Structural Organization Of G-protein Coupled Signaling

G蛋白介导的信号转导通路涉及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。反应的性质可能同样不同，从基因转录的变化到离子通道动力学的改变。当激动剂选择性地与其七螺旋受体(7TM)结合时，G蛋白介导的信号转导发生，导致异源三聚体G蛋白的激活。这些G蛋白由α、β和伽马亚基组成，当被激活时，它们能够调节特定的效应器的活性，如腺苷环化酶(AC)或G蛋白偶联的内向整流K(Kir3)通道。7TM、G蛋白和效应器都是膜相关蛋白，几十年来，两种对立的假说一直在争相接受。最主要的假设是，这些蛋白质在膜上相互独立地运动，当它们作为随机碰撞的结果相遇时，信号转导发生。有争议的假设是，信号是通过这些蛋白质的有组织的复合体来传播的。我们采用了两种基于荧光的检测和成像技术，目的是确定这些假设中哪一种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)，可以用来确定蛋白质是否在复合体中相关联，它们可以提供关于这些复合体的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(例如。GFP或YFP)。当LUC标签的生物发光能量被转移到使其发光的荧光标签时，发生BRET。这只有在标签并列(间隔小于100埃)的情况下才会发生，因为融合蛋白结合在一起形成一个复合体。BIFC是基于这样的观察，即由YFP的氨基酸1-158或159-238组成的多肽片段(分别为YFP(1-158)和YFP(159-238))在共表达时不是荧光的，但如果两个片段能够通过将它们与结合形成复合体的蛋白质融合在一起，则可以通过恢复其荧光性质来重组YFP。用GFP或YFP的互补片段(GFP-GGamma2、YFP(1-158)-Gbeta1和YFP(159-238)-GGamma2)标记G蛋白亚基(Gbeta1和GGamma2)，用Luc(B2AR-Luc、AC-Luc和Kir3.1-Luc)标记B2AR、AC和Kir3.1。标记的信号分子保留了它们的生物活性。当GFP-GGamma2与Luc标记的效应物或与B2AR-Luc共表达时，BRET发生，表明这些蛋白质相互形成复合体。Ac和Kir3.1也被证明与B2AR形成稳定的络合物。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迅速增加(T1/2小于300毫秒)，而GFP-GGamma2与其Luc标记的伴侣的亲和力没有明显变化。这表明，由受体激活引起的构象变化，而不是G蛋白的募集，对效应器的调节起作用。在激动剂诱导的BRET升高之后，BRET的下降相对缓慢，这与受体脱敏导致的难治状态相吻合。 Gbeta与GGamma异源二聚的倾向导致YFP(1-158)-Gbeta1和YFP(159-238-GGamma2)共表达细胞中YFP荧光的重建。当在共表达重组YFP标记的Gbeta-γ异源二聚体和AC-Luc的细胞中观察到Bret时，证明在同一复合体中同时存在三种单独的蛋白质的直接证据，并且与前述结果一致，异丙肾上腺素处理细胞使Bret增加。 在没有共表达Kir3.4亚基的情况下，Kir3.1不针对细胞表面。尽管Kir3.1-Luc和GFP-GGamma2之间有很强的Bret，但它不受膜不通透性激动剂异丙肾上腺素的影响。然而，当使用膜通透性β-肾上腺素能激动剂西马特罗时，激动剂诱导的BRET增加。如果Kir3.4与Kir3.1-Luc和GFP-GGamma2共表达，Kir3通道被靶向细胞表面，并且异丙肾上腺素可以增加Bret。综上所述，这些结果表明，B2AR、G蛋白和效应器在被运输到质膜之前被组装成一个功能复合体。此外，无论信号转导通路是否被激动剂激活，这些复合体都会持续存在，从而显著提高G蛋白介导的信号转导的特异性和有效性。

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.