Receptor binding and signaling of the cardioprotective peptide Adrenomedullin 2/Intermedin.

心脏保护肽肾上腺髓质素 2/Intermedin 的受体结合和信号传导。

基本信息

批准号：
10020190
负责人：
Amanda Roehrkasse
金额：
$ 4.76万
依托单位：
UNIVERSITY OF OKLAHOMA HLTH SCIENCES CTR
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-08-12 至 2021-08-11
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10020190
关键词：
Adopted Affinity Affinity Chromatography Agonist Binding Biochemical Biological Assay Blood Vessels C-terminal Calcitonin Gene-Related Peptide Cardiovascular Diseases Cardiovascular Physiology Cardiovascular system Cell Line Cells Chimeric Proteins Clinical Complex Coupling Crystallization Cyclic AMP Detergents Development Disease Drug Design Drug Receptors Drug Targeting Engineering Exhibits Extracellular Domain Family Fluorescence Anisotropy Future G-Protein-Coupled Receptors GTP-Binding Protein alpha Subunits, Gs GTP-Binding Proteins Gel Heterodimerization Human Human Cell Line Hypoxia Immobilization Investigation Kidney Length Ligands Link Measures Mediating Melanocyte stimulating hormone Methods Migraine Molecular Molecular Conformation Mutagenesis N-terminal Outcome Pathologic Neovascularization Pathologic Processes Pattern Peptide Conformation Peptide Receptor Peptides Pharmaceutical Preparations Pharmacology Pharmacology Study Physiologic Neovascularization Physiology Plant Resins Proteins RAMP1 RAMP2 RAMP3 Receptor Signaling Reperfusion Injury Resolution Role Sepsis Signal Pathway Signal Transduction Structure System Testing Therapeutic Transmembrane Domain Vasodilation Work adrenomedullin base calcitonin receptor-like receptor cardioprotection drug development insight novel peptide analog peptide hormone preference protective effect protein activation receptor receptor binding receptor-activity-modifying protein response side effect tool

项目摘要

PROJECT SUMMARY ABSTRACT The vasoactive peptide adrenomedullin 2/intermedin (AM2/IMD) has important actions in human physiology and disease such as vasodilation, physiologic and pathologic angiogenesis as well as potent protective effects in the cardiovascular and renal systems. Actions of AM2/IMD have been attributed to activation of several signaling intermediates including cAMP and Ca2+ downstream of its G protein-coupled receptor, the calcitonin receptor-like receptor (CLR). Unfortunately, there is little mechanistic insight into how AM2/IMD binds and activates CLR. CLR pharmacology is complicated because it heterodimerizes with any one of three receptor activity-modifying proteins (RAMP1, -2, or -3) that modulate its response to AM2/IMD and the related vasoactive peptides calcitonin gene-related peptide (CGRP) and adrenomedullin (AM). How AM2/IMD, CGRP, and AM act through shared RAMP:CLR receptor complexes to promote their unique signaling outcomes remains unclear. This limits our understanding of how AM2/IMD elicits its broad range of actions in human physiology and hinders our ability to exploit AM2/IMD signaling for drug development. I will test the hypothesis that AM2/IMD adopts a unique receptor-bound conformation and that it promotes a pattern of biased G protein activation at RAMP:CLR complexes that is distinct from those of AM and CGRP. I will test this using rigorous biochemical, pharmacological, and structural methods in two aims: 1) Define the molecular basis for AM2/IMD recognition by soluble RAMP:CLR extracellular domain (ECD) complexes, and 2) Define the G protein-coupling preferences of each full-length receptor complex promoted by AM2/IMD as compared to CGRP and AM. For Aim 1 I purified each of the three ECD complexes as tethered RAMP ECD-CLR ECD fusion constructs and found that AM2/IMD exhibited binding preferences that were distinct from those of CGRP and AM. I solved a 2.05 Å resolution crystal structure that demonstrated a strikingly unique triple b-turn structure of AM2/IMD bound to the RAMP1-CLR ECD. I will determine an AM2/IMD-bound crystal structure of the RAMP3-CLR ECD to fully understand how AM2/IMD binds the different receptor ECDs, and provide crucial insights into how RAMP3 modulates CLR. For Aim 2 we determined conditions to co-express and solubilize the three full-length RAMP:CLR complexes, which form detergent-stable ligand-free complexes. This provides a unique opportunity to study how the three peptides promote coupling of different G-proteins. We will use a native-PAGE method to determine coupling preferences to unpurified receptor complexes and we will purify the ligand-free complexes to study G-protein coupling using a fluorescence anisotropy assay. These biochemical studies will be correlated with pharmacological studies of cAMP and Ca2+ signaling bias in human cell lines that express the RAMP1:CLR (SK-N-MC) or RAMP2:CLR (HUVEC). Successful completion of these aims will provide crucial insights into AM2/IMD function that will enable AM2/IMD-based drug development.!

