Elucidating The Structural Organization Of G-protein Cou

阐明 G 蛋白 Cou 的结构组织

• 批准号: 7299405
• 负责人: ROBERT VICTOR REBOIS
• 金额: --
• 依托单位: NATIONAL INSTITUTE ON DEAFNESS AND OTHER COMMUNICATION DISORDERS
• 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/7299405
• 关键词: Elucidating; Structural; Organization; protein; Cou

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. 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 (Ga), beta (Gb) and gamma (Gg) subunits, and when activated they are able to regulate the activity of specific effectors. Most cells harbor multiple G protein-mediated signaling pathways with the potential to work at cross purposes unless they are appropriately segregated from one another. Mounting evidence suggests that this is achieved by assembling receptors, G proteins and effectors into signaling complexes. Two fluorescence based techniques are being used to investigate when and where these complexes are formed in living cells. These techniques, known as bioluminescent resonance energy transfer (BRET), and bimolecular fluorescence complementation (BiFC), can provide both spatial 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 moiety. The fluorescent moiety can be a fluorescent protein, such as GFP or YFP, or the peptide motif CCPGCC that is capable of binding biarsenical derivatives of fluorescent compound (ie. FlAsH). 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 complementary N- and C-terminal fragments of YFP (YN and YC, respectively) are not themselves fluorescent, but will reconstitute a fluorescent YFP molecule if they are brought together by being fused to proteins that associate to form a complex. The heptahelical beta2 adrenergic receptor (b2AR) and the dopamine D4.2 receptor (D4.2R) trigger 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 D4.2R forms as complex with AC. Because the D4.2R is inactivated by fusion with either Luc or a fluorescent protein these experiments require that the CCPGCC motif be incorporated into the receptor. FlAsH binding this motif makes the D4.2R an acceptor for resonance energy transfer from AC-Luc in BRET experiments. BRET experiments also showed 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 as well as co-immunoprecipitation experiments were 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. Gb and Gg form a stable heterodimer (Gbg). BiFC occurs when Gb tagged with YN and Gg tagged with YC heterodimerize to bring YN and YC together so that a fluorescent YPF is reconstituted. When this BiFC pair is co-expressed with either the b2AR-Luc or a Luc-tagged effector (AC-Luc or Kir3.1-Luc) BRET occurred indicating the simultaneous presence of three different proteins in the same signaling complex. The technique of combining BiFC and BRET is now being used to show that b2AR, G protein subunits and effectors are all simultaneously part of the same complex in living cells. Experimental evidence also supports the hypothesis that G protein-mediated signaling complexes are formed before they reach the plasma membrane. BRET together with subcellular fractionation demonstrated that a complex of AC and the b2AR are present on intracellular membranes. Further, dominant-negative (DN) Rab1 and Sar1 GTPases which block anterograde trafficking out of the endoplasmic reticulum (ER) have no effect on either AC/b2AR or AC/Gbg protein interactions. However, DN Rab1 and Sar1 constructs (but not DN Rabs 2, 6, 8 or 11) prevent the inclusion of Ga subunits in AC signaling complexes suggesting Ga becomes part of the complex at some point beyond the ER. In summary our data support the hypothesis that the heptahelical receptors, G proteins and effectors are assembled into complexes before being transported to the plasma membrane, and that these complexes persist when the signal transduction pathway is activated by an agonist. This arrangement helps to 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。当激动剂选择性结合其七螺旋受体导致异三聚体G蛋白激活时，就会发生G蛋白介导的信号转导。这些G蛋白由α（Ga）、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。大多数细胞具有多个G蛋白介导的信号通路，除非它们彼此适当地分离，否则它们可能以交叉目的工作。越来越多的证据表明，这是通过将受体、G蛋白和效应器组装成信号复合物来实现的。两种基于荧光的技术被用来研究这些复合物在活细胞中何时何地形成。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）或荧光部分标记的融合蛋白的外源表达。荧光部分可以是荧光蛋白，例如GFP或YFP，或能够结合荧光化合物的双砷衍生物（即，FlAsH）。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YFP的互补N-和C-末端片段（分别为YN和YC）本身不发荧光，但如果它们通过融合到蛋白质上形成复合物而融合在一起，则会重建荧光YFP分子。 七螺旋β 2肾上腺素能受体（b2 AR）和多巴胺D4.2受体（D4.2R）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）通道的效应物的调节。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表明D4.2R与AC形成复合物。由于D4.2R通过与Luc或荧光蛋白融合而失活，因此这些实验需要将CCPGCC基序掺入受体中。FlAsH结合该基序使得D4.2R成为BRET实验中从AC-Luc共振能量转移的受体。BRET实验还表明，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以及免疫共沉淀实验也被用于显示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。 Gb和Gg形成稳定的异二聚体（Gbg）。当用YN标记的Gb和用YC标记的Gg异源二聚化以使YN和YC在一起使得荧光YPF重构时，发生BiFC。当该BiFC对与b2 AR-Luc或Luc-taged效应物（AC-Luc或Kir3.1-Luc）共表达时，发生BRET，表明在相同的信号传导复合物中同时存在三种不同的蛋白质。BiFC和BRET结合的技术现在被用于显示b2 AR、G蛋白亚基和效应子都同时是活细胞中相同复合物的一部分。 实验证据也支持G蛋白介导的信号复合物在到达质膜之前形成的假设。BRET与亚细胞分级分离一起证明AC和b2 AR的复合物存在于细胞内膜上。此外，显性负性（DN）Rab 1和Sar 1 GTP酶，阻止顺行运输的内质网（ER）对AC/b2 AR或AC/Gbg蛋白的相互作用没有影响。然而，DN Rab 1和Sar 1构建体（但不是DN Rabs 2、6、8或11）防止在AC信号传导复合物中包含Ga亚基，表明Ga在ER以外的某个点成为复合物的一部分。总之，我们的数据支持这样的假设，即七螺旋受体，G蛋白和效应器组装成复合物，然后被运输到质膜，这些复合物持续存在时，信号转导通路被激活的激动剂。这种排列有助于解释在G蛋白介导的信号转导过程中经常观察到的特异性和功效。