RAMP-altered class B GPCR hormone binding and signaling

摘要/摘要： 人类的三种受体活性修饰蛋白(RAMP1-3)与B-G类蛋白异源二聚体- 偶联受体(GPCRs)、降钙素受体(CTR)和降钙素样受体(CLR)及其调控 降钙素(CT)、胰淀素、降钙素基因相关肽(CGRP)和两种肾上腺髓质素(AM)的反应 多肽激素。它们在骨骼重塑(CT)、调节食物摄入量和血糖方面具有功能 (胰淀素)和调节血液和淋巴管功能(CGRP和AM)通过激活一个或多个 有7种受体来自CTR、CLR和RAMP(仅有6种异源二聚体CTR)。受体是 治疗骨质疏松症(CTR)、糖尿病和肥胖(胰淀素受体)、偏头痛(CGRP)的药物靶点 受体)和心血管和淋巴系统疾病(AM受体)。坡道如何调制CTR和 CLR的配体结合和信号传递尚不完全清楚。这限制了我们对分子的理解 为多肽在体内的作用奠定了基础，阻碍了药物的开发。值得注意的是，有越来越多的证据表明 坡道与许多GPCR相互作用，因此理解这一模式系统将具有广泛的影响。在……里面 在之前的资助周期中，我们使用生物化学、药理学和X射线结晶学来揭示CT、CGRP、 AM多肽与其受体胞外区(ECDs)结合，以及RAMP1/2ECDs如何改变CLR ECD多肽选择性。在这些结果的指导下，我们开发了超高亲和力的CGRP和AM变体 包括皮摩尔亲和力拮抗剂和显示持续cAMP信号的持续信号激动剂。 在这里，我们继续定义RAMP调节CTR和CLR配体结合的机制和 并进一步鉴定我们的新型多肽变体，它们可能具有治疗潜力。在目标1中 我们定义了CGRP和AM受体上的多肽结合动力学(CLR：RAMPS)及其与cAMP的关系 使用生化和药理学分析(SPR和Bret动力学方法)的信号动力学。在AIM 2 我们继续定义与CLR和CTR ECDs结合的肽的RAMP调节的结构基础 使用先进的分子动力学模拟和X射线结晶学。在《目标3》中，我们使用了一部小说 生化分析确定RAMP如何调节激动剂和G蛋白与CTR的偶联以使胰淀素成为可能 发信号。这个项目的完成将产生以下结果：1)了解多肽是如何 结合动力学影响CGRP和AM受体的cAMP信号动力学及其分子基础 超高亲和力变异体的持续信号传递，2)近乎完全的高分辨率结构理解 降钙素家族多肽与其受体ECDs的结合包括CGRP和AM多肽结合途径 和解结合，以及3)澄清长期存在的淀粉素受体药理歧义和描述 坡道如何调制CTR。这项工作将促进我们对附属膜蛋白的理解 调节GPCRs，并帮助开发针对CTR/CLR：RAMP复合体的治疗药物。

SUMMARY/ABSTRACT: Three receptor activity-modifying proteins (RAMP1-3) in humans heterodimerize with the class B G protein- coupled receptors (GPCRs) calcitonin receptor (CTR) and calcitonin-like receptor (CLR) and modulate their responses to calcitonin (CT), amylin, calcitonin gene-related peptide (CGRP), and two adrenomedullin (AM) peptide hormones. These have functions in bone remodeling (CT), regulation of food intake and blood glucose (amylin), and regulation of blood and lymphatic vasculature function (CGRP and AM) by activating one or more of seven receptors arising from CTR, CLR, and the RAMPs (6 heterodimers + CTR alone). The receptors are drug targets for osteoporosis (CTR), diabetes and obesity (amylin receptors), migraine headache (CGRP receptor), and cardiovascular and lymphatic system disorders (AM receptors). How RAMPs modulate CTR and CLR ligand binding and signaling is incompletely understood. This limits our understanding of the molecular basis for the diverse peptide actions in vivo and hinders drug development. Notably, there is growing evidence that RAMPs interact with numerous GPCRs so understanding this model system will have broad impact. In prior funding cycles we used biochemistry, pharmacology, and X-ray crystallography to reveal how CT, CGRP, and the AM peptides bind their receptor extracellular domains (ECDs) and how the RAMP1/2 ECDs alter CLR ECD peptide selectivity. Guided by these results we developed ultra high affinity CGRP and AM variants including picomolar affinity antagonists and sustained signaling agonists that exhibit persistent cAMP signaling. Herein we continue to define the mechanisms of RAMP modulation of CTR and CLR ligand binding and signaling and further characterize our novel peptide variants, which may have therapeutic potential. In Aim 1 we define the kinetics of peptide binding at CGRP and AM receptors (CLR:RAMPs) and its relation to cAMP signaling kinetics using biochemical and pharmacological assays (SPR and BRET kinetic methods). In Aim 2 we continue to define the structural bases for RAMP modulation of peptide binding to the CLR and CTR ECDs using advanced molecular dynamics simulations and X-ray crystallography. In Aim 3 we employ a novel biochemical assay to define how RAMPs modulate agonist and G protein coupling to CTR to enable amylin signaling. Completion of this project will yield the following outcomes: 1) an understanding of how peptide binding kinetics affects cAMP signaling kinetics for CGRP and AM receptors and the molecular basis for sustained signaling of ultra high affinity variants, 2) a near complete high-resolution structural understanding of calcitonin family peptide binding to their receptor ECDs including pathways of CGRP and AM peptide binding and unbinding, and 3) clarification of longstanding amylin receptor pharmacology ambiguities and delineation of how RAMPs modulate CTR. This project will advance our understanding of accessory membrane protein modulation of GPCRs and aid the development of therapeutics targeting CTR/CLR:RAMP complexes.