Elucidating The Structural Organization Of G-protein Cou

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(GB)和伽马(GG)亚基组成，当被激活时，它们能够调节特定效应器的活动。两种基于荧光的技术被用来解决受体、G蛋白和效应器是否以蛋白质复合体的形式存在于活细胞中的问题。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)，可以提供关于蛋白质复合体形成和溶解的空间和时间信息。BRET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是基于这样一个事实，即由氨基酸1-158或159-238组成的YFP的多肽片段在共表达时不是荧光的，但如果它们通过将它们与结合形成复合体的蛋白质融合在一起，就会重新构成荧光YFP。 七螺旋β2肾上腺素能受体(B2AR)可激活G蛋白，调节腺苷环化酶(AC)和G蛋白偶联的内向整流钾(Kir3)通道等效应器。当这些蛋白质被标记用于Bret和BIFC实验时，它们保留了它们的生物活性。BRET用于证明B2AR与AC和Kir3通道亚基Kir3.1形成复合体。这些复合体在没有信号转导的情况下存在，并在信号转导过程中持续存在。BRET还表明G蛋白亚基与B2AR、AC和Kir3.1形成络合物。G蛋白亚基和这些信号蛋白之间的Bret受到受体激动剂的影响。旨在探索激动剂诱导效应的性质的实验表明，它们是由保持完好的蛋白质复合体内的构象变化引起的。实验还表明，这些激动剂敏感的复合体在到达质膜之前就形成了。BIFC与BRET相结合，以确定是否可以检测到同一复合体中同时存在三种信号蛋白。因此，我们已经鉴定出B2AR或效应器与GB和GG亚基的复合体。结合BIFC和BRET的技术现在被用来证明B2AR、G蛋白亚基和效应器在活细胞中都是同一复合体的一部分。综上所述，我们的数据支持这样的假设，即B2AR、G蛋白和效应器在被运输到质膜之前被组装成功能复合体，并且无论信号转导途径是否被激动剂激活，这些复合体都存在。这种排列可能解释了在G蛋白介导的信号转导过程中经常观察到的特异性和有效性。

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 leading to the activation of a heterotrimeric G protein. These G proteins are composed of alpha, beta (Gb) and gamma (Gg) subunits, and when activated they are able to regulate the activity of specific effectors. Two fluorescence-based techniques are being used to resolve the question of whether or not receptors, G proteins, and effectors are present as protein complexes in the living cell. These techniques, known as bioluminescent resonance energy transfer (BRET), and bimolecular fluorescence complementation (BiFC), can provide both spacial and temporal information about the formation and dissolution of protein complexes. BRET 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 fact that peptide fragments of YFP consisting of amino acids 1-158 or 159-238 are not fluorescent when co-expressed, but will reconstitute a fluorescent YFP if they are brought together by fusing them to proteins that associate to form a complex. The heptahelical beta2-adrenergic receptor (b2AR) triggers the activation of G proteins leading to the regulation of effectors including adenylyl cyclase (AC) and G protein-coupled inwardly rectifying K+ (Kir3) channels. When these proteins were tagged for BRET and BiFC experiments they retained their biological activity. BRET was used to show that the b2AR forms a complex with AC and with the Kir3 channel subunit, Kir3.1. These complexes exist in the absence of signal transduction and persist during signal transduction. BRET was also used to show that G protein subunits form complexes with the b2AR, AC and Kir3.1. BRET between the G protein subunits and these signaling proteins was affected by a receptor agonist. Experiments designed to probe the nature of the agonist-induced effects indicated that they were caused by altered conformations within a protein complex that remains intact. Experiments also indicate that these agonist sensitive complexes are formed before they reach the plasma membrane. BiFC was combined with BRET to determine if the simultaneous presence of three signaling proteins within the same complex could be detected. As a result we have identified complexes of either b2AR or effectors with both Gb and Gg subunits. The technique of combining BiFC and BRET is now being used to show that b2AR, G protein subunits and effectors are all part of the same complex in living cells. In summary our data support the hypothesis that the b2AR, G proteins and effectors are assembled into functional complexes before being transported to the plasma membrane, and that these complexes exist regardless of whether or not the signal transduction pathway is activated by an agonist. This arrangement may explain the specificity and efficacy that is often observed during G protein-mediated signal transduction.