Role of RAMPs in Intermedin Signaling

RAMP 在 Intermedin 信号转导中的作用

• 批准号: 7147883
• 负责人: SHEAU-YU Teddy HSU
• 金额: $ 27.74万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-15 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7147883
• 关键词: biological signal transduction; calcitonin; calcitonin gene related peptide; cardiovascular function; genetically modified animals; hormone receptor; laboratory mouse; melanocyte stimulating hormone; peptide hormone; protein protein interaction; protein structure function; receptor expression; transfection; vasodilators

DESCRIPTION (provided by applicant): We have recently discovered and characterized a novel hormone, intermedin/adrenomedullin 2, belonging to the CGRP/adrenomedullin peptide family. Functional studies showed that intermedin signals through a G protein-coupled receptor, calcitonin receptor-like receptor (CLR). CLR is known to mediate the action of CGRP and adrenomedullin, but efficient signaling by these hormones requires one of three coreceptors known as receptor activity-modifying proteins (RAMP1, 2, and 3). CGRP preferentially signals through cells expressing RAMP1 and CLR whereas adrenomedullin exhibits greater potency on cells expressing RAMP2 and CLR, or RAMPS and CLR. In contrast, the newly identified intermedin preferentially activates RAMP1- and RAMP-3 mediated CLR signaling. Therefore, intermedin could regulate novel physiological processes or those previously shown to be regulated by CGRP or adrenomedullin. Furthermore, we have demonstrated that intermedin exhibits potent cardiovascular effects and represents an estrogen-dependent prolactin- releasing hormone in the pituitary. Our preliminary data shows that, 1) a tethered CGRP-RAMP1 chimera constitutively activates CLR in the absence of a ligand, and 2) direct interaction of the tethered CGRP- RAMP1 chimera with CLR results in the formation of a stable complex essential for G protein coupling. Thus, we hypothesize that activation of CLR signaling by CGRP family peptides could involve three sequential steps that lead to the formation of a trimeric complex consisting of the ligand, RAMP, and CLR. Because selective interactions between CGRP family peptides with RAMP proteins are crucial in the selective activation of CLR, in Specific Aim 1 we propose to study the structural-functional characteristics of the CGRP family peptides and generate RAMP-selective agonists and antagonists. In Specific Aim 2, we will investigate the role of ligand-RAMP interactions in the formation of a trimeric ligand-RAMP-CLR complex for signaling using tethered ligand-RAMP fusion proteins. In Specific Aim 3, taking advantage of findings that the tethered CGRP-RAMP1 chimera constitutively activates CLR, we will generate and characterize transgenic mice with the tethered CGRP-RAMP1 chimera using a knock-in strategy. Because the CGRP-RAMP1 transgene is flanked by LoxP sites, we also can generate conditional RAMP1-deficient mice for future studies. Altogether, this proposal aims to answer two of the critical questions in the study of CGRP family peptides: how receptor specificity is determined and what are the physiological roles of individual RAMPs in vivo. By dissecting the molecular mechanisms underling ligand-receptor interactions and specifically investigating the RAMP1- mediated CLR signaling in transgenic mice, these experiments could facilitate the determination of functional motifs in both ligands and RAMPs for CLR activation as well as illustrate the physiological significance of individual RAMP proteins in diverse physiological processes and cardiovascular diseases.

描述(申请人提供)：我们最近发现并鉴定了一种新的激素，中间素/肾上腺髓质素2，属于CGRP/肾上腺髓质素多肽家族。功能研究表明，Intermedin通过G蛋白偶联受体--降钙素受体样受体(CLR)传递信号。已知CLR介导CGRP和肾上腺髓质素的作用，但这些激素的有效信号传递需要三个辅助受体之一，即受体活性修饰蛋白(RAMP1、2和3)。CGRP优先通过表达RAMP1和CLR的细胞发出信号，而肾上腺髓质素在表达RAMP2和CLR或RAMPS和CLR的细胞上显示出更大的效力。相反，新发现的Intermedin优先激活RAMP1和RAMP-3介导的CLR信号。因此，Intermedin可以调节新的生理过程或先前被证明受CGRP或肾上腺髓质素调节的过程。此外，我们还证明了Intermedin具有很强的心血管作用，并且在脑下垂体中代表着一种雌激素依赖性的催乳素释放激素。我们的初步数据表明，1)拴系的CGRP-RAMP1嵌合体在没有配体的情况下结构性地激活CLR，以及2)拴系的CGRP-RAMP1嵌合体与CLR的直接相互作用导致形成G蛋白偶联所必需的稳定的复合体。因此，我们推测，CGRP家族多肽对CLR信号的激活可能包括三个连续的步骤，导致形成由配体、RAMP和CLR组成的三聚体复合体。由于CGRP家族多肽与RAMP蛋白之间的选择性相互作用在CLR的选择性激活中起着至关重要的作用，因此，在特定的目标1中，我们建议研究CGRP家族多肽的结构-功能特性，并产生RAMP选择性激动剂和拮抗剂。在特定的目标2中，我们将研究配体-RAMP相互作用在形成三聚体配体-RAMP-CLR复合体中的作用，以使用拴系配体-RAMP融合蛋白进行信号传递。在具体目标3中，利用系留的CGRP-RAMP1嵌合体结构性激活CLR的发现，我们将使用敲入策略产生并鉴定具有系留的CGRP-RAMP1嵌合体的转基因小鼠。由于CGRP-RAMP1转基因侧翼有loxP位点，我们还可以产生条件性RAMP1缺陷小鼠用于未来的研究。综上所述，该建议旨在回答CGRP家族多肽研究中的两个关键问题：受体的特异性是如何确定的，以及单个RAMP在体内的生理作用是什么。通过剖析配体-受体相互作用的分子机制，以及在转基因小鼠中具体研究RAMP1介导的CLR信号，这些实验可以帮助确定配体和RAMP中激活CLR的功能基序，并阐明单个RAMP蛋白在不同生理过程和心血管疾病中的生理意义。