Structural Organization Of G-protein Coupling Systems

G 蛋白偶联系统的结构组织

• 批准号: 6842472
• 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/6842472
• 关键词: G protein; adenylate cyclase; beta adrenergic receptor; binding proteins; biological signal transduction; bioluminescence; 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; receptor coupling; 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. In order to elucidate the spacial arrangement of the proteins that make up these signaling complexes a technique known as bioluminescence resonance energy transfer (BRET) is being used to investigate protein-protein interactions between the various 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 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 the b2AR, the stimulatory heterotrimeric G protein (Gs), 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, G protein subunits and AC are tagged with GFP or RLuc these signaling molecules retain their biological activity. In HEK 293 cells co-expressing these tagged proteins BRET occurred between GFP-tagged G protein subunits and both the b2AR-RLuc and AC-RLuc. We have also shown that BRET occurs between the b2AR-GFP and AC-RLuc. These date indicate that the b2AR, Gs and AC form a complex in living cells. This complex is present in the absence of hormone stimulation, and BRET persists in the presence of hormone suggesting that these complexes exist in both the basal state and during signal transduction, thus 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 receptor dimers. 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 second and third intracellular loops 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 dimerization 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 second intracellular loop and one that is missing the third intracellular loop) 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、阵风、气味、激素和神经递质。反应的性质可以同样不同，从基因转录的变化到跨膜离子渗透性的改变。该系统的三个核心组成部分是七螺旋受体、异源三聚体G蛋白和效应分子，它们必须相互作用以将信息从一个组成部分传递到下一个组成部分。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，积累的数据提供了证据，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的空间排列，一种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各种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达为用生物发光蛋白荧光素酶（RLuc）或绿色荧光蛋白（GFP）标记的融合蛋白。如果标签通过两个信号分子之间稳定的蛋白质-蛋白质相互作用并置，则BRET发生，因为RLuc标签发射的光将被GFP标签吸收，然后GFP标签发荧光。对于这些研究，我们使用的原型β 2-肾上腺素能受体（b2 AR）信号系统，包括b2 AR，刺激异源三聚体G蛋白（Gs），和效应腺苷酸环化酶（AC）。b2 AR的激动剂刺激导致Gs介导的AC刺激，从而产生环AMP。当b2 AR、G蛋白亚基和AC用GFP或RLuc标记时，这些信号分子保留其生物活性。在共表达这些标记蛋白的HEK 293细胞中，BRET发生在GFP标记的G蛋白亚基与b2 AR-RLuc和AC-RLuc之间。我们还表明BRET发生在b2 AR-GFP和AC-RLuc之间。这些数据表明，b2 AR，Gs和AC在活细胞中形成复合物。这种复合物存在于激素刺激的情况下，BRET持续存在于激素的存在下，这表明这些复合物存在于基础状态和信号转导过程中，从而为不断发展的观点提供支持，即G蛋白介导的信号传导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功效有重要贡献。 除了与下游信号分子形成复合物之外，七螺旋受体彼此相互作用以形成受体二聚体。当受体的多肽链来回穿过质膜时，它产生延伸到细胞外和细胞内空间的环，并将C-末端沉积在细胞质环境中。第二个和第三个胞内环对于与下游信号传导组分的相互作用都是至关重要的，并且缺乏任一结构域使得受体不能介导信号转导，尽管保留了结合配体的能力。据推测，受体二聚化导致分子间相互作用，其中一个受体的一部分与另一个受体的互补部分相关联，从而形成与异源三聚体G蛋白相互作用的结构域。为了检验这一假设，在HEK 293细胞中共表达两种信号传导缺陷型b2 AR（一种缺失第二胞内环，另一种缺失第三胞内环），以确定是否可以从两种突变受体重建全功能b2 AR。