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.!

项目摘要摘要 血管活性肽肾上腺素甲素2/Intermedin（AM2/IMD）在人类中具有重要的作用 生理和疾病，例如血管舒张，生理和病理血管生成以及有效 心血管和肾脏系统中的保护作用。 AM2/IMD的动作已归因于 激活几个信号转导中间体，包括其G蛋白偶联的CAMP和CA2+下游 受体，降钙素受体样受体（CLR）。不幸的是，关于如何的机械洞察力几乎没有 AM2/IMD结合并激活CLR。 CLR药理学很复杂，因为它可以与任何 调节其对AM2/IMD的响应和 相关的血管活性肽降钙素基因相关肽（CGRP）和肾上腺果蛋白（AM）。如何 AM2/IMD，CGRP和AM通过共享坡道：CLR受体复合物来促进其独特 信号传导结果尚不清楚。这限制了我们对AM2/IMD如何引起其广泛范围的理解 人类生理的作用并阻碍了我们利用AM2/IMD信号进行药物开发的能力。我会 检验AM2/IMD采用独特接收器结合的构象的假设，并促进模式 与AM和CGRP不同的CLR复合物处的偏置G蛋白激活的偏置G蛋白激活。我会测试 使用严格的生化，药物和结构方法两个目的：1）定义分子 实心坡道识别AM2/IMD的基础：CLR细胞外域（ECD）配合物，2）定义 与AM2/IMD促进的每个全长受体复合物的G蛋白偶联偏好相比 CGRP和AM。对于AIM 1，我将三个ECD复合物中的每一个纯化为束缚坡道ECD-CLR ECD 融合构建体，发现与CGRP不同的AM2/IMD暴露的结合偏好 和AM。我解决了一个2.05Å分辨率的晶体结构，该结构表现出极为独特的三重b-turn AM2/IMD的结构与RAMP1-CLR ECD结合。我将确定AM2/IMD结合的晶体结构 RAMP3-CLR ECD充分了解AM2/IMD如何绑定不同的接收器ECD，并提供至关重要的 深入了解RAMP3如何调节CLR。对于AIM 2，我们确定条件是共表达和可溶的 三个全长坡道：CLR复合物，形成了稳定的无配体配合物。这提供了 研究三个宠物如何促进不同G蛋白的耦合的独特机会。我们将使用一个 本机页方法确定对未纯正接收器配合物的耦合偏好的偏好，我们将净化 使用荧光各向异性测定法研究G蛋白偶联的无配体复合物。这些 生化研究将与人类的CAMC和CA2+信号传导偏见有关 表达RAMP1的细胞系：CLR（SK-N-MC）或RAMP2：CLR（HUVEC）。这些成功完成 AIMS将为AM2/IMD功能提供至关重要的见解，该功能将促进基于AM2/IMD的药物开发。