RAMP-altered class B GPCR hormone recognition

人类降钙素家族多肽激素降钙素(CT)、胰淀素(AMY)、降钙素基因相关肽 降钙素基因相关肽(CGRP)、肾上腺髓质素(AM)和肾上腺髓质素2/间质(AM2/IMD)表现出不同的作用，包括 骨重建(CT)、血管扩张和心脏保护的调节(CGRP、AM、AM2/IMD)及其调节 通过激活细胞表面B-G蛋白偶联受体调节血糖和食物摄入(AMY) (GPCRs)降钙素受体(CTR)和类降钙素受体(CLR)。三种受体活性修饰 蛋白质(RAMP1-3)与CLR和CTR异源二聚，并确定它们的多肽偏好。七人组 由此产生的受体(仅有六种异源二聚体和CTR)是已被证实或有望成为治疗以下疾病的药物靶点 是人类健康的重大负担，包括偏头痛(CGRP受体；CLR：RAMP1)， 糖尿病和肥胖(AMY受体；CTR：RAMP1/2/3)、骨质疏松症(CT受体；CTR)和心血管 疾病(AM受体；CLR：RAMP2/3)。这些复杂的受体是调节gpcr的范例。 副膜蛋白的药理作用，但我们对其选择性多肽识别的理解 机制和独特的生物功能是不完整的。我们的结构研究强调了关键 受体胞外区(ECDs)在多肽选择性中的作用 RAMP1和RAMP2如何改变CGRP和AM的CLR选择性在此，我们继续调查 斜坡调节CLR和CTR的机制。我们将检验变构调节的假设 斜坡对CLR构象的选择性是重要的，基于我们的发现，CTR N- 糖基化使其多肽亲和力提高10倍，我们将检验这一效应也是变构的假设 大自然。我们将通过提供AM2/IMD、CT和AMY的晶体结构来填补我们知识中的关键空白 与其受体ECD复合体结合。我们使用生化、药理学和X射线结晶学 针对每个具体目标的方法。在目标1中，我们将探讨变构在斜坡改变的CLR中的作用 通过使用新的可变选择性拮抗剂CGRP和AM变异体来实现多肽选择性 通过合理设计和筛选合成肽组合文库。在目标2中，我们将确定 AM2/IMD如何与CLR：RAMP1和CLR：RAMP3 ECD复合体结合，以及RAMP3如何调节CLR。在AIM 3我们将确定RAMPS和CTR ECD的N-糖基化如何调节CT和AMY结合。成功 该项目的完成将导致以下成果：1)更好地了解每个CT如何 家族多肽结合其受体，2)描述变构在RAMP功能中的作用，3)阐明 N-糖链和RAMP调节CTR多肽结合的机制，以及4)新的 具有增强的亲和力和改变的选择性的多肽拮抗剂，可能具有药理价值 用于询问受体生物学的工具，并可能为针对受体的药物开发提供信息。

The human calcitonin family peptide hormones calcitonin (CT), amylin (Amy), calcitonin-gene related peptide (CGRP), adrenomedullin (AM), and adrenomedullin 2/intermedin (AM2/IMD) exhibit diverse actions including regulation of bone remodeling (CT), vasodilation and cardioprotection (CGRP, AM, AM2/IMD), and regulation of blood glucose and food intake (Amy) by activating the cell surface class B G protein-coupled receptors (GPCRs) calcitonin receptor (CTR) and calcitonin-like receptor (CLR). Three receptor activity-modifying proteins (RAMP1-3) heterodimerize with CLR and CTR and determine their peptide preferences. The seven resulting receptors (six heterodimers and CTR alone) are proven or promising drug targets for diseases that are a significant burden on human health including migraine headache (CGRP receptor; CLR:RAMP1), diabetes and obesity (Amy receptors; CTR:RAMP1/2/3), osteoporosis (CT receptor; CTR), and cardiovascular disorders (AM receptors; CLR:RAMP2/3). These complex receptors are a paradigm for modulation of GPCR pharmacology by accessory membrane proteins, but our understanding of their selective peptide recognition mechanisms and distinct biological functions is incomplete. Our structural studies have highlighted the critical role of the receptor extracellular domains (ECDs) for peptide selectivity and provided important insights into how RAMP1 and RAMP2 alter CLR selectivity for CGRP and AM. Herein we continue to investigate the mechanisms by which RAMPs modulate CLR and CTR. We will test the hypothesis that allosteric modulation of CLR conformation by RAMPs is important for selectivity and based on our discovery that CTR N- glycosylation enhances its peptide affinity 10-fold we will test the hypothesis that this effect is also allosteric in nature. We will fill critical gaps in our knowledge by providing crystal structures of AM2/IMD, CT, and Amy bound to their receptor ECD complexes. We use biochemical, pharmacological, and X-ray crystallographic approaches for each of the specific aims. In Aim 1 we will probe the role of allostery in RAMP-altered CLR peptide selectivity through the use of novel altered selectivity antagonist CGRP and AM variants developed through rational design and screening of synthetic peptide combinatorial libraries. In Aim 2 we will determine how AM2/IMD binds CLR:RAMP1 and CLR:RAMP3 ECD complexes and how RAMP3 modulates CLR. In Aim 3 we will define how RAMPs and N-glycosylation of CTR ECD modulate CT and Amy binding. Successful completion of this project will lead to the following outcomes: 1) a better understanding of how each of the CT family peptides bind their receptors, 2) delineation of the role of allostery in RAMP function, 3) elucidation of the mechanisms by which N-glycans and RAMPs modulate CTR peptide binding, and 4) development of novel peptide antagonists with enhanced affinities and altered selectivities that may be of value as pharmacological tools for interrogating receptor biology and may inform drug development targeting the receptors.