REGULATORY DOMAINS OF G PROTEIN COUPLED RECEPTORS

G蛋白偶联受体是一类细胞表面受体， 调节G蛋白信号通路对多种 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、磷酸化级联和 离子通道的调节。控制活动对象的生命周期 受体通过一个称为脱敏的过程是一个关键方面 G蛋白信号通路的调控。视紫红质， 脊椎动物的感光器杆状细胞，已被用作结构 研究G蛋白偶联相互作用的模型 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阻滞素。 此前，我们的实验室发现了几个不重叠的结构域 视紫红质的表面参与GT的激活、相互作用 含有视紫红质激酶和arrestin。本提案的目的是 为了提供对分子作用的深入了解， 我们已经确定的受体调节中的结构域 脱敏。为了实现这一目标，聚簇丙氨酸 突变将被分离成单个丙氨酸点突变和 测试它们被磷酸化和结合arrestin的能力。 将在这些位置进行额外的突变，以确定 对电荷、疏水性、大小或特定次级的要求 结构。影响磷酸化的突变体将被分析以 确定这些位点对结合视紫红质是否重要 调节其活性，或两者兼而有之。影响芳香蛋白的突变体 将检查绑定以确定它们是否是高- 视紫红质表面的亲和结合部位或它们是否 在调节arrestin从低到高的转变中起重要作用 亲和力形式。高度保守的氨基酸Arg-135的突变体 我们的实验室证明是被磷酸化的，并将arrestin结合在 11顺式视网膜缺失。额外的替代诱变将是 用于分析特定氨基酸的需求或 这一地点的二级结构。视网膜的后果-- 独立的磷酸化和arrestin结合能力对这些 激活GT的突变体也将被执行。更好地理解 视紫红质胞质结构域控制的机制 G蛋白的激活和脱敏将有助于确定潜力 G蛋白偶联缺陷引起的疾病机制 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.