BIOPHYSICS OF RECEPTOR/G-PROTEIN INTERACTIONS

本项目主要研究通过G蛋白偶联受体进行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。这一知识的实际应用对于开发针对G蛋白偶联受体和G蛋白的更具体的疗法将是重要的，因为据估计，使用中的药物中有40%以上作用于G蛋白偶联受体。受体究竟是如何激活G蛋白的尚不清楚，因为在获得受体/G蛋白复合体的高分辨率结构信息方面存在普遍的技术困难。这一建议旨在解决膜受体/G-蛋白复合体中催化信号传递的一个主要问题，即G-蛋白β-亚基复合体在受体催化的α-亚基活化中的分子作用。我们建议检验一种假设，即视紫红质主动利用β-亚基复合体来控制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，其中视紫红质相互作用结构域，转导蛋白伽马亚基的C末端，被丙氨酸替换、缺失和序列逆转突变所靶向。我们已经证明，一些突变严重影响与光激活的视紫红质的偶联。我们将通过检测视紫红质-转导蛋白相互作用和转导蛋白激活的不同步骤来广泛考察这些突变体的功能特性。最近，为了用核磁共振波谱研究视紫红质-转导蛋白界面的动力学，我们开发了来源于G蛋白表面结构域的模拟多肽模型。我们将研究失活突变和缺乏法尼化对光激活视紫红质结合状态下伽玛亚单位C-末端结构域结构特征的影响。我们将确定伽马亚基中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多肽平行获得的结构数据将提供关于β-淀粉-亚单位复合体在视紫红质催化的转导蛋白激活中所起作用的有价值的信息。

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.