RAMP-altered class B GPCR hormone binding and signaling

RAMP 改变的 B 类 GPCR 激素结合和信号转导

基本信息

批准号：
10298293
负责人：
Augen A Pioszak
金额：
$ 34.08万
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 2025-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10298293
关键词：
Address Affect Affinity Agonist Area Binding Biochemical Biochemistry Biological Assay Biological Models Biosensor Blood Blood Glucose Blood Vessels Bone remodeling Calcitonin Calcitonin Gene-Related Peptide Calcitonin Receptor Calcitonin-Gene Related Peptide Receptor Cardiovascular Diseases Cardiovascular system Cell surface Cells Chimera organism Collaborations Complex Computational algorithm Coupled Coupling Crystallization Cyclic AMP Detergents Diabetes Mellitus Disease Drug Receptors Drug Targeting Eating Electrophoretic Mobility Shift Assay Event Exhibits Extracellular Domain Family Food Intake Regulation Funding G-Protein-Coupled Receptors GTP-Binding Proteins Goals Hand Health Heterodimerization Hormone Receptor Hormones Human Kinetics Knowledge Ligand Binding Lymphatic System Mammalian Cell Measures Mediating Membrane Membrane Proteins Methods Migraine Molecular Molecular Conformation Molecular Structure Obesity Osteoporosis Outcome Pathway interactions Peptide Signal Sequences Peptides Pharmaceutical Preparations Pharmacology Physiology Property RAMP1 RAMP3 Regulation Resolution Role Salmon Signal Transduction Structure System Techniques Tertiary Protein Structure Testing Therapeutic Time Transmembrane Domain Variant Vasodilation X-Ray Crystallography adrenomedullin adrenomedullin receptor amylin receptor analog base calcitonin receptor-like receptor cardioprotection design drug development in vivo insight islet amyloid polypeptide lymphatic vasculature member molecular dynamics novel overexpression peptide drug peptide hormone preference receptor receptor internalization receptor-activity-modifying protein residence response stem structural biology therapeutic development therapeutic target tool

项目摘要

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.

摘要/摘要：人类中的三种受体活性调节蛋白（ramp1-3）与B级蛋白质的异二聚二聚体耦合受体（GPCR）降钙素受体（CTR）和降钙素样受体（CLR），并调节其对降钙素（CT），淀粉素，降钙素基因相关肽（CGRP）和两个肾上腺素肌蛋白（AM）的反应肽激素。这些在骨骼重塑（CT），食物摄入和血糖的调节中具有功能通过激活一个或多个来调节血液和淋巴脉管功能（CGRP和AM）的调节由CTR，CLR和坡道引起的七个受体（单独使用6个异二聚体 + CTR）。受体是骨质疏松症（CTR），糖尿病和肥胖症（淀粉蛋白酶受体），偏头痛的药物靶标（CGRP）受体）以及心血管和淋巴系统疾病（AM受体）。坡道如何调节CTR和 CLR配体结合和信号传导尚不完全理解。这限制了我们对分子的理解在体内采取多种肽作用的基础，并阻碍了药物开发。值得注意的是，越来越多的证据坡道与众多GPCR相互作用，因此了解该模型系统将产生广泛的影响。在先前的资金周期我们使用生物化学，药理学和X射线晶体学来揭示CT，CGRP如何 AM肽结合其受体细胞外结构域（ECD）以及RAMP1/2 ECD如何改变CLR ECD肽的选择性。在这些结果的指导下，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的cAMP信号传导的持续信号激动剂。在此，我们继续定义CTR和CLR配体结合的坡道调节机制信号传导并进一步表征我们的新型肽变体，可能具有治疗潜力。在目标1中我们定义了CGRP和AM受体（CLR：坡道）的肽结合的动力学及其与CAMP的关系使用生化和药理学测定（SPR和BRET动力学方法）的信号动力学。在目标2中我们继续定义用于调节肽与CLR和CTR ECD的结构碱基使用高级分子动力学模拟和X射线晶体学。在目标3中，我们采用小说生化测定方法以定义坡道如何调节激动剂和G蛋白偶联到CTR以启用淀粉蛋白信号。该项目的完成将产生以下结果：1）了解肽的理解结合动力学会影响CGRP和AM受体的CAMP信号动力学，以及分子基础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肽结合的途径和未连接，以及3）澄清长期存在的淀粉纤维受体药理学的歧义和描述坡道如何调节CTR。该项目将提高我们对附件膜蛋白的理解 GPCR的调节并有助于针对CTR/CLR的治疗剂的发展：坡道复合物。