BIOPHYSICS OF RECEPTOR/G-PROTEIN INTERACTIONS

受体/G 蛋白相互作用的生物物理学

• 批准号: 6730563
• 负责人: OLEG G KISSELEV
• 金额: $ 18.38万
• 依托单位: SAINT LOUIS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-01 至 2007-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6730563
• 关键词: G protein; alanine; binding sites; biological signal transduction; biomimetics; biophysics; cell membrane; colorimetry; fluorescence spectrometry; guanosine diphosphate; high throughput technology; nuclear magnetic resonance spectroscopy; physical model; protein protein interaction; receptor coupling; receptor expression; rhodopsin; transducin; ultraviolet spectrometry

This project focuses on the basic molecular mechanisms of transmembrane signal transduction via G-protein coupled receptors. Understanding of how these plasma membrane receptors interact with G-proteins is crucial for a broad range of cellular effects triggered by hormones, neurotransmitters, odorants and light. Practical applications of this knowledge will be important for the development of more specific therapeutics that target G-protein coupled receptors and G-proteins, because it's estimated that more than 40 percent of drugs in use work on G- protein coupled receptors. How exactly receptors activate G- proteins remains unclear, because of the universal technical difficulties in obtaining the high-resolution structural information in receptor/G-protein complexes. This proposal aims at resolving a major question about catalytic signal relay in membrane receptor/G-protein complexes, namely, the molecular role for the G-protein betagamma-subunit complex in the receptor catalyzed activation of the alpha-subunit. We propose to test a hypothesis that the betagamma-subunit complex is actively employed by rhodopsin in order to control the nucleotide-binding site on the alpha-subunit. We have prepared a set of mutant G- proteins in which a rhodopsin interaction domain, the C-terminus of transducin gamma-subunit, is targeted by Alanine replacements, deletions, and sequence reversal mutations. We have shown that some of the mutations severely affect coupling with the light- activated rhodopsin. We will survey extensively the functional properties of these mutants by examining various individual steps of rhodopsin-transducin interactions and transducin activation. Recently, we have developed the model mimetic peptides derived from the surface domains of G-proteins in order to study the dynamics of rhodopsin-transducin interface by NMR spectroscopy. We will study the effect of the inactivating mutations and the lack of farnesylation on the structural features of the C- terminal domain of the gamma-subunit in the light activated rhodopsin-bound state. We will determine whether the conformational switch in the gamma-subunit is affected by mutations. Functional studies of transducin mutants and structural data obtained in parallel with model mimetic peptides will yield valuable information on the role of the betagamma- subunit complex in rhodopsin-catalyzed activation of transducin.

该项目的重点是通过G蛋白偶联受体转导跨膜信号转导的基本分子机制。 了解这些质膜受体如何与G蛋白相互作用对于由激素，神经递质，气味和光引发的广泛细胞作用至关重要。 该知识的实际应用对于靶向G蛋白偶联受体和G蛋白的更具体的治疗剂的开发将很重要，因为据估计，在G蛋白偶联受体上使用的药物中有40％以上的药物。 由于在获得受体/G蛋白复合物中高分辨率的结构信息方面存在普遍的技术困难，因此如何确切地激活G蛋白仍然不清楚。 该建议旨在解决有关膜受体/G蛋白复合物中催化信号继电器的主要问题，即，G蛋白Betagamma-Subunit复合物在受体催化α-亚基的激活中的分子作用。 我们建议检验一个假设，即视紫红蛋白积极利用Betagamma-Subunit复合物来控制α-亚基上的核苷酸结合位点。 我们已经准备了一组突变的g-蛋白，其中丙氨酸置换量，缺失和序列反转突变旨在靶向变性蛋白γ-亚基的C-末端，即透明蛋白γ-亚基的C末端。 我们已经表明，某些突变严重影响与光活化的视紫红质的耦合。 我们将通过检查视紫红质 - 透明蛋白相互作用和跨丁蛋白激活的各个单个步骤，对这些突变体的功能特性进行广泛的调查。最近，我们开发了从G蛋白表面结构域衍生的模拟模拟肽，以研究NMR光谱法研究视紫红质 - 透明蛋白界面的动力学。我们将研究灭活突变的影响以及缺乏法尼化对在光激活的杜鹃蛋白结合状态下γ-亚基c-末端结构域的结构特征的影响。 我们将确定γ-亚基中的构象开关是否受到突变的影响。 与模型模拟肽并行获得的透明蛋白突变体和结构数据的功能研究将产生有关β-亚基复合物在旋淡蛋白催化的传dendducin激活中的作用的宝贵信息。