RAMP-altered class B GPCR hormone recognition

人降钙素家族肽类激素降钙素（CT）、胰淀素（Amy）、降钙素基因相关肽 肾上腺髓质素（AM）和肾上腺髓质素2/中间肽（AM2/IMD）表现出不同的作用，包括 调节骨重建（CT）、血管舒张和心脏保护（CGRP、AM、AM 2/IMD），以及调节 通过激活细胞表面B G类蛋白偶联受体， （GPCR）降钙素受体（CTR）和降钙素样受体（CGRP）。三受体活性调节 蛋白质（RAMP 1 - 3）异源二聚体化，并确定它们的肽偏好。七 所产生的受体（六种异二聚体和单独的CTR）是已被证明或有希望的疾病的药物靶点， 是人类健康的重大负担，包括偏头痛（CGRP受体; RAMP1）， 糖尿病和肥胖症（Amy受体; CTR：RAMP1/2/3）、骨质疏松症（CT受体; CTR）和心血管疾病 疾病（AM受体; RAMP2/3）。这些复合受体是调节GPCR的范例 药理学的辅助膜蛋白，但我们的理解，他们的选择性肽识别 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 受体胞外结构域（ECD）对肽选择性的作用，并提供了重要的见解， RAMP1和RAMP2如何改变CGRP和AM的选择性。在此，我们继续调查 RAMPs通过什么机制来调节转录因子和CTR。我们将检验变构调节 通过RAMPs对CTR构象的改变对于选择性是重要的，并且基于我们的发现，CTR N- 糖基化使其肽亲和力提高10倍，我们将检验这种作用在 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 与受体ECD复合物结合。我们用生化学，药理学，和X射线晶体学 针对每一个具体目标。在目的1中，我们将探讨变构在RAMP改变的胰腺中的作用。 肽选择性通过使用新的改变选择性拮抗剂CGRP和AM变体开发 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如何结合RAMP1和RAMP3 ECD复合物以及RAMP3如何调节RAMP3。在aim中 我们将定义RAMP和CTR ECD的N-糖基化如何调节CT和Amy结合。成功 本项目的完成将导致以下成果：1）更好地了解每个CT 家族肽结合其受体，2）描绘变构在RAMP功能中的作用，3）阐明 N-聚糖和RAMP调节CTR肽结合的机制，以及4）开发新的 具有增强的亲和力和改变的选择性的肽拮抗剂， 用于询问受体生物学的工具，并且可以为靶向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.