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。当激动剂选择性地与其七螺旋受体结合，导致异源三聚体G蛋白激活时，G蛋白介导的信号转导就发生了。这些G蛋白由α(GA)、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。大多数细胞都有多个G蛋白介导的信号通路，除非它们彼此适当地分离，否则它们可能会在不同的目的下工作。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体来实现的。两种基于荧光的技术正被用来调查这些复合体在活细胞中形成的时间和地点。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，如GFP或YFP，或能够结合荧光化合物的双砷衍生物的肽基序CCPGCC(即，Flash)。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YFP的N-末端和C-末端互补片段(分别为YN和YC)本身不是荧光的，但如果它们通过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分子。 七螺旋β2肾上腺素能受体(B2AR)和多巴胺D4.2受体(D4.2R)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。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分析表明，D4.2R与AC形成络合物。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基序整合到受体中。闪光结合这个基序使D4.2R成为Bret实验中AC-Luc共振能量转移的受体。BRET实验还表明，B2AR与AC和Kir3通道亚单位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和免疫共沉淀实验也表明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。 GB和GG形成稳定的杂二聚体(GBG)。当标记YN的GB和标记YC的GG发生异二聚，使YN和YC结合在一起，从而重组荧光YPF时，BIFC就发生了。当这对BIFC与B2AR-Luc或Luc标记的效应子(AC-Luc或Kir3.1-Luc)共表达时，出现Bret，表明同一信号复合体中同时存在三种不同的蛋白质。结合BIFC和BRET的技术现在被用来表明，在活细胞中，B2AR、G蛋白亚基和效应器都是同一复合体的一部分。 实验证据也支持G蛋白介导的信号复合体在到达质膜之前就形成的假说。BRET结合亚细胞分离表明，胞膜上存在AC和B2AR的复合体。此外，阻断内质网(ER)顺行转运的显性-负性(DN)Rab1和Sar1 GTP酶对AC/B2AR或AC/GBG蛋白相互作用都没有影响。然而，DNRab1和Sar1结构(而不是DNRAB2、6、8或11)阻止在AC信号复合体中包含Ga亚基，这表明在内质网以外的某个时间点，Ga成为复合体的一部分。综上所述，我们的数据支持这样的假设，即七螺旋受体、G蛋白和效应器在被运输到质膜之前被组装成复合体，并且当信号转导途径被激动剂激活时，这些复合体持续存在。这种安排有助于解释在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. 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.