Arrestins in G protein-coupled receptor trafficking

描述(由申请人提供)： G蛋白偶联受体(GPCRs)介导许多重要的生理反应。阻滞素通过作为蛋白骨架与AP2接头复合体，调节激动剂占据的GPCRs的脱敏和内吞作用。此外，arrestin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-arrestin2的N-末端的一小段区域与AP2的MU2-Adaptin亚基结合。MU2-Adaptin亚基在许多具有酪氨酸磷酸化基序的非GPCR蛋白的网状蛋白包被的小孔中，对于AP2介导的内吞作用是至关重要的。这项建议的目的是利用光学成像和生化技术来表征Mu-Adaptin亚单位和酪氨酸激酶在arrestin介导的GPCR内吞调节中的作用。该提案的具体目的是：(I)通过鉴定AP2的MU2亚基在arrestin介导的GPCR胞吞作用和信号转导中起关键作用的假设，确定相互作用的序列并测试它们在GPCR胞吞作用和arrestin介导的信号转导中的重要性。(Ii)检验arrestin通过与c-Src等酪氨酸激酶相互作用受酪氨酸磷酸化调控的假说：(A)使用arrestin衍生的多肽、纯化的阻滞素和纯化的酪氨酸激酶确定arrestin序列中发生酪氨酸磷酸化的位置，并评估这些残基在arrestin介导的GPCR调控中的重要性。这一建议将通过描述arrestin生化的新领域，调控至少两个主要信号通路，从而极大地帮助我们理解正常健康的细胞生理学。

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.