Characterization of Neuroendocrine CGRP Receptors

描述（由申请人提供）：我们发现了一种名为降钙素基因相关肽(CGRP)-受体成分蛋白（RCP）的148aa蛋白，它是神经肽CGRP受体上G蛋白偶联信号转导所必需的。在之前的项目期间，我们确定RCP与其他两种蛋白质一起工作，构成功能性CGRP受体：降钙素受体样受体（CRLR）和受体活性修饰蛋白（RAMP1）。CRLR具有典型的G蛋白偶联受体的7-跨膜拓扑结构，需要RAMP1转运到细胞表面和配体特异性，需要RCP偶联到细胞信号转导途径。CRLR在抑制RCP表达的反义细胞系中不起作用，并且CRLR与RCP共免疫沉淀，表明这两种蛋白在复合物中一起存在。本文拟验证的具体假设是，一个功能性CGRP受体至少由CRLR、RCP和RAMP1三种蛋白组成，而辅助蛋白RCP和RAMP1可以调节CRLR的功能。这项建议的具体目的是：1)利用酵母双杂交实验和细胞培养确定功能CGRP受体所需的RCP和CRLR相互作用位点，2)确定RAMP1或RCP过表达是否可以增加细胞培养中CGRP受体功能，3)确定同源重组缺失RCP是否会增加细胞培养中CGRP受体功能的抑制作用，4)确定调节CGRP受体复合物的细胞环境。重点研究脂筏及其对膜内受体功能和分布的影响。

DESCRIPTION (provided by applicant): We have discovered a 148 aa protein named calcitonin gene-related peptide (CGRP)-receptor component protein (RCP) that is required for G protein-coupled signal transduction at receptors for the neuropeptide CGRP. During the previous project period we determined that RCP works in conjunction with two other proteins to constitute a functional CGRP receptor: calcitonin receptor-like receptor (CRLR) and receptor activity modifying protein (RAMP1). CRLR has the stereotypical 7-transmembrane topology of a G protein-coupled receptor, and requires RAMP1 for trafficking to the cell surface and for ligand specificity, and requires RCP for coupling to the cellular signal transduction pathway. CRLR did not function in antisense cell lines that inhibited RCP expression, and CRLR co-immunoprecipitated with RCP suggesting that the two proteins were together in complex. The specific hypotheses to be tested in this proposal are that a functional CGRP receptor is composed of at least three proteins: CRLR, RCP, and RAMP1, and that the accessory proteins RCP and RAMP1 can modulate CRLR function. The specific aims of this proposal are to: 1) determine the sites of interaction between RCP and CRLR required for a functional CGRP receptor, using yeast two-hybrid assay and cell culture, 2) determine if overexpression of RAMP1 or RCP can increase CGRP receptor function in cell culture, 3) determine if loss of RCP by homologous recombination has increased inhibitory effects on CGRP receptor function in cell culture, 4) identify the cellular environment that regulates the CGRP receptor complex, focusing on lipid rafts and their effect on receptor function and receptor distribution in the membrane. Recent studies have found that in hypertension the vasculature is hyper-responsive to CGRP, suggesting regulation of CGRP receptor function. Thus, the role of RCP in regulating CGRP receptor function is important both for fundamental studies on G protein-mediated signal transduction in neuroendocrine cells, but also for future therapeutic strategies for vascular disorders.