Co-operation of Gsalpha and Gbetagamma in maintaining G2 arrest in Xenopus oocytes.
Progesterone-induced oocyte maturation is thought to involve the inhibition of an oocyte adenylyl cyclase and reduction of intracellular cAMP. Our previous studies demonstrated that injection of inhibitors of G protein betagamma complex induces hormone-independent oocyte maturation. In contrast, over-expression of Xenopus Gbeta1 (xGbeta1), alone or together with bovine Ggamma2, elevates oocyte cAMP and inhibits progesterone-induced oocyte maturation. To further investigate the mechanism of Gbetagamma-induced oocyte maturation, we generated a mutant xGbeta1, substituting Asp-228 for Gly (D228G). An equivalent mutation in the mammalian Gbeta1 results in the loss of its ability to activate adenylyl cyclases. Indeed, co-injection of xGbeta1D228G with Ggamma2 failed to increase oocyte cAMP or inhibit progesterone-induced oocyte maturation. To directly demonstrate that oocytes contained a Gbetagamma-regulated adenylyl cyclase, we analyzed cAMP formation in vitro by using oocyte membrane preparations. Purified brain Gbetagamma complexes significantly activated membrane-bound adenylyl cyclase activities. Multiple adenylyl cyclase isoforms were identified in frog oocytes by PCR using degenerate primers corresponding to highly conserved catalytic amino acid sequences. Among these we identified a partial Xenopus adenylyl cyclase 7 (xAC7) that was 65% identical in amino acid sequence to human AC7. A dominant-negative mutant of xAC7 induced hormone-independent oocyte maturation and accelerated progesterone-induced oocyte maturation. Theses findings suggest that xAC7 is a major component of the G2 arrest mechanism in Xenopus oocytes.
Sheng
Yinglun
Y
Ottawa Health Research Institute, Ottawa Hospital, 1053 Carling Avenue, Ottawa, Canada.
Montplaisir
Véronique
V
Liu
X Johné
XJ
D000262:Adenylyl Cyclases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D002454:Cell Differentiation / Q000187:drug effects / Q000235:genetics*; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism; D017124:Conserved Sequence / Q000235:genetics; D000242:Cyclic AMP / Q000096:biosynthesis; D018076:DNA, Complementary / Q000032:analysis / Q000235:genetics; D005260:Female; D016195:G2 Phase / Q000187:drug effects / Q000235:genetics*; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000235:genetics / Q000378:metabolism*; D044387:GTP-Binding Protein beta Subunits / Q000235:genetics / Q000378:metabolism*; D044388:GTP-Binding Protein gamma Subunits / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009865:Oocytes / Q000187:drug effects / Q000254:growth & development* / Q000378:metabolism; D011374:Progesterone / Q000378:metabolism / Q000494:pharmacology; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D013347:Subcellular Fractions; D015854:Up-Regulation / Q000235:genetics; D014982:Xenopus laevis
D018797:Cell Cycle Proteins; D018076:DNA, Complementary; C118981:G-protein Beta gamma; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D020033:Protein Isoforms; D011374:Progesterone; D000242:Cyclic AMP; D019205:GTP-Binding Protein alpha Subunits, Gs; D000262:Adenylyl Cyclases