INVESTIGATION OF HETEROTRIMERIC GUANINE NUCLEOTIDE BINDING PROTEIN ACTIVATION

异三聚鸟嘌呤核苷酸结合蛋白激活的研究

• 批准号: 6111865
• 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
• 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/6111865
• 关键词: ADP ribosylation; G protein; adenylate cyclase; binding proteins; brain cell; chemical association; cow; enzyme activity; genetic transcription; genetic translation; guanine nucleoside; guanosine diphosphate; guanosine triphosphate; guanosinetriphosphatases; immunoprecipitation; membrane reconstitution /synthesis; nucleotide analog; protein structure; recombinant DNA

Heterotrimeric (alpha-beta-gamma) Gs mediates agonist-induced stimulation of adenylyl cyclase (AC). The alpha subunit (Gs-alpha) has intrinsic GTPase activity. Gs is activated when Gs-alpha binds GTP or a GTP analog (e.g. GTPgammaS) and it is inactivated when GTP is hydrolyzed. Gs-alpha is composed of two domains, one a ras-like GTPase domain and the other a predominately alpha helical domain, whose function remains largely unclear. We have found through mutagenesis that a non-conserved arginine in a region of the ras-like domain known as switch 3 forms a bond with a conserved glutamine in the helical domain. These two amino acids create a "lid' over the active site providing direct evidence that interaction between the two domains is important for high affinity, guanine nucleotide binding, and consequently the stability of Gs-alpha. We have also demonstrated by mutagenesis that a strictly conserved glutamate in switch 3 is important in the activation mechanism because it forms contacts with a region known as switch 2, thereby permitting efficient binding of the transition state that occurs when GTP is hydrolyzed. Under certain conditions the activation of Gs in solution can be accompanied by dissociation of Gs-alpha from the G protein beta-gamma subunit complex (G-beta-gamma). Nevertheless, we have produced compelling evidence that subunit dissociation does not necessarily occur when Gs is activated in solution. We are continuing these investigations by studying the kinetics of G protein subunit dissociation and association under different conditions using surface plasmon resonance spectroscopy. However, what happens when membrane bound Gs is activated is the more physiologically relevant as well as the more difficult question to answer, and this has been a focus of current research. Cholera toxin (CTx) activates AC by ADP-ribosylating Gs-alpha. In solution Gs-alpha is a substrate for CTx only when it is associated with G-beta-gamma. To determine if the substrate specificity for CTx is the same when Gs-alpha is membrane bound, Gs-alpha deficient cyc- membranes were "striped" of G-beta-gamma (cyc-/-) with a solution containing the detergent CHAPS. When Gs-alpha was incorporated into cyc-/-, it was a substrate for CTx only when G-beta-gamma was also incorporated indicating that the Gs heterotrimer but not free Gs-alpha is the substrate for CTx in cell membranes. The stimulation of AC by GTPgammaS-activated Gs-alpha in cyc-/- was also dependent upon the simultaneous incorporation of G-beta-gamma. Furthermore, there was a correlation between the dose dependent effects of G-beta-gamma on the stimulation of AC by GTPgammaS-activated Gs-alpha and the CTx catalyzed ADP-ribosylation of GTPgammaS-liganded Gs-alpha in cyc-/-. These data suggest that AC is stimulated by an activated Gs heterotrimer in cell membranes.

异三聚体(α-β-伽马)Gs介导 激动剂对腺酰环化酶(AC)的刺激作用。阿尔法 亚基(Gs-α)具有固有的GTP酶活性。GS已激活 当Gs-α与GTP或GTP类似物(例如GTP-GammaS)结合时 当GTP被水解时，它被灭活。GS-Alpha由以下组成 两个结构域，一个是类似ras的GTP酶结构域，另一个是 主要是α螺旋区，其功能在很大程度上仍然 不清楚。我们通过突变发现了一种非保守的 Ras样结构域中称为Switch 3的区域中的精氨酸形成 在螺旋结构域中与保守的谷氨酰胺形成的键。这两个 氨基酸在活性部位上形成一个“盖子”，直接提供 这两个领域之间的交互对于 高亲和力，鸟嘌呤核苷酸结合，因此 Gs-α的稳定性。我们也已经通过突变证明了 在Switch 3中严格保守的谷氨酸在 激活机制，因为它与一个区域形成接触 称为开关2，从而允许有效地绑定 GTP被水解时出现的过渡态。在某些情况下 在溶液中激活Gs的条件可以伴随着 Gs-α与G蛋白β-γ亚基的解离 复合体(G-β-伽马)。尽管如此，我们还是制作了 令人信服的证据表明亚基解离并不一定 在溶液中激活Gs时发生。我们将继续这样做 G蛋白亚基的动力学研究 表面在不同条件下的解离和缔合 等离子体共振光谱学。然而，当 膜结合Gs被激活越具有生理性 以及更难回答的问题，而这 一直是当前研究的焦点。霍乱毒素(CTX)激活 ADP-核糖化Gs-α。在溶液中，Gs-α是一个 仅当CTX与G-β-伽马相关时才用于CTX底物。 以确定环磷酰胺的底物专一性是否相同 GS-α是膜结合的，Gs-α缺乏环状膜 用含有以下物质的溶液将G-β-伽马(Cyc-/-)“剥离” 洗涤剂伙计们。当Gs-Alpha被合并到 CyC-/-，只有当G-β-伽马被 还加入了Gs杂三聚体，但不是游离的 GS-α是CTX在细胞膜中的底物。这个 GTP-GammaS激活Gs-α对Cyc-/-Was细胞AC的刺激作用 也取决于同时合并 G-β-伽马。此外，两者之间存在相关性。 G-β-γ对AC刺激的剂量依赖性效应 由GTP-GammaS激活的Gs-α和催化的CTX GTP-GammaS连接的Gs-α在cyc-/-中的ADP-核糖基化。 这些数据表明AC是由激活的Gs刺激的 细胞膜中的杂三聚体。