Structural Organization Of G-protein Coupling Systems

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的空间排列，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各种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，该系统由b2AR、刺激性异三聚体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AMP的产生。当b2AR、G蛋白亚基和AC被GFP或RLuc标记时，这些信号分子保持其生物活性。在共表达这些标记蛋白的HEK 293细胞中，gfp标记的G蛋白亚基与b2AR-RLuc和AC-RLuc之间发生BRET。我们还发现，BRET发生在b2AR-GFP和AC-RLuc之间。这些数据表明b2AR， Gs和AC在活细胞中形成一个复合物。该复合物在没有激素刺激的情况下存在，而BRET在激素存在的情况下仍然存在，这表明这些复合物在基础状态和信号转导过程中都存在，从而支持了G蛋白介导的信号系统作为有组织复合物存在的观点，这些复合物对信号转导过程的特异性和有效性有重要贡献。

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.