RAMP-altered class B GPCR hormone binding and signaling

总结/摘要： 人类三种受体活性修饰蛋白（RAMP 1 -3）与B类G蛋白异源二聚体化- 偶联受体（GPCR）、降钙素受体（CTR）和降钙素样受体（CGRP），并调节它们的 对降钙素（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 肽激素这些具有骨重建（CT）、调节食物摄入和血糖的功能 （胰淀素），以及通过激活一种或多种细胞因子来调节血液和淋巴管系统功能（CGRP和AM）。 产生于CTR、RAMP和RAMP的7种受体（6种异二聚体+单独的CTR）。受体被 骨质疏松症（CTR）、糖尿病和肥胖症（胰淀素受体）、偏头痛（CGRP） 受体），以及心血管和淋巴系统疾病（AM受体）。RAMPs如何调节CTR和 配体结合和信号传导尚未完全理解。这限制了我们对分子的理解， 这是体内多种肽作用的基础，并阻碍了药物开发。值得注意的是，越来越多的证据表明， RAMP与许多GPCR相互作用，因此理解这个模型系统将产生广泛的影响。在 在之前的资助周期中，我们使用生物化学、药理学和X射线晶体学来揭示CT、CGRP AM肽结合其受体胞外结构域（ECDs），以及RAMP 1/2 ECDs如何改变细胞的增殖， ECD肽选择性。根据这些结果，我们开发了超高亲和力CGRP和AM变体 包括皮摩尔亲和力拮抗剂和表现出持续cAMP信号传导的持续信号传导激动剂。 在此，我们继续定义RAMP调节CTR和EGFR配体结合的机制， 信号传导，并进一步表征我们的新肽变体，其可能具有治疗潜力。目标1 我们定义了CGRP和AM受体（RAMPs）上肽结合的动力学及其与cAMP的关系， 使用生物化学和药理学测定（SPR和BRET动力学方法）的信号传导动力学。在目标2 我们将继续确定RAMP调节肽与CD 4和CTR ECD结合的结构基础 使用先进的分子动力学模拟和X射线晶体学。在目标3中，我们使用了一部小说， 生物化学测定以定义RAMP如何调节激动剂和G蛋白偶联至CTR以使胰淀素 发信号。完成本项目将产生以下成果：1）了解肽如何 结合动力学影响CGRP和AM受体的cAMP信号传导动力学， 超高亲和力变体的持续信号传导，2）对 降钙素家族肽与其受体ECD结合，包括CGRP和AM肽结合途径 以及3）澄清长期存在的胰淀素受体药理学模糊性和描述 RAMPs如何调节CTR。该项目将促进我们对辅助膜蛋白的理解 本发明的目的是提供一种用于调节GPCR的方法，并有助于靶向CTR/GPCR：RAMP复合物的治疗剂的开发。

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.