Mechanisms of GPCR Signaling

GPCR 信号传导机制

基本信息

批准号：
10240655
负责人：
STEVEN Owen SMITH
金额：
$ 33.15万
依托单位：
STATE UNIVERSITY NEW YORK STONY BROOK
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-09-15 至 2023-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10240655
关键词：
11 cis Retinal Address Adopted Binding Binding Proteins Binding Sites Biochemical Communication Consensus Coupled Couples Crystallization Dependence Development Disease Electrostatics Elements Environment FDA approved Family Family member Foundations G Protein-Coupled Receptor Signaling G-Protein-Coupled Receptors GTP-Binding Protein alpha Subunits, Gs GTP-Binding Proteins Goals Head Heart Hydrogen Bonding Indoles Lead Ligand Binding Ligands Light Lipids Liquid substance Measurement Mediating Membrane Methods Modeling Molecular Molecular Conformation Motion Movement Mutation NMR Spectroscopy Nature Night Blindness Optics Pathway interactions Pharmacologic Substance Phase Phenylalanine Photoreceptors Positioning Attribute Protons Reaction Receptor Activation Resolution Retina Retinal Diseases Retinitis Pigmentosa Rhodopsin Rod Outer Segments Role Rotation SWI1 Schiff Bases Series Side Site Structure Surface Transducin Tryptophan Vision Visual Work chromophore deprotonation disease-causing mutation extracellular flexibility inhibitor/antagonist insight member mutant protonation receptor scaffold solid state nuclear magnetic resonance

项目摘要

The light-activated visual receptor rhodopsin has provided the foundation for understanding the structure and mechanism of G protein-coupled receptors (GPCRs). Nevertheless, there remain fundamental unanswered questions about how these receptors work. Here, we target several basic questions that are relevant for understanding their mechanism(s) of activation. The approach is primarily through structural measurements using solid-state NMR spectroscopy. The existing crystal structures of these receptors provide a high- resolution framework to study in detail the role of specific residues and motifs in receptor activation. Because of the high conservation of residues between the visual and ligand-activated GPCRs, the emerging consensus is that rather than being unique, the visual receptors provide a basis for understanding the common structural and dynamic elements in these receptors. The general experimental strategy is to use solid-state NMR spectroscopy in combination with mutational, optical and biochemical methods to target specific regions in the inactive and active states of the dim-light receptor, rhodopsin. The goal is to understand in atomic detail the interplay between specific signature, group-conserved and subfamily-conserved motifs in the activation mechanism of rhodopsin and establish a common basis for the activation of other GPCRs. Four specific aims address structure-function questions involving regions on the extracellular side of rhodopsin (Aim 1) and within the transmembrane (TM) core and on the intracellular side of the receptor (Aim 2). In Aim 1, we will establish the role of Trp6.48 – a key residue that mediates retinal isomerization and Schiff base deprotonation with the conserved TM core of the receptor. In Aim 2, we address how retinal Schiff base deprotonation leads to activation. The working model is that there are two triggers, one electrostatic and one steric in nature. We target the conserved TM core of rhodopsin composed of interlocking signature and group- conserved residues. The working model is that the TM core is composed of two packing clusters and two activation switches. These provide stable and flexible elements to the receptor, respectively. In Aim 3, we focus on the G protein and its interactions with residues on the intracellular surface of the active Meta II intermediate. In this aim, we address the role of the membrane environment in receptor stability and activation. Finally, in Aim 4 we use the information garnered above and from past studies to determine the basis for two retinal diseases, congenital stationary night blindness and autosomal dominant retinitis pigmentosa.

光激活视觉受体视紫红质为理解其结构和然而，G 蛋白偶联受体 (GPCR) 的机制仍然存在根本性的未解之谜。关于这些受体如何工作的问题在这里，我们针对与以下相关的几个基本问题。了解它们的激活机制该方法主要是通过结构测量。使用固态核磁共振波谱法，这些受体的现有晶体结构提供了高- 分辨率框架详细研究特定残基和基序在受体激活中的作用。视觉 GPCR 和配体激活 GPCR 之间残基的高度保守性，正在形成的共识视觉感受器并不是独一无二的，而是为理解共同的结构提供了基础这些受体中的动态元素和动态元素的一般实验策略是使用固态核磁共振。光谱学与突变、光学和生化方法相结合，以针对特定区域弱光受体视紫红质的非活性和活性状态的目标是了解原子细节。激活过程中特定特征、群体保守和亚家族保守基序之间的相互作用视紫红质机制，并为其他 GPCR 的激活建立共同基础。四个特定区域的目标是解决涉及视紫红质细胞外侧的结构功能问题（目标 1）以及跨膜 (TM) 核心内和受体的细胞内侧（目标 1）。我们将确定 Trp6.48 的作用——介导视网膜异构化和席夫碱的关键残基在目标 2 中，我们讨论了视网膜希夫碱如何与受体的保守 TM 核心进行去质子化。去质子化导致激活的工作模型是有两种触发器，一种是静电触发器，另一种是静电触发器。我们的目标是由互锁特征和基团组成的视紫红质的保守TM核心。工作模型是TM核心由两个堆积簇和两个组成。这些激活开关分别为受体提供了稳定和灵活的元件，在目标 3 中，我们。重点关注 G 蛋白及其与活性 Meta II 细胞内表面残基的相互作用为此，我们研究了膜环境在受体稳定性和激活中的作用。最后，在目标 4 中，我们使用上面收集的信息和过去的研究来确定两个目标的基础视网膜疾病、先天性静止性夜盲症和常染色体显性视网膜色素变性。