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（由申请人提供）：我们最近发现并表征了一种新型激素，介导蛋白/肾上腺素蛋白2，属于CGRP/肾上腺肾上腺素肽家族。功能研究表明，通过G蛋白偶联受体，降钙素受体样受体（CLR）信号。已知CLR介导CGRP和肾上腺素蛋白的作用，但是这些激素有效信号传导需要三个被称为受体活性修饰蛋白（RAMP1、2和3）的共肽之一。 CGRP优先通过表达RAMP1和CLR的细胞发出信号，而肾上腺果蛋白对表达RAMP2和CLR的细胞表现出更大的效力，或坡道和CLR。相比之下，新鉴定的中间素优先激活了RAMP1-和RAMP-3介导的CLR信号传导。因此，中间素可以调节新型生理过程或先前被CGRP或肾上腺素蛋白调节的生理过程。此外，我们已经证明了中间素表现出有效的心血管效应，并代表了垂体中雌激素依赖性的催乳素激素。我们的初步数据表明，1）在没有配体的情况下，束缚的CGRP-RAMP1嵌合体组成性地激活CLR，以及2）束缚的CGRP-RAMP1嵌合体与CLR与CLR的直接相互作用会导致形成G蛋白质稳定复合物对于G蛋白的稳定复合物。因此，我们假设CGRP家族肽对CLR信号的激活可能涉及三个顺序步骤，从而导致形成由配体，坡道和CLR组成的三聚体复合物。由于CGRP家族肽与坡道蛋白之间的选择性相互作用对于CLR的选择性激活至关重要，因此在特定的目标1中，我们建议研究CGRP家族肽的结构功能特征，并产生坡道选择性的激动剂和拮抗剂。在特定的目标2中，我们将研究配体 - 刺激相互作用在形成三聚体配体-CLR复合物中使用系列配体 - 刺激融合蛋白的信号传导的作用。在特定的目标3中，利用了束缚的CGRP-RAMP1嵌合体组成性激活CLR的发现，我们将使用敲入策略使用束缚的CGRP-ramp1嵌合体生成和表征转基因小鼠。由于CGRP-RAMP1转基因是LOXP位点的两侧，因此我们还可以生成有条件的RAMP1缺陷型小鼠以进行将来的研究。总的来说，该提案旨在回答CGRP家族肽研究中的两个关键问题：如何确定受体特异性以及个体坡道在体内的生理作用是什么。 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.