Structure and Dynamics of Beta 2 Adrenoceptor Coupling to Gs

Beta 2 肾上腺素受体与 Gs 偶联的结构和动力学

基本信息

批准号：
8102237
负责人：
Brian K Kobilka
金额：
$ 6.6万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-08-01 至 2011-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8102237
关键词：
Adenylate Cyclase Adrenergic Receptor Agonist Alzheimer&apos s Disease Apolipoprotein A-I Behavior Disorders Binding Biochemical Biological Models Cardiovascular Diseases Catecholamines Cell physiology Cells Collaborations Complex Computer Simulation Coupling Cryoelectron Microscopy Cysteine Development Diabetes Mellitus Dihydroalprenolol Disease Dopamine Drug Design Energy Transfer Environment Environment and Public Health Epinephrine Fab Immunoglobulins Family Fluorescence Resonance Energy Transfer G Protein-Coupled Receptor Signaling G-Protein-Coupled Receptors GTP-Binding Proteins Goals High Density Lipoproteins Hormones Human Genome Image Inflammation Laboratories Ligands Lipids Lung diseases Membrane Proteins Methodology Mutagenesis Neurotransmitters Norepinephrine Obesity Pharmaceutical Preparations Phospholipids Process Proteins Recombinants Research Resolution Screening procedure Signal Pathway Structure System X-Ray Crystallography base beta-2 Adrenergic Receptors drug development drug discovery fluorophore improved interest member monoamine monomer particle public health relevance receptor reconstitution

项目摘要

DESCRIPTION (provided by applicant): The majority of hormones and neurotransmitters communicate information to cells via G protein-coupled receptors (GPCRs), and GPCRs represent the largest group of targets for drug development. The 22 adrenoceptor (22AR) has been one of the most extensively characterized members of the GPCR family. It responds to the catecholamine neurotransmitters epinephrine, norepinephrine and dopamine; and much is known about its agonist binding and G protein coupling domains from extensive mutagenesis studies. The coupling of the 22AR to Gs, the stimulatory protein for adenylyl cyclase, was one of the first hormone activated signaling pathways to be discovered and serves as a paradigm of GPCR signaling. During the past several years the Kobilka lab has made significant progress towards characterizing the structural changes associated with agonist activation, and has recently obtained a crystal structure of the wild type 22AR in complex with a Fab fragment, as well as a crystal structure of a 22AR that has been modified to improve its structural stability. The Sunahara lab recently developed recombinant HDL phospholipid particles as an ideal biochemical system for studying structural interactions between the 22AR and Gs. This proposal represents a close collaboration that combines the expertise of these two labs to characterize the structural interactions between the 22AR and Gs. The findings will likely apply to the large number of closely related monoamine receptors and to GPCRs in general. Moreover, the methodologies developed for characterizing 22AR coupling to Gs will be applicable to other GPCRs. A better understanding of the structure and mechanism of activation of the 22AR-Gs complex will further the potential for structure-based drug design and in silico screening for GPCR targets, leading to more rapid development of highly selective and effective drugs. the specific aims are: aim 1. characterize the structural dynamics of 22ar coupling to Gs. We will use biophysical approaches to examine the structural basis of functional cooperativity observed in the 22AR-Gs complex. We will examine the effect of Gs on the structure of the 22AR, and the effect of the 22AR on the structure of Gs. We will examine coupling of Gs to 22AR monomers and oligomers. In addition, we will study the effects of ligands having different efficacies on interactions between the 22AR and Gs. Aim 2. Determine the structure of the 22ar-gs complex. We will take several complementary approaches to obtain a high-resolution crystal structure of the 22AR-Gs complex. We will also use single particle imaging by cryoelectron microscopy to study the structure of the 22AR-Gs complex in a native lipid environment. Public Health Relevance: The goal of this proposal is to determine the mechanism by which G protein coupled receptors (GPCRs) respond to hormones and neurotransmitters, and modify the function of cells. This information will facilitate the process of drug discovery for GPCRs, which are the largest family of membrane proteins in the human genome. Drugs acting on GPCRs can have an impact on a broad spectrum of diseases including: cardiovascular disease, pulmonary disease, inflammation, diabetes and obesity, behavioral disorders and Alzheimer's disease.

描述（由申请人提供）：大多数激素和神经递质通过 G 蛋白偶联受体 (GPCR) 向细胞传达信息，GPCR 代表了药物开发的最大一组靶标。 22 肾上腺素受体 (22AR) 是 GPCR 家族中特征最广泛的成员之一。它对儿茶酚胺神经递质肾上腺素、去甲肾上腺素和多巴胺有反应；通过广泛的诱变研究，人们对它的激动剂结合和 G 蛋白偶联结构域有了很多了解。 22AR 与 Gs（腺苷酸环化酶的刺激蛋白）的偶联是最早被发现的激素激活信号传导途径之一，并可作为 GPCR 信号传导的范例。在过去的几年中，Kobilka 实验室在表征与激动剂激活相关的结构变化方面取得了重大进展，最近获得了与 Fab 片段复合的野生型 22AR 的晶体结构，以及 22AR 的晶体结构。经过修改以提高其结构稳定性。 Sunahara 实验室最近开发了重组 HDL 磷脂颗粒，作为研究 22AR 和 Gs 之间结构相互作用的理想生化系统。该提案代表了密切合作，结合了这两个实验室的专业知识来表征 22AR 和 G 之间的结构相互作用。这些发现可能适用于大量密切相关的单胺受体和一般 GPCR。此外，为表征 22AR 与 G 偶联而开发的方法将适用于其他 GPCR。更好地了解 22AR-Gs 复合物的结构和激活机制将进一步发挥基于结构的药物设计和 GPCR 靶点计算机筛选的潜力，从而更快地开发高选择性和有效的药物。具体目标是：目标 1. 表征 22ar 与 Gs 偶联的结构动力学。我们将使用生物物理学方法来检查 22AR-Gs 复合物中观察到的功能协同性的结构基础。我们将研究Gs对22AR结构的影响，以及22AR对Gs结构的影响。我们将检查 G 与 22AR 单体和低聚物的偶联。此外，我们还将研究具有不同功效的配体对22AR和Gs之间相互作用的影响。目标 2. 确定 22ar-gs 复合物的结构。我们将采取几种互补的方法来获得 22AR-Gs 复合物的高分辨率晶体结构。我们还将使用冷冻电子显微镜的单粒子成像来研究天然脂质环境中 22AR-Gs 复合物的结构。公共健康相关性：该提案的目标是确定 G 蛋白偶联受体 (GPCR) 响应激素和神经递质并改变细胞功能的机制。这些信息将促进 GPCR 的药物发现过程，GPCR 是人类基因组中最大的膜蛋白家族。作用于 GPCR 的药物可以对多种疾病产生影响，包括：心血管疾病、肺部疾病、炎症、糖尿病和肥胖、行为障碍和阿尔茨海默病。