Role of RAMPs in Intermedin Signaling

RAMP 在 Intermedin 信号转导中的作用

• 批准号: 7771528
• 负责人: SHEAU-YU Teddy HSU
• 金额: $ 0.16万
• 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/7771528
• 关键词: Agonist; Animals; Calcitonin; Calcitonin Gene-Related Peptide; 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; Signal Pathway; Signal Transduction; Site; Specificity; Surface; Transgenes; Transgenic Mice; adrenomedullin; base; bone; calcitonin receptor-like receptor; gain of function; 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型激素，intermedin/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，intermedin 通过 G 蛋白偶联受体、降钙素受体样受体 (CLR) 发出信号。已知 CLR 可介导 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（RAMP1、2 和 3）的三种辅助受体之一。 CGRP 优先通过表达 RAMP1 和 CLR 的细胞发出信号，而肾上腺髓质素对表达 RAMP2 和 CLR 或 RAMPS 和 CLR 的细胞表现出更大的效力。相比之下，新鉴定的 intermedin 优先激活 RAMP1 和 RAMP-3 介导的 CLR 信号传导。因此，intermedin 可以调节新的生理过程或先前显示由 CGRP 或肾上腺髓质素调节的生理过程。此外，我们已经证明，intermedin 表现出强大的心血管作用，并且代表垂体中的雌激素依赖性催乳素释放激素。我们的初步数据表明，1) 系留的 CGRP-RAMP1 嵌合体在没有配体的情况下组成型激活 CLR，2) 系留的 CGRP-RAMP1 嵌合体与 CLR 的直接相互作用导致形成 G 蛋白必需的稳定复合物耦合。因此，我们假设 CGRP 家族肽激活 CLR 信号传导可能涉及三个连续步骤，导致形成由配体、RAMP 和 CLR 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对于 CLR 的选择性激活至关重要，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生成 RAMP 选择性激动剂和拮抗剂。在具体目标 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，该复合物用于使用拴系配体-RAMP 融合蛋白进行信号传导。在具体目标 3 中，利用系留 CGRP-RAMP1 嵌合体组成型激活 CLR 的发现，我们将使用敲入策略生成并表征带有系留 CGRP-RAMP1 嵌合体的转基因小鼠。由于 CGRP-RAMP1 转基因两侧是 LoxP 位点，因此我们还可以生成有条件的 RAMP1 缺陷小鼠用于未来的研究。总而言之，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各个 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，并专门研究转基因小鼠中 RAMP1 介导的 CLR 信号传导，这些实验可以促进配体和 RAMP 中 CLR 激活的功能基序的确定，并说明各个 RAMP 的生理意义多种生理过程和心血管疾病中的蛋白质。