Molecular mechanisms of arrestin function

描述(逐字摘自申请者摘要)：单元格减少 对持续刺激的反应，通常被称为脱敏，是一种 广泛存在的生物现象。由各种各样的G发出信号 蛋白质偶联受体(GPCRs)通过两步机制减弱： 受体被特定的激酶磷酸化，紧接着与一个 Arrestin蛋白激活的磷酸化受体。Arrestin结合 通过G蛋白终止信号，标记受体进行内化，并 在某些情况下，通过Src激酶启动额外的信号级联反应。 内化的受体要么循环回到质膜 (再增敏)或运输到溶酶体并降解(下调)。 众所周知，拦阻蛋白在脱敏和脱敏中起关键作用。 贩运各种GPCR。然而，决定这一现象的分子机制 抑制素对激活的磷酸化受体具有显著的选择性， 确定不同arrestin蛋白的受体特异性，并调节 逮捕素与细胞内其他各种伙伴的互动仍将继续 将被澄清。 本提案的目标是确定b-arrestin和barrestin的成分 抑制素3参与它们与受体的相互作用，并确定 这些因素决定了抑制素的受体特异性。我们建议澄清 芦荟素从基础失活状态转变为 高亲和力受体结合状态。一种突变的组合， 将使用定点定向自旋标记和X射线结晶学来实现这一点 目的。各种arrestin突变体将在体外、细胞培养和 在非洲爪哇的卵母细胞中。具有特殊功能特性的arrestin突变体 将被构建，例如具有约束力的“构成活性”逮捕 磷酸化和非磷酸化受体，以及增强的阻滞剂 某些受体的专一性。这些突变体将被用来研究 细胞内受体转运的机制。某些人发出了过多的信号 GPCRs导致各种疾病，包括几种形式的癌症。 对这些受体具有增强的特异性和增强的arrestin突变体 减弱这种错误信令的能力有望成为有用的工具 用于这些疾病的基因治疗。

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.