项目概要摘要血管活性肽肾上腺髓质素 2/intermedin (AM2/IMD) 在人体中具有重要作用生理学和疾病，如血管舒张、生理和病理性血管生成以及强效 AM2/IMD 对心血管和肾脏系统的保护作用。激活多种信号传导中间体，包括其 G 蛋白偶联下游的 cAMP 和 Ca2+ 不幸的是，人们对降钙素受体样受体（CLR）的机制了解甚少。 AM2/IMD 结合并激活 CLR 药理学很复杂，因为它可以与任何物质形成异二聚体。三种受体活性修饰蛋白（RAMP1、-2 或 -3）之一，调节其对 AM2/IMD 的反应，相关的血管活性肽有降钙素基因相关肽（CGRP）和肾上腺髓质素（AM）。 AM2/IMD、CGRP 和 AM 通过共享的 RAMP:CLR 受体复合物发挥作用，以促进其独特的作用信号传导结果仍不清楚，这限制了我们对 AM2/IMD 如何引发其广泛作用的理解。我会的，这会影响人类生理学的行为，并阻碍我们利用 AM2/IMD 信号传导进行药物开发的能力。检验 AM2/IMD 采用独特的受体结合构象并促进某种模式的假设我将测试 RAMP:CLR 复合物中与 AM 和 CGRP 不同的偏向 G 蛋白激活。使用严格的生化、药理学和结构方法来实现两个目标：1）定义分子可溶性 RAMP:CLR 胞外域 (ECD) 复合物识别 AM2/IMD 的基础，以及 2) 定义 AM2/IMD 促进的每个全长受体复合物的 G 蛋白偶联偏好与对于目标 1，我将三种 ECD 复合物分别纯化为束缚 RAMP ECD-CLR ECD。融合构建体，发现 AM2/IMD 显示出与 CGRP 不同的结合偏好我解决了 2.05 Å 分辨率的晶体结构，展示了惊人独特的三重 b 转角。 AM2/IMD 与 RAMP1-CLR ECD 结合的结构我将确定 AM2/IMD 结合的晶体结构。 RAMP3-CLR ECD 可以充分了解 AM2/IMD 如何结合不同的受体 ECD，并提供关键的信息对于目标 2，我们确定了 RAMP3 如何调节 CLR 的条件。三个全长 RAMP:CLR 复合物，形成洗涤剂稳定的无配体复合物。这是研究三种肽如何促进不同 G 蛋白偶联的独特机会。天然 PAGE 方法来确定与未纯化受体复合物的偶联偏好，我们将纯化使用荧光各向异性测定研究 G 蛋白偶联的无配体复合物。生化研究将与人类 cAMP 和 Ca2+ 信号偏向的药理学研究相关联表达 RAMP1:CLR (SK-N-MC) 或 RAMP2:CLR (HUVEC) 的细胞系成功完成这些。目标将为 AM2/IMD 功能提供重要见解，从而实现基于 AM2/IMD 的药物开发。