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。

• 批准号: 9761234
• 负责人: Amanda Roehrkasse
• 金额: $ 3.35万
• 依托单位: UNIVERSITY OF OKLAHOMA HLTH SCIENCES CTR
• 依托单位国家: 美国
• 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/9761234
• 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

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.!

项目摘要 血管活性肽肾上腺髓质素2/中间肽（AM 2/IMD）在人体内具有重要的生理功能， 生理学和疾病如血管舒张、生理和病理性血管生成以及有效 对心血管和肾脏系统有保护作用。AM 2/IMD的行动归因于 几种信号传导中间体的激活，包括其G蛋白偶联的cAMP和Ca 2+下游 降钙素受体样受体（Calcitonin receptor-like receptor，简称CGRP）。不幸的是，很少有机械的洞察力， AM 2/IMD结合并激活AM 2/IMD。药理学是复杂的，因为它与任何 调节其对AM 2/IMD的反应的三种受体活性修饰蛋白（RAMP 1、-2或-3）之一， 相关血管活性肽降钙素基因相关肽（CGRP）和肾上腺髓质素（AM）。如何 AM 2/IMD、CGRP和AM通过共享的RAMP：RAMP受体复合物起作用，以促进其独特的 信号结果仍不清楚。这限制了我们对AM 2/IMD如何扩展其广泛应用范围的理解。 这一发现阻碍了我们利用AM 2/IMD信号进行药物开发的能力。我会 检验AM 2/IMD采用独特的受体结合构象并促进模式的假设 在RAMP：CGRP复合物中的偏向G蛋白激活与AM和CGRP不同。我将测试 本研究使用严格的生物化学、药理学和结构学方法，目的有二：1）定义分子 通过可溶性RAMP：ECD复合物识别AM 2/IMD的基础，和2）定义 AM 2/IMD促进的每个全长受体复合物的G蛋白偶联偏好与 CGRP和AM。对于目标1，I将三种ECD复合物中的每一种纯化为系留RAMP ECD-ECD 发现AM 2/IMD表现出与CGRP不同的结合偏好 和AM。我解出了一个2.05 μ m分辨率的晶体结构，它展示了一个惊人的独特的三重b转弯 AM 2/IMD的结构结合到RAMP 1-BLECD。我将确定AM 2/IMD结合的晶体结构， RAMP 3-EECDs完全了解AM 2/IMD如何结合不同的受体ECDs，并提供关键的 深入了解RAMP 3如何调节CLR。对于目标2，我们确定了共表达和溶解 三种全长RAMP：RAMP复合物，其形成洗涤剂稳定的无配体复合物。这提供了 这是研究这三种肽如何促进不同G蛋白偶联的独特机会。我们将使用一个 天然PAGE方法，以确定耦合偏好未纯化的受体复合物，我们将纯化 使用荧光各向异性测定法研究无配体复合物与G蛋白的偶联。这些 生物化学研究将与人类cAMP和Ca 2+信号偏差的药理学研究相关联 表达RAMP 1：CRP 1（SK-N-MC）或RAMP 2：CRP 2（HUVEC）的细胞系。成功完成这些 aims将提供对AM 2/IMD功能的重要见解，这将使基于AM 2/IMD的药物开发成为可能。