RAMP-altered class B GPCR hormone recognition

RAMP 改变的 B 类 GPCR 激素识别

基本信息

批准号：
9384071
负责人：
Augen A Pioszak
金额：
$ 32.99万
依托单位：
UNIVERSITY OF OKLAHOMA HLTH SCIENCES CTR
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-09-26 至 2021-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9384071
关键词：
Affinity Agonist Alpha Cell Amino Acid Receptors Area Binding Binding Sites Biochemical Biochemistry Biological Assay Biological Process Biology Blood Glucose Blood Vessels Bone remodeling Calcitonin Calcitonin Gene-Related Peptide Calcitonin Receptor Calcitonin-Gene Related Peptide Receptor Cardiovascular Diseases Cell surface Cells Clinical Complex Crystallization Development Diabetes Mellitus Disease Drug Receptors Drug Targeting Eating Exhibits Extracellular Domain Family Food Intake Regulation G-Protein-Coupled Receptors Goals Hand Health Heterodimerization Hormone Receptor Hormones Human Knowledge Libraries Mammalian Cell Melanocyte stimulating hormone Membrane Proteins Methods Migraine Molecular Conformation N Domain Nature Obesity Osteoporosis Outcome Peptide Receptor Peptides Pharmacology Physiology Polysaccharides Production Property Proteins RAMP1 RAMP2 RAMP3 Ramp Regulation Resolution Roentgen Rays Role Series Shapes Signal Transduction Site Specificity Structure Techniques Tertiary Protein Structure Testing Variant Vasodilation adrenomedullin adrenomedullin receptor amylin receptor analog base blood glucose regulation calcitonin receptor-like receptor design drug development glycosylation insight islet amyloid polypeptide member mutant novel peptide hormone peptide structure preference receptor receptor binding receptor-activity-modifying protein screening structural biology synthetic peptide combinatorial library therapeutic development therapeutic target tool

项目摘要

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.

人降钙素家族肽激素降钙素（CT），淀粉胺（AMY），降钙素与基因相关的肽（CGRP），肾上腺素肌蛋白（AM）和肾上腺素蛋白2/Intermedin（AM2/IMD）表现出多种作用，包括调节骨骼重塑（CT），血管舒张和心脏保护（CGRP，AM，AM2/IMD）和调节通过激活细胞表面B G蛋白偶联受体的血糖和食物摄入量（AMY）（GPCR）降钙素受体（CTR）和降钙素样受体（CLR）。三个受体活性调节蛋白质（RAMP1-3）与CLR和CTR异二聚体，并确定其肽偏好。七个由此产生的受体（六个异二聚体和单独的CTR）被证明或有希望的药物靶标的疾病是人类健康的重大负担，包括偏头痛（CGRP受体； CLR：RAMP1），糖尿病和肥胖症（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的范例配件膜蛋白的药理学，但我们对它们的选择性肽识别的理解机制和不同的生物学功能是不完整的。我们的结构研究强调了关键受体细胞外结构域（ECD）的作用在肽选择性方面的作用，并提供了重要的见解 RAMP1和RAMP2如何改变CGRP和AM的CLR选择性。在这里，我们继续调查坡道调节CLR和CTR的机制。我们将测试变构调节的假设坡道的CLR构象对选择性很重要，并且基于我们发现Ctr n- 糖基化增强其肽亲和力10倍，我们将检验以下假设：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的知识的关键空白与他们的受体ECD复合物结合。我们使用生化，药理学和X射线晶体学每个特定目标的方法。在AIM 1中，我们将探测变构在改变坡道的CLR中的作用肽选择性通过使用新颖的选择性拮抗剂CGRP和AM变体的选择性选择性通过合理设计和合成肽组合文库的筛选。在AIM 2中，我们将确定 AM2/IMD如何结合CLR：RAMP1和CLR：RAMP3 ECD复合物以及RAMP3如何调节CLR。目标 3我们将定义CTR ECD的坡道和N-糖基化如何调节CT和AMY结合。成功的该项目的完成将导致以下结果：1）更好地了解每个CT 家族肽结合其受体，2）划定变构作用在坡道功能中的作用，3）阐明 N-聚糖和坡道调节CTR肽结合的机制，以及4）新型的发展肽拮抗剂具有增强的亲和力和变化的选择性，这可能是有价值的询问受体生物学的工具，并可能告知针对受体的药物开发。