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涉及生物体及其组成细胞对多种刺激的反应，包括光、味觉剂、气味剂、激素和神经递质。当激动剂选择性结合其七螺旋受体时，G 蛋白介导的信号转导就会发生，从而导致异源三聚体 G 蛋白的激活。这些 G 蛋白由 α (Ga)、β (Gb) 和 γ (Gg) 亚基组成，激活后它们能够调节特定效应蛋白的活性。大多数细胞都具有多种 G 蛋白信号传导途径，除非它们彼此适当隔离，否则可能会产生交叉作用。越来越多的证据表明，这是通过将受体、G 蛋白和效应子组装成信号复合物来实现的。几种不同的基于荧光的技术被用来研究信号复合物何时、何地以及在什么情况下在活细胞中形成和溶解。这些技术被称为共振能量转移（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。荧光部分可以是荧光蛋白，例如绿色(GFP)或黄色荧光蛋白(YFP)，或者是能够结合荧光化合物的双砷衍生物(即FlAsH)的四半胱氨酸基序CCPGCC。当一种蛋白质上的 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），导致受体发出荧光时，就会发生 RET。仅当供体和受体标签并置（相距小于 100 埃）时才会发生这种情况，因为它们融合在一起的蛋白质形成复合物。 BiFC 基于以下事实：YFP 的互补 N 端和 C 端片段（分别为 YN 和 YC）本身不具有荧光性，但如果通过与相关蛋白质融合而将它们重新构成荧光 YFP 分子。形成复合体。 D4.2 多巴胺受体 (D4.2R) 通过激活抑制性异三聚体 G 蛋白 Gi 来抑制效应蛋白腺苷酸环化酶 (AC)。 D4.2R 与 Luc 或荧光蛋白的融合蛋白无活性。为了创建可用于 RET 实验的荧光 D4.2R，将 CCPGCC 基序添加到 C 末端 (D4.2R-PGCC) 或第三个细胞内环内的两个不同位置（D4.2R-G259C 和 D4.2R-G259C）。 2R-G275C）。四半胱氨酸基序不影响细胞表面表达、配体与受体的结合或激动剂介导的 AC 抑制，并且与该基序结合的 FlAsH 产生荧光 D4.2R，可用作 RET 实验的受体。当 D4.2R-G257C 或 D4.2R-G275C 在 HEK 293 细胞中与 Luc 标记的 AC (AC-Luc) 共表达时，会发生 RET。 AC-Luc 和 D4.2R PGCC 之间没有显着的 RET。 RET 也发生在标记的 D4.2R 和 Luc 标记的 Gg 之间。这些数据表明 G 蛋白和 AC 都是 D4.2R 信号传导复合物的一部分。 β2-肾上腺素能受体 (b2AR) 通过激活刺激性异三聚体 G 蛋白 Gs 来刺激 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，这表明参与 AC 双重调节的刺激受体和抑制受体存在于同一细胞中。信号复合体。 b2AR-Luc 和 D4.2R-PGCC 之间没有显着的 RET，尽管它在功能上与野生型 D4.2R 无法区分。这可能是由于供体和受体标签距离太远或方向错误而无法进行 RET 的结果。 Luc 标记的信号蛋白和 CCPGCC 标记的 D4.2R 之间的 RET 在没有信号传导的情况下发生，并且不受激动剂介导的信号传导的影响。 BiFC 与 RET 结合证明信号复合物中同时存在三种不同的蛋白质。当用 YC 标记的 b2AR (b2AR-YC) 和用 YN 标记的 Gg (YN-Gg) 在 HEK 293 细胞中共表达时，会发生 BiFC。当 AC-Luc 与 b2AR-YC 和 YN-Gg 共表达时发生 RET，表明受体、G 蛋白和效应子是同一信号复合物的一部分。实验证据还支持 G 蛋白介导的信号复合物在到达质膜之前形成的假设。 RET 与亚细胞分级分离证明 AC 和 b2AR 的复合物存在于细胞内膜上。此外，显性失活 (DN) GTP 酶（Rab1 和 Sar1）可阻止内质网 (ER) 顺行运输，对 b2AR/AC、Gg/AC 或 b2AR/Gg 相互作用没有影响。然而，DN Rab1 和 Sar1 构建体（但不是 DN Rabs 2、6、8 或 11）阻止 Ga 亚基包含在 AC 信号复合物中，这表明 Ga 在 ER 之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：七螺旋受体、G蛋白和效应子在被转运到其靶膜之前组装成复合物，并且当信号转导途径被激动剂激活时这些复合物持续存在。这种排列有助于解释 G 蛋白介导的信号转导过程中经常观察到的特异性和功效。