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.

光激活的视觉受体视紫红质为理解其结构和功能提供了基础。 G蛋白偶联受体（GPCRs）。尽管如此， 这些受体是如何工作的在这里，我们针对几个基本问题， 了解其激活机制。该方法主要是通过结构测量 使用固态核磁共振光谱法。这些受体的现有晶体结构提供了高- 解析框架，以详细研究特定残基和基序在受体活化中的作用。因为 视觉和配体激活的GPCR之间残基的高度保守性， 视觉感受器并不是独一无二的，它为理解视觉系统的共同结构提供了基础。 和动态元素。一般的实验策略是使用固态核磁共振 光谱学与突变、光学和生物化学方法相结合，以靶向细胞中的特定区域。 弱光受体视紫红质的非活性和活性状态。我们的目标是了解原子细节， 在激活过程中，特异性签名、基团保守和亚家族保守基序之间的相互作用 研究视紫红质的作用机制，为其他GPCR的激活建立共同的基础。 四个具体的目标解决结构-功能的问题，涉及区域的细胞外侧的视紫红质 (Aim 1）和跨膜（TM）核心内以及受体的细胞内侧（Aim 2）。在目标1中， 我们将确定Trp6.48的作用-一个关键的残基，介导的视网膜异构化和希夫碱 去质子化与保守的TM核心的受体。在目标2中，我们解决了视网膜席夫碱如何 去质子化导致活化。工作模型是有两个触发器，一个是静电触发器， 在性质上是立体的。我们的目标是保守的TM核心的视紫红质组成的联锁签名和基团- 保守残基工作模型是TM核由两个堆积团簇和两个 激活开关。这些分别为接收器提供稳定和柔性元件。在目标3中，我们 专注于G蛋白及其与活性Meta II细胞内表面残基的相互作用 中间体在这个目标中，我们解决的作用，膜环境中的受体的稳定性和激活。 最后，在目标4中，我们使用上述信息和过去的研究来确定两个基础 视网膜疾病、先天性静止性夜盲和常染色体显性视网膜色素变性。