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

项目总结摘要 血管活性多肽肾上腺髓质素2/中介素(AM2/IMD)在人体内具有重要作用 生理和疾病，如血管扩张，生理性和病理性血管生成以及 对心血管和肾脏系统的保护作用。AM2/IMD的行动被归因于 G蛋白偶联下游包括cAMP和钙离子在内的多种信号中间产物的激活 受体，降钙素受体样受体(CLR)。不幸的是，几乎没有机械性的洞察力来了解 AM2/IMD结合并激活CLR。CLR药理很复杂，因为它与任何 三种受体活性修饰蛋白(RAMP1、-2或-3)之一，可调节其对AM2/IMD和 相关血管活性多肽降钙素基因相关肽(CGRP)和肾上腺髓质素(AM)。多么 AM2/IMD、CGRP和AM通过共享的RAMP：CLR受体复合体促进其独特的 信号结果仍不清楚。这限制了我们对AM2/IMD如何引发其广泛的 在人体生理学中的作用，并阻碍我们利用AM2/IMD信号进行药物开发的能力。这就做 检验AM2/IMD采用独特的受体结合构象以及它促进一种模式的假设 与AM和CGRP不同的RAMP：CLR复合体的偏向G蛋白激活。我要测试一下 这使用严格的生化、药理和结构方法有两个目的：1)定义分子 可溶性RAMP：CLR胞外区(ECD)复合体识别AM2/IMD的基础，以及2)定义 AM2/IMD促进的每个全长受体复合体的G蛋白偶联偏好 CGRP与AM对于目标1，我将三个ECD复合体分别提纯为拴系斜带ECD-CLR ECD 融合构建，发现AM2/IMD表现出与CGRP不同的结合偏好 和AM我解决了一个2.05？分辨率的晶体结构，它展示了一个非常独特的三个B转弯 与RAMP1-CLR ECD结合的AM2/IMD结构。我将确定AM2/IMD结合的晶体结构 RAMP3-CLR ECD，以充分了解AM2/IMD如何结合不同的受体ECD，并提供至关重要的 深入了解RAMP3如何调节CLR。对于目标2，我们确定了共表达和增溶的条件 三个全长斜面：CLR络合物，形成稳定的洗涤剂-无配体络合物。这提供了一种 这是研究这三种多肽如何促进不同G蛋白偶联的难得机会。我们将使用 Native-PAGE方法确定未纯化受体复合体的偶联偏好，我们将纯化 用荧光各向异性分析研究G蛋白偶联的无配体复合体。这些 生化研究将与人体内cAMP和钙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。成功完成这些任务 AIMS将提供对AM2/IMD功能的重要见解，使基于AM2/IMD的药物开发成为可能。

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.!