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-beta-gamma）GS介导 激动剂诱导的腺苷环酶刺激（AC）。 alpha 亚基（GS-Alpha）具有内在的GTPase活性。 GS被激活 当GS-Alpha结合GTP或GTP类似物（例如GTPGAMMA）和 当GTP水解时，它被灭活。 GS-Alpha由 两个域，一个域，一个类似于Ras的GTPase域，另一个是A 主要的α螺旋结构域，其功能在很大程度上保持 不清楚。我们通过诱变发现了一个未保存的 精氨酸在类似于Ras的域的区域中称为Switch 3形式 在螺旋结构域中具有保守谷氨酰胺的键。这两个 氨基酸在可直接提供直接的活动位置上产生一个“盖” 证据表明两个领域之间的相互作用对于 高亲和力，鸟嘌呤核苷酸结合，因此 GS-Alpha的稳定性。我们也通过诱变证明了 开关3中严格保守的谷氨酸在 激活机制，因为它与区域形成接触 称为开关2，从而允许有效的结合 GTP水解时发生的过渡状态。在一定程度上 条件溶液中GS的激活可以伴随 GS-Alpha与G蛋白β-GAMMA亚基的解离 复杂（G-Beta-Gamma）。尽管如此，我们已经生产了 令人信服的证据表明亚基解离不一定是 当在溶液中激活GS时发生。我们正在继续这些 通过研究G蛋白亚基的动力学来进行研究 在不同条件下使用表面解离和关联 等离子体共振光谱。但是，当 激活膜绑定的GS是生理上越多的 相关和更难回答的问题，这 一直是当前研究的重点。霍乱毒素（CTX）激活 AC通过ADP-核糖基化GS-Alpha。在解决方案中，GS-Alpha是 仅当CTX与G-Beta-Gamma相关时，仅用于CTX。 确定CTX的底物特异性是否相同 GS-Alpha是膜结合的，GS-Alpha缺乏Cyc-膜 用包含溶液的G-Beta-Gamma（CYC - / - ）为“条纹” 洗涤剂粉碎机。当将GS-Alpha纳入 CYC - / - ，仅当G-Beta-gamma是CTX的底物 还合并表明GS异三聚体但不是免费的 GS-Alpha是细胞膜中CTX的底物。这 通过GTPGAMMAS激活的GS-Alpha刺激AC - / - 也取决于同时合并 G-Beta-Gamma。此外， G-Beta-Gamma对AC刺激的剂量依赖性影响 通过GTPGAMMAS激活的GS-Alpha和CTX催化 gtpgammas-ligand的GS-α在CYC-/ - 中的ADP-核糖基化。 这些数据表明AC被激活的GS刺激 细胞膜中的异三聚体。