Elucidating The Structural Organization Of G-protein Coupled Signaling Systems

阐明 G 蛋白偶联信号系统的结构组织

• 批准号: 7593343
• 负责人: John K Northup
• 金额: $ 48.12万
• 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/7593343
• 关键词: Adenylate Cyclase; Adrenergic Receptor; Affect; Agonist; Binding; C-terminal; Cell Fractionation; Cell membrane; Cell surface; Cells; Chimeric Proteins; Complex; Coupled; Data; Dominant-Negative Mutation; Dopamine Receptor; Endoplasmic Reticulum; Energy Transfer; Fluorescence; Fusion Protein Expression; G-Protein Signaling Pathway; GTP-Binding Proteins; Goals; Green Fluorescent Proteins; Guanosine Triphosphate Phosphohydrolases; Heterotrimeric GTP-Binding Proteins; Hormones; Intracellular Membranes; Lead; Life; Ligand Binding; Ligands; Light; Luciferases; Mediating; Membrane; Neurotransmitters; Organism; Pathway interactions; Pharmaceutical Preparations; Pharmacologic Substance; Positioning Attribute; Process; Proteins; Purpose; Regulation; Signal Transduction; Signal Transduction Pathway; Signaling Protein; Site; Specificity; Stimulus; System; Techniques; Work; base; human disease; next generation; organizational structure; prevent; receptor; reconstitution; research study; response; trafficking

