Elucidating The Structural Organization Of G-protein Cou

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。为了阐明活细胞中构成这些信号复合物的蛋白质的空间排列，我使用了生物发光共振能量转移（BRET）。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。在这些研究中，我们使用了原型β 2-肾上腺素能受体（b2 AR）信号系统，该系统由b2 AR、刺激性异源三聚体G蛋白（Gs）（由α（G-α）、β（G-β）和γ亚基组成）和效应器腺苷酸环化酶（AC）组成。b2 AR的激动剂刺激导致Gs介导的AC刺激，从而产生环AMP。当用GFP或AC与RLuc标记b2 AR时，这些信号传导分子保留其生物活性。当b2 AR-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，这种复合物存在于激素刺激的情况下，表明它存在于基础状态。目前，G-alpha-GFP和G-beta-RLuc正被用于研究G蛋白亚基之间以及与受体（即受体）之间的相互作用。b2 AR-GFP和b2 AR-RLuc）和效应子（即，AC-RLuc）蛋白。这些数据提供了支持的不断发展的观点，G蛋白介导的信号系统存在的组织复合物，显着的特异性和有效性的信号转导过程。 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同源多聚体（最可能是同源二聚体）。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。第三胞内环和C-末端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，受体寡聚化导致分子间相互作用，其中一种受体的一部分与另一种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（一种缺失第三胞内环，另一种缺失C末端氨基酸序列），以确定是否可以从两种突变受体重建全功能b2 AR

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