BIOPHYSICS OF RECEPTOR/G-PROTEIN INTERACTIONS

本项目主要研究g蛋白偶联受体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，对于激素、神经递质、气味和光线引发的广泛细胞效应至关重要。这些知识的实际应用对于开发针对G蛋白偶联受体和G蛋白的更具体的治疗方法非常重要，因为据估计，超过40%的正在使用的药物对G蛋白偶联受体起作用。由于在获得受体/G蛋白复合物的高分辨率结构信息方面存在普遍的技术困难，受体究竟如何激活G蛋白仍不清楚。本提案旨在解决膜受体/ g蛋白复合物催化信号传递的主要问题，即g蛋白β - γ -亚基复合物在受体催化α -亚基活化中的分子作用。我们提出验证一种假设，即β -亚基复合体被视紫红质积极利用，以控制α -亚基上的核苷酸结合位点。我们制备了一组突变G-蛋白，其中一个视紫红质相互作用域，即转导蛋白γ亚基的c端，是丙氨酸替换、缺失和序列逆转突变的目标。我们已经证明，一些突变严重影响与光激活视紫红质的偶联。我们将通过检查视紫红质-转导素相互作用和转导素激活的各个单独步骤，广泛地调查这些突变体的功能特性。最近，我们开发了源自g蛋白表面结构域的模型模拟肽，以便通过核磁共振光谱研究视紫红质-转导素界面的动力学。我们将研究失活突变和法尼化缺失对光激活视紫红质结合状态下γ -亚基C端结构域结构特征的影响。我们将确定γ -亚基的构象开关是否受到突变的影响。对转导蛋白突变体的功能研究和与模型模拟肽并行获得的结构数据将为β - γ -亚基复合物在紫红质催化的转导蛋白活化中的作用提供有价值的信息。

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.