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Elucidating The Structural Organization Of G-protein Cou

阐明 G 蛋白 Cou 的结构组织

基本信息

批准号：
7143854
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
项目类别：
财政年份：
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/7143854
关键词：
G protein adenylate cyclase beta adrenergic receptor biological signal transduction bioluminescence fluorescence resonance energy transfer fluorescent dye /probe green fluorescent proteins human tissue intermolecular interaction luciferin monooxygenase molecular assembly /self assembly potassium channel protein localization protein structure function receptor coupling tissue /cell culture

项目摘要

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.

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、阵风、气味、激素和神经递质。反应的性质可以同样不同，从基因转录的变化到改变的离子通道动力学。G蛋白介导的信号转导发生在激动剂选择性结合其七螺旋受体，导致异源三聚体G蛋白的激活时。这些G蛋白由α、β（Gb）和γ（Gg）亚基组成，当被激活时，它们能够调节特定效应物的活性。两种基于荧光的技术被用来解决受体，G蛋白和效应器是否作为蛋白质复合物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），可以提供有关蛋白质复合物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）。BRET发生在Luc标签的生物发光能量转移到荧光标签，使其发出荧光时。这仅在标签并置（相距小于100埃）时发生，因为融合蛋白缔合形成复合物。BiFC基于以下事实：由氨基酸1-158或159-238组成的YFP的肽片段在共表达时不发荧光，但如果通过将它们融合到缔合形成复合物的蛋白质上而将它们聚集在一起，则将重构荧光YFP。七螺旋β 2-肾上腺素能受体（b2 AR）触发G蛋白的活化，导致包括腺苷酸环化酶（AC）和G蛋白偶联的内向整流K+（Kir 3）通道的效应器的调节。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用于显示b2 AR与AC和Kir 3通道亚基Kir3.1形成复合物。这些复合物在没有信号转导的情况下存在，并在信号转导期间持续存在。BRET还用于显示G蛋白亚基与b2 AR、AC和Kir3.1形成复合物。BRET之间的G蛋白亚基和这些信号蛋白的受体激动剂的影响。旨在探索激动剂诱导效应的性质的实验表明，它们是由保持完整的蛋白质复合物内的构象改变引起的。实验还表明，这些激动剂敏感的复合物在它们到达质膜之前形成。将BiFC与BRET组合以确定是否可以检测到同一复合物内同时存在三种信号蛋白。因此，我们已经确定了复合物的b2 AR或效应与Gb和Gg亚基。BiFC和BRET结合的技术现在被用来证明b2 AR、G蛋白亚基和效应子都是活细胞中同一复合物的一部分。总之，我们的数据支持这样的假设，即b2 AR，G蛋白和效应物在被转运到质膜之前组装成功能复合物，并且这些复合物存在，而不管信号转导途径是否被激动剂激活。这种排列可以解释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。