Regulation of GPCR Recycling at the Plasma Membrane

描述（由申请人提供）：G蛋白偶联受体（GPCR）是将外部信号传递到细胞中的整合质膜蛋白。GPCR介导中枢神经系统（CMS）中许多重要的信号传导功能，并介导许多治疗和滥用药物的作用。调节这些受体的基本机制是通过配体诱导的内吞作用。这一过程减少了细胞表面活化受体的数量，从而调节受体信号传导。虽然内吞作用受到生理配体和药物的强烈调节，但通常认为再循环是一个不受调节的过程。来自我们实验室和其他实验室的新证据正在挑战气相化学还原再循环的这一默认假设。我们已经开发了一种技术，调查单插入事件在细胞表面结合活全反射显微镜（TIRF）与pH敏感的胞外标签融合到胞外末端的GPCR。我们的初步结果表明，受体回收是一个严格的调节过程中参与的稳态反应细胞外刺激。此外，再循环的调节依赖于受体活化，其最终降低再循环事件的频率，从而减少细胞表面的受体数量。我建议扩展和进一步发展这种新的方法来研究：a）介导受体再循环的囊泡的特征，B）鉴定融合过程中涉及的机制，及其通过受体活性的可能调节，c）研究受体再循环的调节是否是GPCR的一般过程，d）定义和研究新插入受体的功能以及与重新插入受体相关的分子复合物的可能存在。这项工作将使用实验上有利的异源细胞模型进行，随后将在海马神经元中检查结果。通过研究受体插入细胞表面的调节，我们的工作有可能为可能与各种生理和病理过程相关的关键细胞事件提供基本的新见解。获得K99/ROO奖将是一个非凡的机会，从我的博士后研究过渡到大学/研究机构的独立职位。

DESCRIPTION (provided by applicant): G protein-coupled receptors (GPCR) are integral plasma membrane proteins that transduce external signals into the cell. GPCRs mediate many important signaling functions in the central nervous system (CMS) and mediate the effects of many therapeutic and abused drugs. A basic mechanism by which these receptors are regulated is by ligand-induced endocytosis. This process reduces the number of activated receptors at the cell surface therefore regulating receptor signaling. While endocytosis is strongly regulated by physiological ligands and drugs, it is generally believed that recycling is a nonregulated process. New evidence from our laboratory and others are challenging this default hypothesis of GPCR recycling. We have developed a technique to investigate single insertion events at the cell surface by combining live total reflection microscopy (TIRF) with a pH sensitive extracelullar tag fused to the extracelullar terminus of GPCRs. Our preliminary results indicate that receptor recycling is a tightly regulated process involved in the homeostatic response to extracelullar stimuli. Furthermore, regulation of recycling is dependent on receptor activation, which ultimately decreases the frequency of recycling events reducing receptor number at the cell surface. I propose to extend and further develop this novel approach to examine: a) The characteristics of the vesicles mediating receptor recycling, b) Identify the machinery involved in the fusion process, and its possible regulation by receptor activity, c) Investigate if the regulation of receptor recycling is a general process for GPCRs, d) Define and investigate the functionality of newly inserted receptors and the possible existence of a molecular complex associated to reinserted receptors. This work will be perfomed using an experimentally advantageous heterologous cell model, and the results will be later examined in hippocamapal neurons. By investigating the regulation of receptor insertion to the cell surface, our work has the potential to provide fundamental new insight to key cellular events likely to be relevant to a wide variety of physiological and pathological processes. Obtaining the K99/ROO award will be an extraordinary opportunity to transition from my postdoctoral studies to an independent position in a university/research institution.