REGULATORY DOMAINS OF G PROTEIN COUPLED RECEPTORS

G蛋白偶联受体是细胞表面受体家族， 调节G蛋白信号通路，以响应各种 环境刺激 这些事件对于控制 真核细胞的生长、分化和代谢， 第二信使的刺激，磷酸化级联反应和 离子通道的调节。 控制活性物质的寿命 通过一个被称为脱敏的过程对受体的影响是一个关键的方面， G蛋白信号通路的调节。 视紫红质 脊椎动物视杆细胞的光感受器，已被用作结构 研究G蛋白偶联蛋白之间相互作用的模型 受体及其G蛋白，以及G蛋白偶联 介导受体脱敏的受体激酶和抑制蛋白。 以前，我们的实验室确定了几个不重叠的领域， 涉及Gt活化、相互作用 视紫红质激酶和抑制蛋白。 本建议的目的是 深入了解分子作用， 我们已经确定的受体调节区域 脱敏。 为了实现这一目标，聚集的丙氨酸 突变将被分成单独的丙氨酸点突变体， 测试它们被磷酸化和结合抑制蛋白的能力。 将在这些位点进行额外的突变以确定 对电荷、疏水性、尺寸或特定次级的要求 结构 将分析影响磷酸化的突变体， 确定这些位点对结合视紫红质是否重要 激酶，调节其活性，或两者。 影响抑制蛋白的突变体 结合将被检查，以确定他们是否是一个高- 视紫红质表面上的亲和结合位点，或者它们是否 在调节抑制蛋白从低到高的转变中很重要 亲和力形式 高度保守的氨基酸Arg-135的突变体， 我们的实验室显示，它是磷酸化的，并结合抑制蛋白， 缺乏11-顺式-视黄醇。另外的取代诱变将是 分析特定氨基酸的需求，或 二级结构在这个网站上。 视网膜病变的后果- 独立的磷酸化和抑制蛋白结合的能力， 还将进行激活Gt的突变体。 更好地理解 视紫红质的胞质结构域控制 G蛋白激活和脱敏将有助于确定潜在的 G蛋白偶联缺陷导致的疾病机制 受体信号通路和设计治疗策略。

G protein-coupled receptors are a family of cell surface receptors that regulate G protein signaling pathways in response to a variety of environmental stimuli. These events are important in the control of growth, differentiation and metabolism in eukaryotic cells through the stimulation of second messengers, phosphorylation cascades and the regulation of ion channels. Control of the lifetime of an active receptor through a process known as desensitization is a critical aspect of the regulation of G protein signaling pathways. Rhodopsin, the photoreceptor of the vertebrate rod cell, has been used as a structural model for investigating the interactions between G protein-coupled receptors and their G proteins, as well as the G protein-coupled receptor kinases and arrestins that mediate receptor desensitization. Previously, our laboratory identified several nonoverlapping domains on the surface of rhodopsin that are involved in Gt activation, interaction with rhodopsin kinase and arrestin. The aim of the present proposal is to provide an in-depth understanding of the molecular roles played by domains that we have identified in the regulation of receptor desensitization. In order to achieve this goal, clustered alanine mutations will be separated into individual alanine point mutants and tested for their ability to be phosphorylated and to bind arrestin. Additional mutations will be made at these sites to determine the requirement for charge, hydrophobicity, size or specific secondary structure. The mutants that affect phosphorylation will be analyzed to determine whether these sites are important for binding rhodopsin kinase, regulating its activity, or both. Mutants that affect arrestin binding will be examined to determine whether they are part of a high- affinity binding site on the surface of rhodopsin or whether they are important in regulating the transition of arrestin from a low to a high affinity form. Mutants of the highly conserved amino acid Arg-135, was shown by our laboratory to be phosphorylated and to bind arrestin in the absence of 11-cis-retinal. Additional substitution mutagenesis will be performed to analyze the requirement for specific amino acids or secondary structure at this site. The consequences of retinal- independent phosphorylation and arrestin binding on the ability of these mutants to activate Gt will also be performed. A better understanding of the mechanisms by which the cytoplasmic domains of rhodopsin control G protein activation and desensitization will aid in defining potential mechanisms of disease resulting from defects in G protein-coupled receptor signaling pathways and in designing therapeutic strategies.