Regulation of GPCR Recycling at the Plasma Membrane

描述(申请人提供)：G蛋白偶联受体(GPCR)是一种完整的质膜蛋白，将外部信号传递到细胞内。GPCRs在中枢神经系统(CMS)中调节许多重要的信号功能，并调节许多治疗和滥用药物的效果。调节这些受体的一个基本机制是配体诱导的内吞作用。这一过程减少了细胞表面激活的受体数量，从而调节受体信号。虽然内吞作用受到生理配体和药物的强烈调控，但人们普遍认为循环是一个非调控的过程。来自我们实验室和其他实验室的新证据正在挑战这一默认的GPCR回收假说。我们开发了一种技术，通过结合活的全反射显微镜(TIRF)和融合到GPCRs胞外末端的pH敏感的胞外标签来研究细胞表面的单插入事件。我们的初步结果表明，受体回收是一个严格调控的过程，参与了对细胞外刺激的动态平衡反应。此外，循环的调节依赖于受体的激活，这最终减少了循环事件的频率，减少了细胞表面的受体数量。我建议扩展和发展这一新的方法来检查：a)介导受体循环的囊泡的特征，b)确定参与融合过程的机制，以及它可能通过受体活性进行调节，c)调查受体循环的调节是否是GPCRs的一般过程，d)定义和研究新插入的受体的功能以及与重新插入的受体相关的可能的分子复合体的存在。这项工作将使用实验上有利的异种细胞模型来完成，结果将在稍后的海马神经元中进行检验。通过研究受体插入到细胞表面的调节，我们的工作有可能为可能与广泛的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奖将是我从博士后学习过渡到大学/研究机构的独立职位的一个非同寻常的机会。

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.