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 148 个氨基酸的蛋白质，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 G 蛋白偶联受体的典型 7 跨膜拓扑结构，需要 RAMP1 才能运输到细胞表面并实现配体特异性，并且需要 RCP 才能与细胞信号转导途径偶联。 CRLR 在抑制 RCP 表达的反义细胞系中不起作用，并且 CRLR 与 RCP 进行免疫共沉淀，表明这两种蛋白以复合物形式存在在一起。该提案要测试的具体假设是，功能性CGRP受体至少由三种蛋白组成：CRLR、RCP和RAMP1，并且辅助蛋白RCP和RAMP1可以调节CRLR功能。该提案的具体目的是：1) 使用酵母双杂交测定和细胞培养确定功能性 CGRP 受体所需的 RCP 和 CRLR 之间的相互作用位点，2) 确定 RAMP1 或 RCP 的过度表达是否可以增加细胞培养中的 CGRP 受体功能，3) 确定同源重组导致的 RCP 丢失是否会增加对细胞培养中 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，重点关注脂筏及其对膜中受体功能和受体分布的影响。 最近的研究发现，高血压患者的脉管系统对 CGRP 反应过度，表明 CGRP 受体功能受到调节。因此，RCP在调节CGRP受体功能中的作用对于神经内分泌细胞中G蛋白介导的信号转导的基础研究以及未来血管疾病的治疗策略都很重要。

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.