Molecular mechanisms of arrestin function

描述（来自申请人摘要的逐字描述）： 对持续刺激的反应，通常称为脱敏，是一种 普遍的生物现象。各种各样的G 蛋白偶联受体（GPCR）通过两步机制减弱： 受体磷酸化的特定激酶，随后紧密结合的一个 抑制蛋白与活化的磷酸化受体的结合。抑制蛋白结合 终止通过G蛋白的信号传导，标记受体用于内化，和 在某些情况下通过Src激酶启动额外的信号级联。 内化的受体要么再循环回到质膜 （再致敏）或转运至溶酶体并降解（下调）。 已经确定，抑制蛋白在脱敏中起关键作用， 贩运各种GPCR。然而，决定性的分子机制 抑制蛋白对活化的磷酸化受体的显著选择性， 确定不同抑制蛋白的受体特异性，并调节 抑制蛋白与细胞中其他多种伙伴的相互作用仍然存在， 被阐明。 本提案的目的是确定b-抑制素的成分， 抑制蛋白3参与它们与受体的相互作用，并决定哪些 这些元件决定了抑制蛋白的受体特异性。我们建议澄清 arrestin从其基本的非活性状态转变为 高亲和力受体结合状态。结合诱变， 定点自旋标记和X射线晶体学将用于此 目的.各种抑制蛋白突变体将在体外、在细胞培养物中进行测试， 在非洲爪蟾卵母细胞中。具有特殊功能特性的抑制蛋白突变体 将被构建，如“组成型活性”抑制蛋白，其结合至 磷酸化和非磷酸化受体，以及具有增强的 对某些受体的特异性。这些突变体将用于研究 细胞中受体运输的机制。某些人发出过多的信号 GPCR导致多种疾病，包括几种形式的癌症。 抑制蛋白突变体对这些受体的特异性增强， 减弱这种错误信号的能力有望成为有用的工具 基因治疗的方法。

DESCRIPTION (Verbatim from the Applicant's Abstract): The decrease of cell responsiveness to a persistent stimulus, usually termed desensitization, is a widespread biological phenomenon. Signaling by a wide variety of G protein-coupled receptors (GPCRs) is attenuated by a two-step mechanism: receptor phosphorylation by a specific kinase, followed by tight binding of an arrestin protein to activated phosphorylated receptor. Arrestin binding terminates the signaling via G protein, tags receptor for internalization, and in some cases initiates an additional signaling cascade via Src kinase. Internalized receptor is either recycled back to the plasma membrane (resensitization) or transported to lysosomes and degraded (down-regulation). It is well established that arrestins play a key role in desensitization and trafficking of various GPCRs. However, the molecular mechanisms that dictate arrestins' remarkable selectivity toward activated phosphorylated receptors, determine receptor specificity of different arrestin proteins, and regulate arrestins' interaction with a variety of other partners in the cell remain to be elucidated. The objectives of this proposal are to identify the elements of b-arrestin and arrestin3 involved in their interaction with receptors and determine which of these elements dictate arrestins' receptor specificity. We propose to elucidate the mechanism of arrestin transition from its basal inactive state into high-affinity receptor binding state. A combination of mutagenesis, site-directed spin labeling, and X-ray crystallography will be used for this purpose. Various arrestin mutants will be tested in vitro, in cell culture, and in Xenopus oocytes. Arrestin mutants with special functional characteristics will be constructed, such as "constitutively active" arrestins that bind to phosphorylated and unphosphorylated receptors, and arrestins with enhanced specificity for certain receptors. These mutants will be used to study the mechanisms of receptor trafficking in cells. Excessive signaling by certain GPCRs causes a variety of disorders, including several forms of cancer. Arrestin mutants with enhanced specificity for these receptors and enhanced capability to attenuate such faulty signaling promise to become useful tools for gene therapy of these disorders.