Structure And Functions Of Signal-transducing G-proteins

信号转导 G 蛋白的结构和功能

• 批准号: 7130185
• 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/7130185
• 关键词: G protein; G protein coupled receptor kinase; biochemistry; biological signal transduction; biophysics; calcium; glutamate receptor; molecular cloning; protein protein interaction; receptor expression; serotonin receptor; surface plasmon resonance; taste buds

We extended our work on the human 5-HT receptors to understand the apparent discrepancies among investigations determining G-protein selectivity for GPCR structures in heterologous cell expression models. To examine this we compared the apparent G-protein selectivity for two human 5-HT receptors, 5HT2c and 5HT1A, activating Gq and Gi respectively. Examined by co-expression of the receptors and G-proteins in Sf9 cells, both 5HT2c and 5HT1A appeared to activate Gi significantly more than Gq. However, when receptors and G-proteins were expressed separately and examined by in vitro reconstitution, the 5HT2c displayed the expected preference for Gq, while 5HT1A preferred Gi. These experiments reveal a significant artifact of co-expression of GPCR and G-proteins in heterologous cells as a method to examine receptor signaling. Therefore, we have re-examined the structural determinants of GPCR-selectivity for chimeric G-protein alpha subunits composed of sequence from Gq and Gi proteins using in vitro assays. As opposed to earlier investigations using cell-based assays, our studies have identified the region of Gq which is selectively recognized by the 5HT2c as residues 301-322 and not the C-terminal 5 amino acids (350-353) as previously reported. In collaboration with Dr. Susan Sullivan, we have identified, cloned and expressed the entire repertoire of human bitter taste receptor (hT2R) genes. We have used the expressed hT2Rs to identify both the bitter compounds recognized by several of these receptors and the G-protein signaling pathways activated in response to bitter substances. Because the 25 hT2R genes must recognize a very large number of toxic substances, we had predicted a broad specificity with each of the hT2Rs recognizing a number of bitter compounds. While two of the h2TR genes encoding broadly tuned bitter receptors, surprisingly, two of the structures respond uniquely to a single compound of the some 60 tested. These investigations are providing fundamental insights into the molecular basis for human chemosensation. We have characterized a novel compound developed as a regulator of calcium homeostasis acting on the unique calcium sensing receptor (hCaR). Martial Ruat and colleagues from the CNRS, France, have provided the compound Calindol for our testing with transmembrane core domain (7TM) constructs we have produced from hCaR in examination of the fundamental mechanism for signaling by this unique receptor. We have established that Calindol acts both as a synergist for the natural calcium activation of the unmodified hCaR and as a potent agonist of the 7TM construct which lacks calcium ion sensing. Our data favor a new model for the molecular regulation of the hCaR which may pertain to the entire family of similar structures encoded in the human genome, including the receptors for the neurotransmitters GABBA and glutamate. Our data also provide an approach for studying the signaling properties of the more than 30 uncharacterized similar receptors identified in the human genome. Lastly, R. Victor Rebois has joined the laboratory and continued a project establishing resonance energy transfer-based methods for investigating the cellular localization and protein interactions of GPCRs, G-proteins and cellular effectors. We have initiated a research project to validate the protein-interactions of novel fluorescent-constructs for GPCRs and G-protein subunits using our in vitro biochemical and biophysical methods. Our initial study has focused on the D4 Dopamine receptor, as this GPCR cannot be labeled by the jellyfish fluorescent proteins.

我们扩展了对人类5-HT受体的工作，以了解确定异源细胞表达模型中GPCR结构的G蛋白选择性的研究之间的明显差异。为了检查这一点，我们比较了两个人类5HT受体（5HT2C和5HT1A）的明显G蛋白选择性，分别激活GQ和GI。通过在SF9细胞中共表达受体和G蛋白的共表达，5HT2C和5HT1A似乎都比GQ激活了GI的激活明显大。但是，当受体和G蛋白分别表达并通过体外重构进行检查时，5HT2C显示了对GQ的预期偏好，而5HT1A则首选GI。这些实验揭示了GPCR和G蛋白在异源细胞中的共表达的显着伪像，作为检查受体信号传导的一种方法。因此，使用体外测定法，我们重新检查了由GQ和GI蛋白序列组成的嵌合GPCR选择性的结构决定因素。与使用基于细胞的测定的早期研究相反，我们的研究确定了GQ的区域，该区域被5HT2C选择性地识别为残基301-322，而不是C-末端5氨基酸（350-353），如前所述。 我们与苏珊·沙利文（Susan Sullivan）博士合作，确定，克隆并表达了人类苦味受体（HT2R）基因的整个曲目。我们已经使用表达的HT2RS来识别这些受体中的几种识别的苦味化合物，以及响应于苦味物质而激活的G蛋白信号传导途径。由于25种HT2R基因必须识别大量的有毒物质，因此我们预测了每种HT2Rs都识别出许多苦涩化合物的广泛特异性。虽然两个编码广泛调谐苦味受体的H2TR基因令人惊讶，但其中两个结构对大约60种测试的单个化合物的响应唯一。这些研究为人类化学一致化的分子基础提供了基本见解。 我们已经表征了一种新型化合物，该化合物是作用于独特的钙感应受体（HCAR）的钙稳态调节剂。来自法国CNRS的武术RUAT和同事为我们从HCAR生产的跨膜核心结构域（7TM）构建体提供了复合Calindol，以检查该独特受体信号传导的基本机制。我们已经确定Calindol既充当未修饰的HCAR自然钙激活的协同作用，也是缺乏钙离子传感的7TM构建体的有效激动剂。我们的数据有利于HCAR分子调节的新模型，该模型可能与人类基因组中编码的整个类似结构的家族有关，包括神经递质GABBA和谷氨酸的受体。我们的数据还提供了一种研究人类基因组中鉴定出的30多个未表征的类似受体的信号传导特性的方法。 最后，R。VictorRebois加入了实验室，并继续进行了一个项目，以建立基于共振能量转移的方法，以研究GPCR，G蛋白和细胞效应子的细胞定位和蛋白质相互作用。我们启动了一个研究项目，旨在验证使用我们的体外生化和生物物理方法的GPCR和GPROTEN亚基的新型荧光结构的蛋白质交流。我们的最初研究集中在D4多巴胺受体上，因为该GPCR不能用水母荧光蛋白标记。