Characterization of Neuroendocrine CGRP Receptors

神经内分泌 CGRP 受体的表征

• 批准号: 6578702
• 负责人: IAN M DICKERSON
• 金额: $ 9.58万
• 依托单位: UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-01-01 至 2003-08-18
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6578702
• 关键词: 3T3 cells; biological signal transduction; calcitonin gene related peptide; gene targeting; immunoprecipitation; membrane proteins; neuropeptide receptor; protein protein interaction; protein structure function; receptor binding; receptor coupling; receptor expression; receptor sensitivity; transfection; yeast two hybrid system

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.

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）-受体组分蛋白（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。在上一个项目期间，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（RAMP 1）。CRLR具有G蛋白偶联受体的常规7-跨膜拓扑结构，并且需要RAMP 1用于运输至细胞表面和用于配体特异性，并且需要RCP用于偶联至细胞信号转导途径。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，表明这两种蛋白质在复合物中在一起。在这个提议中要测试的具体假设是，功能性CGRP受体由至少三种蛋白质组成：CRLR、RCP和RAMP 1，并且辅助蛋白RCP和RAMP 1可以调节CRLR功能。这项建议的具体目标是：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RAMP 1或RCP的过表达是否可以增加细胞培养中的CGRP受体功能，3）确定通过同源重组导致的RCP缺失是否增加了细胞培养中对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，重点是脂筏及其对受体功能和受体在膜中分布的影响。 最近的研究发现，在高血压中，血管对CGRP是高反应性的，表明CGRP受体功能的调节。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血管疾病的未来治疗策略都是重要的。