INVESTIGATION OF HETEROTRIMERIC GUANINE NUCLEOTIDE BINDING PROTEIN ACTIVATION

异源三聚体（α-β-γ）Gs介导 激动剂诱导的腺苷酸环化酶（AC）刺激。阿尔法 亚基（Gs-alpha）具有内在的GT3活性。GS被激活 当Gs-α结合GTP或GTP类似物（例如GTP γ S）时， 当GTP被水解时，它被灭活。GS-α由以下成分组成： 两个结构域，一个是ras样GT3结构域，另一个是 主要是α螺旋结构域，其功能在很大程度上保持不变 不清楚我们通过诱变发现， ras样结构域中称为开关3的区域中的精氨酸 与螺旋结构域中保守的谷氨酰胺形成键。这两 氨基酸在活性位点上形成“盖子”， 证据表明，这两个领域之间的相互作用是重要的， 高亲和力，鸟嘌呤核苷酸结合，因此 Gs-alpha的稳定性我们还通过诱变证明了 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，从而允许有效地绑定 当GTP水解时发生的过渡状态。在一定 条件下，溶液中Gs的活化可以伴随着 从G蛋白β-γ亚基上解离Gs-alpha 复合物（G-β-γ）。尽管如此，我们还是制作了 有令人信服的证据表明，亚基解离不一定 当Gs在溶液中被激活时发生。我们继续这些 G蛋白亚基动力学研究 在不同条件下的解离和缔合 等离子体共振光谱然而，当 在生理上，膜结合的Gs被激活的程度越高， 这是一个很难回答的问题，而且 一直是当前研究的焦点。霍乱毒素（CTx）激活 AC通过ADP-核糖基化GS-α。在溶液中，Gs-alpha是 CTx仅在与G-β-γ相关时为CTx的底物。 为了确定CTx的底物特异性是否相同， Gs-alpha是膜结合的，Gs-alpha缺陷的细胞膜 用含有以下物质的溶液“去除”G-β-γ（cyc-/-） 清洁剂CHAPS。当Gs-alpha被整合到 cyc-/-，它是CTx的底物，只有当G-β-γ 还结合表明Gs异源三聚体但不是游离的 Gs-alpha是细胞膜中CTx的底物。的 在cyc-/-洗涤中由GTP γ S-活化Gs-α刺激AC 也取决于同时结合 G-B-G此外， G-β-γ对AC刺激的剂量依赖性效应 通过GTP γ S激活的Gs-alpha和CTx催化的 在cyc-/-中GTP γ S配体的Gs-α的ADP核糖基化。 这些数据表明AC是由激活的Gs刺激的， 细胞膜中的异源三聚体。

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.