Role of RAMPs in Intermedin Signaling

描述（由申请人提供）：我们最近发现并鉴定了一种新的激素，中间体/肾上腺髓质素2，属于CGRP/肾上腺髓质素肽家族。功能研究表明，中间体信号通过G蛋白偶联受体，降钙素受体样受体（CLR）传递。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种称为受体活性修饰蛋白（RAMP1, 2和3）中的一种。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的细胞中表现出更强的效力。相反，新发现的中间体优先激活RAMP1-和RAMP1- 3介导的CLR信号。因此，中间体可以调节新的生理过程或那些以前被证明是由CGRP或肾上腺髓质素调节。此外，我们已经证明中间素具有强大的心血管作用，代表了垂体中雌激素依赖的催乳素释放激素。我们的初步数据表明，1)拴系的CGRP-RAMP1嵌合体在缺乏配体的情况下组成性地激活CLR， 2)拴系的CGRP-RAMP1嵌合体与CLR直接相互作用导致形成G蛋白偶联所必需的稳定复合物。因此，我们假设CGRP家族肽激活CLR信号可能涉及三个连续的步骤，导致由配体、RAMP和CLR组成的三聚体复合物的形成。由于CGRP家族肽与RAMP蛋白之间的选择性相互作用在CLR的选择性激活中至关重要，因此在Specific Aim 1中，我们建议研究CGRP家族肽的结构功能特征，并生成RAMP选择性激动剂和拮抗剂。在Specific Aim 2中，我们将研究配体- ramp相互作用在三聚体配体- ramp - clr复合物形成中的作用，该复合物用于使用栓系配体- ramp融合蛋白进行信号传导。在Specific Aim 3中，利用拴系CGRP-RAMP1嵌合体组成性激活CLR的研究结果，我们将使用敲入策略产生并表征拴系CGRP-RAMP1嵌合体的转基因小鼠。由于CGRP-RAMP1转基因两侧有LoxP位点，我们也可以产生条件ramp1缺陷小鼠，用于未来的研究。总之，本提案旨在回答CGRP家族肽研究中的两个关键问题：受体特异性是如何确定的，以及个体RAMPs在体内的生理作用。通过分析配体-受体相互作用的分子机制，并具体研究转基因小鼠中RAMP1介导的CLR信号传导，这些实验可以帮助确定配体和RAMPs中CLR激活的功能基序，并说明单个RAMP蛋白在不同生理过程和心血管疾病中的生理意义。

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.