Role of RAMPs in Intermedin Signaling

RAMP 在 Intermedin 信号转导中的作用

• 批准号: 7258366
• 负责人: SHEAU-YU Teddy HSU
• 金额: $ 28.21万
• 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/7258366
• 关键词: Agonist; Animals; CALCA gene; Calcitonin; Calcitonin Gene-Related Peptide; Calcitonin-Gene Related Peptide Receptor; Cardiovascular Diseases; Cardiovascular Physiology; Cardiovascular system; Cells; Characteristics; Chimera organism; Chimeric Proteins; Complex; Coupling; Data; Estrogens; Exhibits; Family; Future; G-Protein-Coupled Receptors; GTP-Binding Proteins; Generations; Homeostasis; Hormones; Individual; Kidney; Knock-in Mouse; Laboratories; Lead; Length; Ligand Binding; Ligands; Light; Maintenance; Mediating; Molecular; Morphogenesis; Mus; Neurosecretory Systems; Peptide Receptor; Peptides; Physiological; Physiological Processes; Pituitary Gland; Play; Process; Prolactin-Releasing Hormone; Proteins; RAMP1; RAMP2; Ramp; Receptor Activation; Receptor Signaling; Regulation; Research Personnel; Respiratory System; Role; S100A12 gene; Signal Pathway; Signal Transduction; Site; Specificity; Surface; Transgenes; Transgenic Mice; adrenomedullin; base; bone; calcitonin receptor-like receptor; gain of function; human S100A12 protein; in vivo; islet amyloid polypeptide; mutant; novel; peptide hormone; programs; promoter; receptor; receptor-activity-modifying protein; research study

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的激素，intermedin/adrenomedullin 2，属于CGRP/adrenomedullin肽家族。功能研究表明，中介素信号通过G蛋白偶联受体，降钙素受体样受体（CGRP）。已知CGRP介导CGRP和肾上腺髓质素的作用，但这些激素的有效信号传导需要称为受体活性修饰蛋白（RAMP 1，2和3）的三种辅助受体之一。CGRP优先通过表达RAMP 1和RAMP 2的细胞发出信号，而肾上腺髓质素对表达RAMP 2和RAMP 3或RAMP 3和RAMP 4的细胞表现出更大的效力。相比之下，新鉴定的intermedin优先激活RAMP 1和RAMP-3介导的RAMP信号传导。因此，intermedin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，intermedin表现出强大的心血管作用，并代表了雌激素依赖性催乳素释放激素在垂体。我们的初步数据显示，1）栓系的CGRP-RAMP 1嵌合体在不存在配体的情况下组成型激活CRP 1，和2）栓系的CGRP-RAMP 1嵌合体与CRP 1的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cardiac信号传导可能涉及三个连续步骤，导致形成由配体、RAMP和cardiac组成的三聚体复合物。由于CGRP家族肽与RAMP蛋白之间的选择性相互作用在选择性激活CGRP中至关重要，因此在具体目标1中，我们建议研究CGRP家族肽的结构-功能特征并产生RAMP选择性激动剂和拮抗剂。在特定目标2中，我们将研究配体-RAMP相互作用在使用系留配体-RAMP融合蛋白形成用于信号传导的三聚体配体-RAMP-RAMP复合物中的作用。在具体目标3中，利用栓系的CGRP-RAMP 1嵌合体组成性激活cells的发现，我们将使用敲入策略产生和表征具有栓系的CGRP-RAMP 1嵌合体的转基因小鼠。由于CGRP-RAMP 1转基因两侧是LoxP位点，我们也可以产生条件RAMP 1缺陷小鼠用于未来的研究。总而言之，这项建议旨在回答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，特别是在转基因小鼠中研究RAMP 1介导的RAMP 1信号转导，这些实验可以促进确定配体和RAMP中的功能基序，以激活RAMPs，并说明单个RAMP蛋白在不同的生理过程和心血管疾病中的生理意义。