Functional Determinants in G Protein-coupled Receptors

描述（由申请人提供）：这项工作的目的是揭示G蛋白偶联受体（gpcr）特异性的决定因素。这些受体和由它们控制的途径是比任何其他类型的途径更多的药物的作用点。该方法将计算与实验相结合，并依赖于PI开发的一种新的序列分析策略，该策略使用系统发育树来识别蛋白质中功能重要的残基。要测试的假设是：(1)通过提供大量的进化突变和分析，否则将仍然未被开发，这种进化追踪策略（ET）将使我们能够识别gpcr中的信号转导决定因素。这些决定因素依次应该揭示(2)控制G蛋白激活的通用开关跨膜构象开关，(3)每个受体精确配体和G蛋白特异性的不同结合位点和传递途径。这些假设将首先针对文献中已有的大量GPCR突变进行测试，然后通过视紫红质靶向突变进行测试，旨在可预测地改变配体和G蛋白偶联特异性。具体来说，目标1是开发计算策略和计算工具，将这种进化追踪策略应用于大规模的gpcr。目的2是通过计算确定跨膜域配体诱导信号传导的决定因素，然后通过文献和视紫红质的直接实验进行实验验证。目的3是确定G蛋白偶联和特异性的决定因素，随后进行如上的实验验证。

DESCRIPTION (provided by applicant): The objective of this work is to uncover the determinants of specificity in G protein-coupled receptors (GPCRs). These receptors and the pathways controlled by them are the sites of action of more drugs than any other class of pathways. The approach marries computation with experiment and relies on a novel sequence analysis strategy developed by the PI, that uses phylogenetic trees to identify functionally important residues in proteins. The hypotheses to be tested are that (1) by providing a vast number of evolutionary mutations and assays that would otherwise remain untapped, this Evolutionary Trace strategy (ET) will allow us to identify signal transduction determinants in GPCRs. These determinants in turn should reveal (2) a universal on-off transmembrane conformational switch that controls G protein activation and, (3), distinct binding sites and relay pathways that are specific to the each receptors' precise ligand and G protein(s). These hypotheses will be tested first against the vast body of GPCR mutations already available from the literature, and second through targeted mutations in rhodopsin that aim to predictably change ligand and G protein coupling specificity. Specifically, Aim 1 is to develop the computational strategy and computational tools to apply this Evolutionary Trace strategy to GPCRs on a large-scale. Aim 2 is to identify computationally the determinants of ligand induced signaling in the transmembrane domain, followed by experimental validation through the literature and direct experiments in rhodopsin. Aim 3 is to identify the determinant of G protein coupling and specificity, followed by experimental validation as above. Thus we propose an integrated strategy to study the evolution of sequence, structure and function in GPCRs, closely coupled with experimental proof of principle in rhodopsin. In addition to the important insight into the molecular basis of GPCR function, and of drug activity that these studies should provide, development of an efficient, rational, and general strategy to guide experiments in GPCR would be a significant contribution to an important and difficult problem.