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生物体及其组成细胞对各种刺激的反应，包括光、味觉、气味、激素和神经递质。反应的性质可以同样多样化，从基因转录的变化到跨膜离子通透性的改变。这个系统的三个核心组件是七螺旋受体、异三聚体G蛋白和效应器分子，它们必须相互作用才能将信息从一个组件传递到另一个组件。流行的观点认为，这些相互作用是信号分子之间随机碰撞的结果，这些分子在质膜中自由移动。然而，最近的证据表明，信号分子聚集在细胞表面的小凹等微域中，这表明这些系统比之前认为的更有组织。为了确定这些系统的组织是否延伸到分子水平，一种被称为生物发光共振能量转移(BRET)的技术被用来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。这些信号分子在转基因的哺乳动物细胞中以融合蛋白的形式表达，标记有生物发光蛋白荧光素酶(RLuc)或绿色荧光蛋白的增强变体(EGFP)。如果标签通过两个信号分子之间稳定的蛋白质-蛋白质相互作用而并置，则会发生Bret，因为RLuc标签发出的光将被EGFP标签吸收，然后EGFP标签发出荧光。在这些研究中，我们使用了典型的β2-肾上腺素能受体(B2AR)信号系统，该信号系统由B2AR、刺激性异三聚体G蛋白(Gs)和效应器腺酰环化酶(AC)组成。激动剂刺激B2AR通过Gs产生环状AMP激活AC。构建了Ac-RLuc和B2AR-EGFP，并在HEK 293细胞中表达。它们的功能被增加的配体结合和激动剂介导的环状AMP的产生所证实。在没有激动剂的情况下，B2AR-EGFP和AC-RLuc之间存在Bret。这些数据表明，在活细胞中，即使在基础状态下也存在受体-效应器复合体，并支持G蛋白介导的信号系统作为有组织的复合体存在的不断发展的观点。