Regulation of GPCR Recycling at the Plasma Membrane

描述（由申请人提供）：G蛋白偶联受体（GPCR）是完整的质膜蛋白，将外部信号转导到细胞中。GPCRs介导中枢神经系统（CMS）中许多重要的信号功能，并介导许多治疗药物和滥用药物的作用。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GPCR回收的默认假设。我们开发了一种技术，通过结合活全反射显微镜（TIRF）和融合到gpcr细胞外末端的pH敏感细胞外标签来研究细胞表面的单个插入事件。我们的初步结果表明，受体循环是一个受到严格调控的过程，涉及对细胞外刺激的稳态反应。此外，循环的调节依赖于受体的激活，这最终降低了循环事件的频率，减少了细胞表面的受体数量。我建议扩展和进一步发展这种新方法来检查：a)囊泡介导受体回收的特征，b)确定融合过程中涉及的机制，以及受体活性可能的调节，c)研究受体回收的调节是否是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，结果将在稍后的海马神经元中进行检验。通过研究受体插入细胞表面的调控，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奖将是我从博士后研究过渡到大学/研究机构独立职位的绝佳机会。

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.