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、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，越来越多的数据提供了证据，证明信号分子在细胞表面被组织成大分子复合体。为了阐明组成这些信号复合体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)的技术被用来研究活细胞中不同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，并标记有生物发光蛋白荧光素酶(RLuc)或绿色荧光蛋白(GFP)。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GFP标签吸收，然后GFP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)和效应腺酰环化酶(AC)组成。激动剂对B2AR的刺激导致Gs介导的AC刺激，从而导致环磷酸腺苷的产生。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学活性。在共表达这些标记蛋白的HEK 293细胞中，B2AR-RLuc和AC-RLuc与GFP标记的G蛋白亚基之间发生了Bret。我们还证明了Bret发生在B2AR-GFP和AC-RLuc之间。这些数据表明，B2AR、Gs和AC在活细胞中形成了一个复合体。这种复合体在没有激素刺激的情况下存在，并且Bret在激素的存在下持续存在，这表明这些复合体既存在于基础状态，也存在于信号转导过程中，从而支持了G蛋白介导的信号系统作为有组织的复合体存在的观点，这些复合体对信号转导过程的特异性和有效性做出了重要贡献。 除了与下游信号分子形成复合体外，七螺旋受体还相互作用形成受体二聚体。当受体的多肽链通过质膜来回传递时，它会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。第二个和第三个细胞内环对于与下游信号成分的相互作用都是至关重要的，这两个环中的任何一个的缺失使得受体尽管保留了与配体结合的能力，但仍无法介导信号转导。有人假设，受体二聚化会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，从而形成一个与异源三聚体G蛋白相互作用的结构域。为了验证这一假设，两个信号缺陷的B2AR(一个缺失第二个细胞内环，另一个缺失第三个细胞内环)在HEK 293细胞中共表达，以确定是否可以从两个突变的受体重建完全功能的B2AR。

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.