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.

光激活的视紫红质受体为理解其结构和功能奠定了基础 G蛋白偶联受体的作用机制。然而，仍然有一些根本问题没有得到回答。 关于这些受体如何工作的问题。在这里，我们针对与以下内容相关的几个基本问题 了解他们的激活机制(S)。这种方法主要通过结构测量来实现。 采用固体核磁共振波谱。这些受体的现有晶体结构提供了高度的- 详细研究特定残基和基序在受体激活中的作用的解析框架。因为 视觉和配体激活的GPCRs之间的残基高度保守，正在形成的共识 视觉感受器不是独一无二的，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素。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，以靶向 暗光感受器视紫红质的不活跃和活跃状态。目标是在原子细节上理解 特异特征基序、基团保守基序和亚家族保守基序在激活过程中的相互作用 研究视紫红质的作用机制，并为激活其他GPCRs奠定共同基础。 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)以及在跨膜(TM)核心和受体的细胞内侧(目标2)。在目标1中， 我们将确定Trp6.48的作用，Trp6.48是介导视网膜异构化和Schiff碱基的关键残基 与受体保守的TM核心的去质子化。在目标2中，我们讨论了视网膜Schiff碱是如何 去质子化会导致活化。工作模式是有两个触发器，一个是静电，一个是 在自然界中是立体的。我们针对视紫红质保守的TM核心，由连锁签名和基团组成- 保守残基。工作模式是TM核心由两个堆积簇和两个 激活开关。它们分别为受体提供稳定和灵活的元件。在目标3中，我们 关注G蛋白及其与活性Meta II胞内残基的相互作用 中级的。在这个目标中，我们讨论了膜环境在受体稳定和激活中的作用。 最后，在目标4中，我们使用上面和从过去研究中获得的信息来确定两个 视网膜疾病、先天性静止性夜盲和常染色体显性遗传性视网膜色素变性。