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。当激动剂选择性结合其七螺旋受体导致异三聚体G蛋白的激活时，G蛋白介导的信号转导发生。这些G蛋白由Alpha，beta（GB）和伽马（GG）亚基组成，当激活时，它们能够调节特定效应子的活性。正在使用两种基于荧光的技术来解决活细胞中是否存在作为蛋白质复合物的受体，G蛋白和效应子的问题。这些技术（称为生物发光共振能传递（BRET）和双分子荧光互补（BIFC））可以提供有关蛋白质复合物的形成和溶解的时空信息。 BRET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基于以下事实：氨基酸1-158或159-238的YFP的肽片段在共表达时不会荧光，但如果将它们融合在一起，将它们融合到蛋白质中，将它们聚集在一起，以形成复合物。七螺旋β2-肾上腺素受体（B2AR）触发了G蛋白的激活，导致腺苷酸环酶（AC）和G蛋白偶联的效应子的激活，并偶然偶联地偶联了K+（KIR3）通道。当这些蛋白质被标记为BRET和BIFC实验时，它们会保留其生物学活性。 BRET用于表明B2AR与AC形成复合物，并与KIR3通道亚基（Kir3.1）形成。这些复合物存在在信号转导期间没有信号转导和持续存在的情况下存在。 BRET还用于表明G蛋白亚基与B2AR，AC和KIR3.1形成复合物。 G蛋白亚基和这些信号蛋白之间的BRET受受体激动剂的影响。旨在探测激动剂诱导效应的性质的实验表明，它们是由蛋白质复合物中保持完整的构象改变引起的。实验还表明，这些激动剂敏感的复合物是在到达质膜之前形成的。将BIFC与BRET合并，以确定是否可以在同一复合物中同时存在三种信号蛋白。结果，我们已经确定了B2AR或GB和GG亚基的效应子的复合物。现在使用将BIFC和BRET结合的技术表明B2AR，G蛋白亚基和效应子都是活细胞中同一复合物的一部分。总而言之，我们的数据支持以下假设：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，并且这些复合物存在于信号传递途径是否由agonist激活。这种布置可以解释在G蛋白介导的信号转导期间经常观察到的特异性和功效。