Elucidating The Structural Organization Of G-protein Cou

阐明 G 蛋白 Cou 的结构组织

• 批准号: 6661049
• 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/6661049
• 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, accumulating data has provided evidence that signaling molecules are organized into macromolecular complexes on the cell surface. To elucidate the spatial arrangement of the proteins that make up these signaling complexes in living cells, I have used bioluminescence resonance energy transfer (BRET). The signaling molecules are expressed in transfected mammalian cells as fusion proteins tagged with either the bioluminescent protein luciferase (RLuc) or green fluorescent protein (GFP). 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 GFP 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) which is made up of an alpha (G-alpha), a beta (G-beta) and a gamma subunit, and the effector adenylyl cyclase (AC). Agonist stimulation of the b2AR results in the Gs-mediated stimulation of AC leading to the production of cyclic AMP. When the b2AR is tagged with GFP or AC with RLuc these signaling molecules retain their biological activity. BRET occurred when the b2AR-GFP and AC-RLuc were co-expressed in HEK 293 cells indicating that they form a complex in living cells. BRET experiments indicate that this complex is present in the absence of hormone stimulation suggesting that the it exists in the basal state. Currently, G-alpha-GFP and G-beta-RLuc are being used to investigate the interaction of G protein subunits with each other, and with receptor (ie. b2AR-GFP and b2AR-RLuc) and effector (ie. AC-RLuc) proteins. These data are providing support for the evolving view that G protein-mediated signaling systems exist as organized complexes that contribute significantly to the specificity and efficacy of the signal transduction process. In addition to forming complexes with down stream signaling molecules, heptahelical receptors interact with each other to form homomultimers (most probably homodimers). As the receptor's polypeptide chain passes back and forth through the plasma membrane it creates loops that extend into the extra- and intracellular spaces and deposits the C-terminus within the cytoplasmic milieu. The third intracellular loop and the C-terminus are both critical for interaction with downstream signaling components, and the lack of either domain renders the receptor unable to mediate signal transduction despite retention of the ability to bind ligand. It has been hypothesized that receptor oligomerization results in intermolecular interactions in which a portion of one receptor is associated with the complementary part of the other, thus forming a domain that interacts with the heterotrimeric G proteins. To test this hypothesis two signaling deficient b2ARs (one that is missing the third intracellular loop and one that is missing the C-terminal amino acid sequence) are being co-expressed in HEK 293 cells in order to determine if a fully functional b2AR can be reconstitute from the two mutated receptors

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、气味、激素和神经递质。这种反应的性质也可以是多种多样的，从基因转录的变化到跨膜离子渗透性的改变。该系统的三个核心成分是七螺旋受体、异源三聚体G蛋白和效应分子，它们必须相互作用才能将信息从一个成分传递到另一个成分。主流观点认为，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，越来越多的数据提供了证据，表明信号分子在细胞表面被组织成大分子复合物。为了阐明构成这些信号复合物的蛋白质在活细胞中的空间排列，我使用了生物发光共振能量转移（BRET）。这些信号分子在转染的哺乳动物细胞中以融合蛋白的形式表达，融合蛋白被标记为生物发光蛋白荧光素酶（RLuc）或绿色荧光蛋白（GFP）。如果两个信号分子之间通过稳定的蛋白质-蛋白质相互作用将标签并置，则会发生BRET，因为RLuc标签发出的光将被GFP标签吸收，然后发出荧光。在这些研究中，我们使用了典型的β -肾上腺素能受体（b2AR）信号系统，该信号系统由b2AR、由α (G- α)、β (G- β)和γ亚基组成的刺激异三聚体G蛋白（Gs）和效应腺苷酸环化酶（AC）组成。b2AR的激动剂刺激导致gs介导的AC刺激导致环状AMP的产生。当b2AR被GFP或带有RLuc的AC标记时，这些信号分子保持其生物活性。当b2AR-GFP和AC-RLuc在HEK 293细胞中共表达时，发生BRET，表明它们在活细胞中形成复合物。BRET实验表明，该复合物在没有激素刺激的情况下存在，表明它存在于基础状态。目前，G- α - gfp和G- β - rluc正被用于研究G蛋白亚基之间的相互作用，以及与受体的相互作用。b2AR-GFP和b2AR-RLuc)和效应物(即；AC-RLuc)的蛋白质。这些数据为不断发展的观点提供了支持，即G蛋白介导的信号系统作为有组织的复合物存在，对信号转导过程的特异性和有效性有重要贡献。