Mechanisms of GPCR Signaling

GPCR 信号传导机制

• 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
• 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

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 G 蛋白偶联受体 (GPCR) 的机制。尽管如此，仍有一些根本问题没有得到解答 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。该方法主要通过结构测量 使用固态核磁共振波谱。这些受体现有的晶体结构提供了高 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。因为 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 以及这些受体中的动态元件。一般的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。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。 四个具体目标解决涉及视紫红质细胞外侧区域的结构功能问题 （目标 1）以及跨膜 (TM) 核心内和受体的细胞内侧（目标 2）。在目标 1 中， 我们将确定 Trp6.48 的作用——介导视网膜异构化和希夫碱的关键残基 与受体保守的TM核心去质子化。在目标 2 中，我们讨论视网膜希夫碱如何 去质子化导致激活。工作模型是有两个触发器，一个是静电触发器，一个是 本质上是立体的。我们的目标是由互锁签名和基团组成的视紫红质的保守TM核心 保守的残留物。工作模型是TM核由两个封装簇和两个封装簇组成。 激活开关。它们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 中间的。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，我们使用上面获得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。