BIOPHYSICS OF RECEPTOR/G-PROTEIN INTERACTIONS

本项目主要研究G蛋白偶联受体介导的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蛋白的更特异性的治疗方法将是重要的，因为据估计，超过40%的使用中的药物作用于G蛋白偶联受体。 由于获得受体/G蛋白复合物中的高分辨率结构信息的普遍技术困难，受体究竟如何激活G蛋白仍不清楚。 该提议旨在解决关于膜受体/G蛋白复合物中催化信号传递的主要问题，即G蛋白β-亚基复合物在受体催化的α-亚基活化中的分子作用。 我们建议测试一个假设，即β γ亚基复合物被视紫红质积极利用，以控制α亚基上的核苷酸结合位点。 我们已经制备了一组突变的G蛋白，其中视紫红质相互作用结构域，转导素γ-亚基的C-末端，通过丙氨酸置换、缺失和序列逆转突变靶向。 我们已经表明，一些突变严重影响耦合与光激活视紫红质. 我们将广泛调查这些突变体的功能特性，通过检查各个单独的步骤视紫红质转导相互作用和转导激活。最近，我们开发了G蛋白表面结构域的模拟肽，以研究视紫红质-转导蛋白界面的动力学。我们将研究失活突变和缺乏法尼基化对光激活视紫红质结合状态下γ亚基的C末端结构域的结构特征的影响。 我们将确定γ亚基中的构象转换是否受到突变的影响。 与模型模拟肽平行获得的转导素突变体和结构数据的功能研究将产生有价值的信息的β-亚基复合物在视紫红质催化激活的转导素的作用。

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.