Arrestins in G protein-coupled receptor trafficking

描述（由申请人提供）： G蛋白偶联受体（GPCR）介导许多重要的生理反应。抑制蛋白通过充当网格蛋白和AP 2衔接子复合物的蛋白质支架来调节激动剂占据的GPCR的脱敏和内吞作用。此外，抑制蛋白通过与c-Src酪氨酸激酶的直接相互作用控制生长信号级联的GPCR激活。我们最近发现，β-arrestin 2的N-末端的一个小区域与AP 2的mu 2-adaptin亚基结合。mu 2-适应素亚基对于具有酪氨酸磷酸化基序的许多非GPCR蛋白的网格蛋白包被的凹坑中的AP 2介导的内吞作用是关键的。这是本提案的目标，其特征在于使用光学成像和生物化学技术的μ-适应素亚基和酪氨酸激酶的调节抑制蛋白介导的GPCR内吞作用的作用。 该提案的具体目的是：（I）通过鉴定相互作用序列并测试它们在GPCR内吞作用和抑制蛋白介导的信号传导中的重要性来测试AP 2的μ 2亚基在抑制蛋白介导的GPCR内吞作用和信号传导中起关键作用的假设。 (II)为了检验抑制蛋白通过与酪氨酸激酶如c-Src相互作用而受酪氨酸磷酸化调节的假设，通过（a）使用抑制蛋白衍生的肽、纯化的抑制蛋白和纯化的酪氨酸激酶确定其中酪氨酸磷酸化可以发生的抑制蛋白序列，并评估这些残基在抑制蛋白介导的GPCR调节中的重要性。通过表征抑制蛋白生物化学的新领域，调节至少两个主要的信号通路。

DESCRIPTION (provided by applicant): G protein-coupled receptors (GPCRs) mediate many important physiological responses. Arrestins regulate desensitization and endocytosis of agonist-occupied GPCRs by acting as protein scaffolds for clathrin and the AP2 adaptor complex. Also, arrestin controls GPCR activation of growth signaling cascades through direct interactions with c-Src tyrosine kinase. We have recently discovered that a small region of the N-terminus of beta-arrestin2 binds to the mu2-adaptin subunit of AP2. The mu2-adaptin subunit is critical for the AP2 mediated endocytosis in clathrin-coated pits of many non-GPCR proteins that possess tyrosine phosphorylation motifs. It is the goal of this proposal to characterize using optical imaging and biochemical techniques the role of the mu-adaptin subunit and tyrosine kinases in the regulation of arrestin-mediated GPCR endocytosis. The specific aims of the proposal are: (I) To test the hypothesis that the mu2-subunit of AP2 plays a critical role in arrestin mediated GPCR endocytosis and signaling by identifying the interacting sequences and testing their importance in GPCR endocytosis and arrestin-mediated signaling. (II) To test the hypothesis that arrestin, by interacting with tyrosine kinases such as c-Src is regulated by tyrosine phosphorylation by (a) Determining the arrestin sequence where tyrosine phosphorylation can occur using arrestin derived peptides, purified arrestins and purified tyrosine kinases and assess the importance of these residues in arrestin mediated GPCR regulation This proposal will greatly our understanding of the cell physiology underlying normal health, by characterizing novel areas of arrestin biochemistry that regulate at least two major signaling pathways.