Regulation of adenylyl cyclase by GTP-binding Proteins

GTP结合蛋白(G蛋白)由α、β、γ亚基组成，将信号从膜受体传递到酶或离子通道等效应器。G蛋白在与激动剂-受体复合体相互作用时解离为GTP结合的α和β亚基。在本研究中，我们研究了G蛋白特别是β-亚基对腺苷环化酶和磷脂酰肌醇(PI)3-激酶活性的调节机制。在豚鼠中性粒细胞中，趋化物质甲酰-甲硫氨酸-亮氨酸-苯丙氨酸(FMLP)显著增强了前列腺素(PG)E_1对细胞内cAMP的形成。这种增强作用可被百日咳毒素预先处理的细胞所消除，但不能被阻止fMLP诱导的细胞内Ca~(2+)和Gt~(2+)升高，这表明抑制性G蛋白(G_I)直接参与了fMLP诱导的cAMP生成的增强。负责G_I诱导的增强作用的腺酰环化酶是…部分用Forskolin-琼脂糖柱从GTPGammaS处理的细胞膜中进一步纯化。该环化酶与刺激性G_S蛋白的GTP-γ结合的α亚基(G_SALPHA)呈复杂形式，而与β-亚基不同。β-伽马亚基进一步增强了GTP-GammaS结合的G_Salpha刺激的环化酶活性。这些结果表明，G_1释放的GTP结合的G_Salpha和Betagamma亚基协同刺激中性粒细胞的腺酰环化酶活性。与此相关，我们还发现PI3K的活性被Wortmannin抑制，而几种类型的PI3Ks被BG亚基刺激。有趣的是，含有磷酸化的Tyr和Betagamma亚基的多肽协同激活了某种类型的PI3-激酶。在胰岛素和fMLP刺激下，完整的中性粒细胞也观察到这种协同激活，因此，G_i激活可能通过PI-3-K中的β-amma亚基与Tyr-磷酸化信号通路有串扰。较少

GTP-binding protein (G proteins) consisting of alpha, beta, gamma subunits, carry signals from membrane receptors to effectors such as enzymes or ion channels. G proteins dissociate into the GTP-bound alpha and betagamma subunits upon their interaction with agonist-receptor complex. In the present studies, we investigated mechanisms by which the activities of adenylyl cyclase and phosphatidylinositol (PI) 3-kinase are regulated by G proteins, especially the betagamma subunits.In guinea pig neutrophils, the cellular cAMP formation by prostagladin (PG) E_1 was markedly potentiated by the chemoattractant formyl-Met-Leu-Phe (fMLP). This potentiation was abolished by prior treatment of the cells with pertussis toxin, but not by the prevention of fMLP-induced intracellular Ca^<2+> increase in the cells, indicating the direct involvement of the inhibitory G protein (G_i) in the fMLP-induced potentiation of cAMP formation. Adenylyl cyclase responsible for the G_i-induced potentiation was parti … More ally purified from the GTPgammaS-treated cell membranes with a forskolin-agarose column. The cyclase appeared to be a complex form with the GTPgammaS-bound alpha subunit of the stimulatory G_S protein (G_Salpha), but not with the betagamma subunits. The GTPgammaS-bound G_Salpha-stimulated cyclase activity was further enhanced by betagamma subunits. These results indicate that GTP-bound G_Salpha and betagamma subunits released from G_i synergistically stimulate adenylyl cyclase activity in neutrophils. In relation to this, we also found that the activity of PI 3-kinase is inhibited by wortmannin and that several types of PI 3-kinase are stimulated by bg subunits. Interestingly, a peptide containing phosphorylated Tyr and betagamma subunits synergistically activated a certain type of PI 3-kinase. Such synergistic activation was also observed in intact neutrophil-like cells upon their stimulation with insulin and fMLP.Thus, G_i activation appeared to have a cross-talk with Tyr-phosphorylation signaling pathway via betagamma subunits in PI 3-kinase. Less