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。这些受体和它们控制的途径是比任何其他类型途径更多药物的作用位点。该方法将计算与实验结合起来，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。有待检验的假设是：（1）通过提供大量的进化突变和分析，否则将保持未开发的，这种进化跟踪策略（ET）将使我们能够识别GPCR中的信号转导决定簇。这些决定子反过来应该揭示（2）控制G蛋白活化的通用开关跨膜构象开关，以及（3）对每个受体的精确配体和G蛋白特异的不同结合位点和中继途径。这些假设将首先针对已经从文献中获得的大量GPCR突变进行测试，其次通过视紫红质中的靶向突变进行测试，这些突变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，将这种进化跟踪策略大规模地应用于GPCR。目的2是通过计算确定配体诱导的跨膜结构域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蛋白偶联的决定簇和特异性，然后进行上述实验验证。 因此，我们提出了一个综合的策略来研究GPCR的序列，结构和功能的演变，密切结合在视紫红质的原理的实验证明。除了对GPCR功能的分子基础和这些研究应该提供的药物活性的重要见解之外，开发一种有效的、合理的和通用的策略来指导GPCR实验将是对一个重要而困难的问题的重大贡献。

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.