Structural Organization Of G-protein Coupled Signaling

G 蛋白偶联信号传导的结构组织

• 批准号: 6990044
• 负责人: ROBERT VICTOR REBOIS
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6990044
• 关键词: G protein; beta adrenergic receptor; biological signal transduction; bioluminescence; human tissue; intermolecular interaction; molecular assembly /self assembly; protein localization; protein structure function; receptor coupling; tissue /cell culture; G protein; beta adrenergic receptor; biological signal transduction; bioluminescence; human tissue; intermolecular interaction; molecular assembly /self assembly; protein localization; protein structure function; receptor coupling; tissue /cell culture

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.

G蛋白介导的信号转导途径与生物体及其成分细胞对各种刺激的反应有关，包括光，胶，气味，激素和神经递质。响应的性质可能是从基因转录的变化到改变的离子通道动力学不同的不同。 G蛋白介导的信号转导发生时，当激动剂选择性结合其七螺旋受体（7TM）时，导致异三聚体G蛋白的激活。这些G蛋白由Alpha，beta和Gamma亚基组成，并且在激活时，它们能够调节特定效应子的活性，例如腺苷酸环化酶（AC）或G蛋白偶联，以内部凝结K+（KIR3）通道。 7TM，G蛋白和效应子都是与膜相关的蛋白质，数十年来，有两个相反的假设争夺接受。主要的假设是这些蛋白质在膜上彼此独立地移动，并且信号转导在彼此遇到随机碰撞的结果时发生。竞争的假设是信号传播由这些蛋白质的有组织的复合物传播。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的哪一个最准确地描述了活细胞中G蛋白介导的信号转导的过程。这些称为生物发光共振能传递（BRET）和双分子荧光互补（BIFC）的技术可用于确定蛋白质是否与复合物相关联，它们可以提供有关这些复合物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）标记的融合蛋白的外源表达。当将Luc标签的生物发光能量转移到荧光标签中，导致其荧光时，就会发生BRET。仅当标签与并列（距离远小于100埃）时，才会发生这种情况，因为融合蛋白会形成复合物。 BIFC基于观察结果，即YFP（YFP（1-158）和YFP（1-158）和YFP（159-238）组成的氨基酸1-158或159-238组成的肽片段，在共表达时不会荧光，但如果可以通过将两个碎片融合在一起，以使其与蛋白质相关联，则可以将两个碎片融合在一起，以使其与蛋白质相关联，以使其与蛋白质相关联。 G蛋白亚基（GBETA1和GGAMMA2）用GFP或YFP（GFP-GGAMMA2，YFP（1-158）-GBETA1和YFP（159-238）-GGAMMA2）以及Beta2-beta2-adenrenergic Hosefors（B2ar），AC和KIR3 WAS（B2 artar），AC和Kir3 WAS（B2ar），AC（B2），用GFP（GFP-GGAMMA2，YFP（1-158）-GBETA1和YFP（1-158）-GBETA1和YFP（1-158）-GBETA1和YFP（159-238），B2）标记为GFP（GBETA1和GGAMMA2）。 AC-LUC和KIR3.1-LUC）。标记的信号分子保留了其生物学活性。当GFP- ggamgama2与luc标记的效应子或B2AR-LUC共表达时，发生了BRET。 AC和KIR3.1也已显示与B2AR形成稳定的复合物。 β-肾上腺素激动剂异丙肾上腺素诱导的GFP-GGAMMA2和AC-LUC和AC-LUC和B2AR-LUC之间的BRET迅速增加（T1/2少于300毫秒），其GFP-GGGAMMA2对其Luc-Tagaggaggaggaggaggaggaggaggaggaggaggagma2的亲和力没有明显变化。这表明受体激活诱导的构象变化，而不是募集G蛋白，是效应子调节的原因。激动剂引起的BRET的增加之后，BRET的下降相对较慢，这与受体脱敏引起的难治状态相吻合。 GBETA与GGAMMA异二聚体的倾向导致在YFP（1-158）-GBETA1和YFP共表达的细胞中重构YFP荧光（159-238-GGAMMA2）。当在共表达重建的YFP标记的YFP标记的GBETA-GAMMA异二聚体和AC-Luc的细胞中观察到BRET在同一复合物中同时存在三种单独的蛋白质的直接证据，并且与蛋白酚的细胞治疗增加了BRET。 在没有共表达KIR3.4亚基的情况下，Kir3.1并非针对细胞表面。尽管KIR3.1-LUC和GFP-GGAMMA2之间有强大的BRET，但它不受膜不渗透激动剂异丙肾上腺素的影响。然而，当使用了膜可渗透β-肾上腺素能Cimonaterol时，确实会发生激动剂引起的BRET增加。如果Kir3.4与Kir3.1-Luc和GFP-GGAMMA2共表达，则KiR3通道靶向细胞表面，而Isoproterenol可以增加BRET。总之，这些结果表明B2AR，G蛋白和效应子在运输到质膜之前被组装成功能复合物中。此外，这些复合物持续存在，无论信号转导途径是否被激动剂激活，因此，这样做对G蛋白介导的信号转导的特异性和功效产生了显着贡献。