Structure and Dynamics of Beta 2 Adrenoceptor Coupling to Gs

描述(申请人提供)：大多数激素和神经递质通过G蛋白偶联受体(GPCRs)向细胞传递信息，GPCRs是药物开发的最大靶点。22肾上腺素能受体(22AR)是GPCR家族中特征最为广泛的成员之一。它对儿茶酚胺类神经递质肾上腺素、去甲肾上腺素和多巴胺有反应；从广泛的突变研究中，人们已经知道了它的激动剂结合和G蛋白偶联结构域。22AR与腺酰环化酶刺激蛋白Gs的偶联是最早发现的激素激活的信号通路之一，是GPCR信号转导的典范。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，最近获得了野生型22AR与Fab片段的复合体的晶体结构，以及22AR的晶体结构，该晶体结构已被修改以提高其结构稳定性。Sunahara实验室最近开发了重组高密度脂蛋白磷脂颗粒，作为研究22AR和Gs之间结构相互作用的理想生化系统。这一建议代表了一种密切的合作，它结合了这两个实验室的专业知识，以表征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的GPCRs。此外，为确定22AR与Gs之间的耦合而开发的方法将适用于其他GPCRs。更好地了解22AR-Gs复合体的结构和激活机制将进一步促进基于结构的药物设计和GPCR靶点的电子筛选，从而更快地开发出高选择性和有效的药物。具体目标是：目的1.表征~(22)Ar-Gs耦合的结构动力学。我们将使用生物物理方法来检查在22AR-Gs复合体中观察到的功能协作性的结构基础。我们将考察Gs对22AR结构的影响，以及22AR对Gs结构的影响。我们将研究Gs与22AR单体和低聚物的偶联。此外，我们还将研究不同效率的配体对22AR和Gs相互作用的影响。目的2.确定22Ar-GS复合体的结构。我们将采取几种互补的方法来获得22AR-Gs络合物的高分辨率晶体结构。我们还将使用低温电子显微镜的单粒子成像来研究天然脂环境中的22AR-Gs复合体的结构。 公共卫生相关性：这项提案的目标是确定G蛋白偶联受体(GPCRs)对激素和神经递质做出反应的机制，并改变细胞的功能。这些信息将促进GPCRs的药物发现过程，GPCRs是人类基因组中最大的膜蛋白家族。作用于GPCRs的药物可以对一系列疾病产生影响，包括：心血管疾病、肺部疾病、炎症、糖尿病和肥胖症、行为障碍和阿尔茨海默病。

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.