Elucidating The Structural Organization Of G-protein Cou

阐明 G 蛋白 Cou 的结构组织

• 批准号: 6503234
• 负责人: ROBERT VICTOR REBOIS
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6503234
• 关键词: G protein; adenylate cyclase; beta adrenergic receptor; binding proteins; biological signal transduction; cyclic AMP; enzyme activity; fluorescence resonance energy transfer; genetic transcription; green fluorescent proteins; guanine nucleoside; human tissue; luciferin monooxygenase; membrane reconstitution /synthesis; protein protein interaction; protein structure; tissue /cell culture

G protein-mediated signal transduction systems are involved in the responses of organisms and their constituent cells to a wide variety of stimuli including light, gustants, odorants, hormones, and neurotransmitters. The nature of the response can be equally diverse varying from changes in gene transcription to altered transmembrane ion permeability. The three core components of this system are the heptahelical receptors, heterotrimeric G proteins and effector molecules which must interact in order to convey information from one component to the next. The prevailing view has been that these interactions are the result of random collisions between signaling molecules that move about freely in the plasma membrane. However, recent evidence indicates that signaling molecules are corralled in microdomains such as caveolae on the cell surface suggesting that these systems are more well organized than previously thought. In order to determine if the organization of these systems extends to the molecular level, a technique know as bioluminescence resonance energy transfer (BRET) is being used to investigate protein-protein interactions between these signaling molecules in living cells. The signaling molecules are expressed in transfected mammalian cells as fusion proteins tagged with either the bioluminescent protein luciferase (RLuc) or an enhanced variant of green fluorescent protein (EGFP). If the tags are brought into juxtaposition by a stable protein-protein interaction between two signaling molecules, BRET occurs because light emitted by the RLuc tag will be absorbed by the EGFP tag which then fluoresces. For these studies, we are using the prototypical beta2-adrenergic receptor (b2AR) signaling system that consists of b2AR, the stimulatory heterotrimeric G protein (Gs) and the effector adenylyl cyclase (AC). Agonist stimulation of b2AR activates AC through Gs producing cyclic AMP. AC-RLuc and b2AR-EGFP were constructed and expressed in HEK 293 cells. Their functionality was confirmed by increased ligand binding and agonist-mediated cyclic AMP production. BRET was detected between b2AR-EGFP and AC-RLuc in the absence of agonist. These data suggest that in living cells, a receptor-effector complex exists even in the basal state, and provides support for the evolving view that G protein-mediated signaling systems exist as organized complexes.

G蛋白介导的信号转导系统参与了生物体及其成分细胞对各种刺激的反应，包括光，胶，气味，激素和神经递质。响应的性质可能是从基因转录的变化到变化的跨膜离子渗透性的不同。该系统的三个核心组成部分是七螺旋体受体，异三聚体G蛋白和效应分子，它们必须相互作用才能从一个成分传达到下一个成分。流行的观点是这些相互作用是在质膜中自由移动的信号分子之间随机碰撞的结果。然而，最近的证据表明，在细胞表面上的小窝中，信号分子在小caveolae中被折叠，这表明这些系统的组织性比以前想象的要井井有条。为了确定这些系统的组织是否扩展到分子水平，一种被称为生物发光共振能量转移（BRET）的技术被用于研究活细胞中这些信号分子之间的蛋白质蛋白质相互作用。信号分子在转染的哺乳动物细胞中表示为用生物发光蛋白荧光素酶（RLUC）或增强的绿色荧光蛋白（EGFP）变体的融合蛋白。如果标签通过两个信号分子之间稳定的蛋白质 - 蛋白质相互作用并并置，则会发生BRET，因为RLUC标签发出的光将被EGFP TAG吸收，然后将其吸收。在这些研究中，我们使用的是由B2AR，刺激性异三聚体G蛋白（GS）和效应子腺苷酸环化酶（AC）组成的典型β2-肾上腺素能受体（B2AR）信号传导系统。激动剂刺激B2AR通过产生环状AMP的GS激活AC。在HEK 293细胞中构建并表达AC-rluc和B2AR-EGFP。通过增加的配体结合和激动剂介导的循环AMP产生来证实它们的功能。在没有激动剂的情况下，在B2AR-EGFP和AC-rluc之间检测到BRET。这些数据表明，在活细胞中，即使处于基础状态，也存在一种受体效应络合物复合物，并为不断发展的观点提供了支持G蛋白介导的信号系统作为有组织的复合物存在的观点。