Structural Organization Of G-protein Coupled Signaling

G蛋白介导的信号转导通路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。反应的性质可以同样多样化，从基因转录的变化到离子通道动力学的改变。G蛋白介导的信号转导发生在激动剂选择性地与其七螺旋受体（7TM）结合导致异源三聚体G蛋白的激活时。这些G蛋白由α、β和γ亚基组成，当它们被激活时，它们能够调节特定效应物的活性，如腺苷酸环化酶（AC）或G蛋白偶联的内向纠偏K+ (Kir3)通道。tms、G蛋白和效应蛋白都是膜相关蛋白，几十年来，有两种相反的假设一直在争夺被接受的机会。主要的假设是，这些蛋白质在膜中彼此独立地移动，当它们由于随机碰撞而相遇时，信号转导就发生了。有争议的假设是，信号是由这些蛋白质的有组织的复合体传播的。我们采用了两种基于荧光的检测和成像技术，目的是确定哪一种假设最准确地描述了活细胞中G蛋白介导的信号转导过程。这些技术被称为生物发光共振能量转移（BRET）和双分子荧光互补（BiFC），可用于确定蛋白质是否在复合物中相关，它们可以提供有关这些复合物形成和溶解的空间和时间信息。BRET和BiFC都涉及用荧光素酶（Luc）或荧光蛋白标记的融合蛋白外源表达。GFP或YFP)。BRET发生时，Luc标签的生物发光能量转移到荧光标签，使其发出荧光。这种情况只有在标签并列（相距小于100埃）时才会发生，因为融合蛋白会结合形成复合物。bbifc是基于观察到由YFP的氨基酸1-158或159-238组成的肽片段（分别为YFP（1-158）和YFP(159-238)）在共表达时不具有荧光性，但如果两个片段可以通过将它们融合到相关的蛋白质中形成复合物，则可以重建YFP并恢复其荧光特性。G蛋白亚基（Gbeta1和Ggamma2）用GFP或YFP的互补片段(GFP-Ggamma2、YFP（1-158)-Gbeta1和YFP(159-238)-Ggamma2）标记，β 2-肾上腺素能受体（b2AR）、AC和Kir3.1用Luc标记（b2AR-Luc、AC-Luc和Kir3.1-Luc）。标记的信号分子保留了它们的生物活性。当gfp - gamma2与luc标记的效应物或b2AR-Luc共表达时，BRET发生，表明这些蛋白相互形成复合物。AC和Kir3.1也被证明与b2AR形成稳定的配合物。β -肾上腺素能激动剂异丙肾上腺素诱导GFP-Ggamma2与AC-Luc和b2AR-Luc之间的BRET快速增加（t1/2小于300毫秒），而GFP-Ggamma2对其luc标记的伴侣的亲和力没有明显变化。这表明受体激活引起的构象变化，而不是G蛋白的募集，是效应调节的原因。在激动剂诱导的BRET增加之后，BRET出现相对缓慢的下降，这与受体脱敏引起的难治性状态相吻合。

G protein-mediated signal transduction pathways 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 ion channel kinetics. G protein-mediated signal transduction occurs when an agonist binds selectively to its heptahelical receptor (7TM) leading to the activation of a heterotrimeric G protein. These G proteins are composed of alpha, beta and gamma subunits, and when activated they are able to regulate the activity of specific effectors such as adenylyl cyclase (AC) or G protein-coupled inwardly rectifying K+ (Kir3) channels. 7TMs, G proteins and effectors are all membrane-associated proteins, and for decades two opposing hypotheses have vied for acceptance. The predominant hypothesis has been that these proteins move about independently of one another in membranes, and that signal transduction occurs when they encounter each other as the result of random collisions. The contending hypothesis is that signaling is propagated by an organized complex of these proteins. We have employed two fluorescence-based detection and imaging techniques with the goal of determining which of these hypotheses most accurately describes the process of G protein-mediated signal transduction in a living cell. These techniques known as bioluminescent resonance energy transfer (BRET), and bimolecular fluorescence complementation (BiFC) can be used to determine if proteins are associated in a complex and they can provide both spacial and temporal information about the formation and dissolution of these complexes. Both BRET and BiFC involves the exogenous expression of fusion proteins tagged with either luciferase (Luc) or a fluorescent protein (eg. GFP or YFP). BRET occurs when the bioluminescent energy of the Luc tag is transferred to the fluorescent tag causing it to fluoresce. This only occurs if the tags are juxtaposed (less than 100 angstroms apart) because the fusion proteins associate to form a complex. BiFC is based on the observations that peptide fragments consisting of amino acids 1-158 or 159-238 of YFP (YFP(1-158) and YFP(159-238), respectively) are not fluorescent when co-expressed, but if the two fragments can be brought together by fusing them to proteins that associate to form a complex YFP can be reconstituted with restoration of its fluorescent properties. G protein subunits (Gbeta1 and Ggamma2) were tagged with GFP or with the complementary fragments of YFP (GFP-Ggamma2, YFP(1-158)-Gbeta1 and YFP(159-238)-Ggamma2), and beta2-adrenergic receptors (b2AR), AC and Kir3.1 were tagged with Luc (b2AR-Luc, AC-Luc and Kir3.1-Luc). The tagged signaling molecules retained their biological activity. BRET occurred when GFP-Ggamma2 was co-expressed with either Luc-tagged effectors or with b2AR-Luc indicating that these proteins form complexes with each other. AC and Kir3.1 have also been shown to form stable complexes with the b2AR. The beta-adrenergic agonist, isoproterenol, induced a rapid (t1/2 less than 300 msec) increase in BRET between GFP-Ggamma2 and both AC-Luc and the b2AR-Luc with no apparent change in the affinity of GFP-Ggamma2 for its Luc-tagged partner. This suggests that conformational changes induced by receptor activation, rather than recruitment of G protein, is responsible for effector modulation. The agonist-induced increase in BRET was followed by a relatively slow decline in BRET that coincided with a refractory state caused by receptor desensitization. The proclivity of Gbeta to heterodimerize with Ggamma results in reconstitution of YFP fluorescence in cells co-expressed both YFP(1-158)-Gbeta1 and YFP(159-238-Ggamma2). Direct evidence for the simultaneous presence of three individual proteins in the same complex was demonstrated when BRET was observed in cells co-expressing a reconstituted YFP-tagged Gbeta-gamma heterodimer and AC-Luc, and, consistent with the forgoing results, the BRET was increased by treatment of the cells with isoproterenol. In the absence of co-expressed Kir3.4 subunits, Kir3.1 is not targeted to the cell surface. Although there was a robust BRET between Kir3.1-Luc and GFP-Ggamma2 it was not affected by the membrane impermeable agonist isoproterenol. However, an agonist-induced increase in BRET did occur when the membrane permeable beta-adrenergic agonist cimaterol was used. If Kir3.4 was co-expressed with Kir3.1-Luc and GFP-Ggamma2 the Kir3 channels were targeted to the cell surface, and BRET could be increased by isoproterenol. Taken together these results suggest that the b2AR, G proteins and effectors are assembled into a functional complexes before being transported to the plasma membrane. Furthermore, these complexes persist regardless of whether or not the signal transduction pathway is activated by an agonist, and in so doing contribute significantly to the specificity and efficacy of G protein-mediated signal transduction.