体差异 哺乳动物细胞以及SF9细胞中的细菌病毒表达。 使用这些技术，我们检查了孟买受体 家族（胃蛋白释放肽受体-GRPR，神经蛋白B 受体 - NMBR和bombesin受体亚型3，BRS3）稳定 在鼠标3T3成纤维细胞中表达。 GRPR和NMBR 夫妇专门针对G-Alpha-Q，并显示出相似的选择性 G-Beta-Gamma二聚体。但是，两个受体不同 用于G-Alpha-Q的km中，G-Beta-gamma的K1/2和 VMAX用于催化的GTP交换。 BRS3受体未能 将任何经过测试的G蛋白求助。识别G蛋白 BRS3的耦合伙伴，我们与此相关的SF9单元 受体或GRPR与g- alpha和g-beta-gamma一起 病毒。在这些实验中，我们发现BRS3受体可以 激活，尽管部分是哺乳动物G-Alpha-Q。因此，我们 结论是，密切相关的孟买受体结构具有 独特的G蛋白选择性。这些数据显示了我们的实用性 原位方法研究受体选择性 G蛋白。我们还扩展了对5HT2C的调查 通过表达这两个人类等位基因变体的受体 受体（CYS和SER 23）。这两个受体都表现出很高的 基础活性，这受到经典拮抗剂的抑制 5HT2受体。受体的基础活性通过 高G蛋白浓度。增加G蛋白增加了 部分激动剂MCPP与完整的功效相同 激动剂5HT，在完整的5HT2C-G蛋白饱和受体下 在没有激动剂的情况下完全激活。反向激动剂 甲烯素，酮酸和中硫酸的功效也会提高 通过高G蛋白饱和度，因此在充分饱和时 拮抗剂抑制受体活性30倍。这些研究提供了 分子洞察G蛋白的激活机制 耦合受体，揭示了激活的构象 受体通过G蛋白相互作用稳定。原位重构 因此，为分子分析提供了强大的方法 重要的中枢神经系统受体功能。最后，我们确定了 螺旋结构域（HD）部分的生物学功能 最近溶液晶体揭示的G蛋白α亚基 视网膜G蛋白的结构。我们成功了 来自代表的HD蛋白的自主表达 所有G蛋白家族，我们检查了生化功能 用于使用脊椎动物视觉模型纯化的HD蛋白。这 HD充当目标酶的变构调节剂 视觉转导，环状GMP磷酸二酯酶（PDE）。这个高清 调节具有生物学选择性，只有来自 G蛋白以前已知可以调节PDE 活动。此外，我们还表征了分子基础 高清功能是一种完全新颖的变构机制，我们 建议，可能适用于所有G蛋白调节的酶。