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 effector proteins. Most cells harbor multiple G protein 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. Several different fluorescence based techniques are being used to investigate when, where and under what circumstances signaling complexes are formed and dissolved in living cells. These techniques, known as resonance energy transfer (RET), and bimolecular fluorescence complementation (BiFC), can provide both spatial and temporal information about protein complexes. RET 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 green (GFP) or yellow fluorescent protein (YFP), or a tetracysteine motif CCPGCC that is capable of binding biarsenical derivatives of fluorescent compound (ie. FlAsH). RET occurs when the energy from Luc or a fluorescent tag (the donor) on one protein is transferred to the fluorescent tag (the acceptor) on another protein causing the acceptor to fluoresce. This only occurs if the donor and acceptor tags are juxtaposed (less than 100 angstroms apart) because the proteins they are fused to associated to form a complex. BiFC is based on the fact that the 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 D4.2 dopamine receptor (D4.2R) inhibits the effector protein adenylyl cyclase (AC) by activating the inhibitory heterotrimeric G protein, Gi. Fusion proteins of D4.2R with Luc or a fluorescent protein are inactive. To create a fluorescent D4.2R that could be used in RET experiments a CCPGCC motif was added to the C-terminus (D4.2R-PGCC) or at two different positions within the third intracellular loop (D4.2R-G259C and D4.2R-G275C). The tetracysteine motif did not affect cell surface expression, ligand binding to the receptor, or agonist mediated-inhibition of AC, and FlAsH binding to this motif produced a fluorescent D4.2R that could be used as an acceptor for RET experiments. RET occurs when either D4.2R-G257C or D4.2R-G275C was co-expressed in HEK 293 cells with a Luc tagged AC (AC-Luc). There was no significant RET between AC-Luc and D4.2R PGCC. RET also occurred between the tagged D4.2R and Luc-tagged Gg. These data suggest that both G protein and AC are part of a signaling complex with D4.2R. The beta2-adrenergic receptor (b2AR) stimulates AC by activating the stimulatory heterotrimeric G protein, Gs. RET was observed when the b2AR was tagged with Luc (b2AR-Luc) and co-expressed with D4.2R-G259C or D4.2R-G275C suggesting that both stimulator and inhibitory receptors involved in the dual regulation of AC are present in the same signaling complex. There was no significant RET between b2AR-Luc and D4.2R-PGCC even though it was functionally indistinguishable from wild type D4.2R. This is likely a consequence of the donor and acceptor tags being to too far apart or in the wrong orientation for RET to occur. RET between Luc-tagged signaling proteins and CCPGCC-tagged D4.2R occurred in the absence of signaling, and was not affected by agonist-mediated signaling. BiFC was combined with RET to demonstrate the simultaneous presence of three different protein in a signaling complex. BiFC occurred when b2AR tagged with YC (b2AR-YC) and Gg tagged with YN (YN-Gg) were co-expressed in HEK 293 cells. RET occurred when AC-Luc was co-expressed with b2AR-YC and YN-Gg indicating that receptor, G protein and effector are part of the same signaling complex. Experimental evidence also supports the hypothesis that G protein-mediated signaling complexes are formed before they reach the plasma membrane. RET together with subcellular fractionation demonstrated that a complex of AC and the b2AR are present on intracellular membranes. Further, dominant-negative (DN) GTPases (Rab1 and Sar1) which block anterograde trafficking out of the endoplasmic reticulum (ER) have no effect on either b2AR/AC, Gg/AC or b2AR/Gg 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 heptahelical receptors, G proteins and effectors are assembled into complexes before being transported to their target 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。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（GA），Beta（GB）和Gamma（GG）亚基组成，当激活时，它们能够调节特定效应蛋白的活性。大多数细胞具有多个G蛋白信号通路，除非将它们彼此适当隔离，否则可能会在交叉目的上工作。越来越多的证据表明，这是通过将受体，G蛋白和效应子组装到信号传导复合物中来实现的。正在使用几种不同的基于荧光的技术来调查在何时，何时何地，在哪些情况下以及在活细胞中形成并溶解了信号复合物。这些技术称为共振能传递（RET）和双分子荧光互补（BIFC），可以提供有关蛋白质复合物的空间和时间信息。 RET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。荧光部分可以是一种荧光蛋白，例如绿色（GFP）或黄色荧光蛋白（YFP），也可以是能够结合荧光化合物（即闪光灯）的双囊性基序CCPGCC。当将一种蛋白质上的luc或荧光标签（供体）的能量转移到另一种蛋白质上的荧光标签（受体）上时，就会发生RET。仅当捐赠者和受体标签并列（相距不到100埃）时，才会发生这种情况，因为它们融合到相关的蛋白质形成复合物中。 BIFC是基于以下事实：YFP的互补N和C末端片段（分别为Yn和YC）本身不是荧光，而是将荧光YFP分子重新构成，如果将它们通过融合到蛋白质中融合在一起以形成复合物。 D4.2多巴胺受体（D4.2R）通过激活抑制性异三聚体G蛋白GI抑制效应蛋白腺苷酸环化酶（AC）。 D4.2R的融合蛋白与LUC或荧光蛋白无活性。为了创建可用于RET实验的荧光D4.2R，将CCPGCC基序添加到C-terminus（D4.2R-PGCC）中，或在第三个细胞内环内的两个不同位置（D4.2R-G259C和D4.2R-G275C）。四环素生态安全基序不影响细胞表面表达，与受体的配体结合或激动剂介导的AC抑制作用，而闪光与该基序的结合产生了荧光D4.2R，可用于保留实验的受体。当D4.2R-G257C或D4.2R-G275C在HEK 293细胞中使用LUC标记的AC（AC-LUC）共表达D4.2R-G257C或D4.2R-G275C时，就会发生RET。 AC-LUC和D4.2R PGCC之间没有明显的RET。在标记的D4.2R和Luc标记的GG之间也发生了RET。这些数据表明G蛋白和AC都是D4.2R信号复合物的一部分。 β2-肾上腺素受体（B2AR）通过激活刺激异三聚体G蛋白GS刺激AC。当B2AR用LUC（B2AR-LUC）标记并与D4.2R-G259C或D4.2R-G275C共表达时，观察到RET，这表明与AC双重调控有关的刺激受体和抑制受体都存在于同一信号复合物中。 B2AR-LUC和D4.2R-PGCC之间没有明显的RET，即使它与野生型D4.2R无法区分。这可能是捐赠者和受体标签的结果，要过得太远，或者在错误的方向上以无法进行RET。在没有信号传导的情况下，发生Luc标记的信号蛋白和CCPGCC标记的D4.2R之间的RET，不受激动剂介导的信号传导的影响。 将BIFC与RET结合使用，以证明在信号传导复合物中同时存在三种不同蛋白质。 BIFC发生在用YC（B2AR-YC）标记的B2AR并用Yn（Yn-GG）标记的GG时，会在HEK 293个细胞中共表达。当AC-Luc与B2AR-YC并YN-GG共表达时，发生了RET，表明受体，G蛋白和效应子是同一信号传导复合物的一部分。实验证据还支持以下假设：G蛋白介导的信号传导复合物在达到质膜之前形成。与亚细胞分馏一起的RET表明，AC和B2AR的复合物存在于细胞内膜上。此外，占主导地位的（DN）GTPases（RAB1和SAR1），这些GTPase（RAB1和SAR1）从内质网（ER）中阻塞行进（ER）对B2AR/AC/AC，GG/AC或B2AR/GG相互作用均无影响。但是，DN RAB1和SAR1构建体（但不是DN RABS 2、6、8或11）阻止将GA亚基纳入AC信号传导复合物中，这表明GA在ER之外的某个点成为该复合物的一部分。总而言之，我们的数据支持以下假设：在将信号转导途径被激动剂激活时，在将信号转导途径激活时，将七螺旋体受体，G蛋白和效应子组装成络合物，并且这些复合物持续存在。这种布置有助于解释在G蛋白介导的信号转导期间经常观察到的特异性和功效。