Heterotrimeric G-proteins transduce signals from heptahelical transmembrane receptors to different effector systems, regulating diverse complex intracellular pathways and functions. In brain, facilitation of depolarization-induced neurotransmitter release for synaptic transmission is mediated by Gsalpha and Gqalpha. To identify effectors for Galpha-proteins, we performed a yeast two-hybrid screening of a human brain cDNA library, using the human Galphas protein as a bait. We identified a protein member of the synembryn family as one of the interacting proteins. Extending the study to other Galpha subunits, we found that Gqalpha also interacts with synembryn, and these interactions were confirmed by in vitro pull down studies and by in vivo confocal laser microscopy analysis. Furthermore, synembryn was shown to translocate to the plasma membrane in response to carbachol and isoproterenol. This study supports recent findings in C. elegans where, through genetic studies, synembryn was shown to act together with Gqalpha regulating neuronal transmitter release. Based on these observations, we propose that synembryn is playing a similar role in human neuronal cells.

Progesterone, produced by follicular cells, induces Xenopus laevis oocyte maturation through a very early event that inhibits the activity of the adenylyl cyclase effector system. The participation of a G-protein has been implicated, based on the fact that the inhibitory effect of the steroid is GTP-dependent, and it has been proposed that progesterone acts interfering with G(alpha)s function at the plasma membrane. Here we investigate whether the change in oocyte G(alpha)s levels affects the maturation process induced by progesterone. Overexpression of X. laevis wild type (wt) G(alpha)s and the constitutive activated G(alpha)s(QL) mutant, both blocked progesterone-induced maturation, G(alpha)s(QL) being much more effective than the wt protein. On the other hand, depletion of G(alpha)s, by the use of antisense oligonucleotides, caused spontaneous maturation measured as MAPK activation, indicating clearly that the presence of G(alpha)s is necessary to keep oocytes arrested. Overexpression of three different G-protein coupled receptors (GPCR), the beta2-adrenergic receptor and the m4 and m5 muscarinic receptors, all caused inhibition of MAPK activation induced by progesterone. These receptors, upon their activation with the respective ligands, might be inducing the release of G(beta)gamma from their respective G(alpha), which together with endogenous G(alpha)s-GTP, activate adenylyl cyclase. Our results indicate that G(alpha)s plays an important role in the maturation process and support previous findings of G(beta)gamma participation, suggesting the presence of a mechanism where a constitutively activated G(alpha)s subunit, together with the G(beta)gamma heterodimer, both maintain high levels of intracellular cAMP levels, blocking the G2/M transition.

No abstract

Immature stage VI Xenopus oocytes are arrested at the G(2)/M border of meiosis I until exposed to progesterone, which induces meiotic resumption through a non-genomic mechanism. One of the earliest events produced by this hormone is inhibition of the plasma membrane enzyme adenylyl cyclase (AC), with the concomitant drop in intracellular cAMP levels and reinitiation of the cell cycle. Recently Gsalpha and Gbetagamma have been shown to play an important role as positive regulators of Xenopus oocyte AC, maintaining the oocyte in the arrested state. However, a question that still remains unanswered, is how the activated state of Gsalpha and Gbetagamma is achieved in the immature oocyte, since no receptor or ligand have been found to be required. Here we provide evidence that xRic-8 can act in vitro and in vivo as a GEF for Gsalpha. Overexpression of xRic-8, through mRNA injection, greatly inhibits progesterone induced oocyte maturation and endogenous xRic-8 mRNA depletion, through siRNA microinjection, induces spontaneous oocyte maturation. These results suggest that xRic-8 is participating in the immature oocyte by keeping Gsalpha-Gbetagamma-AC signaling complex in an activated state and therefore maintaining G2 arrest.

Xenopus laevis oocyte maturation is induced by the steroid hormone progesterone through a nongenomic mechanism that implicates the inhibition of the effector system adenylyl cyclase (AC). Recently, it has been shown that the G protein betagamma heterodimer is involved in oocyte maturation arrest. Since AC is the proposed target for Gbetagamma action, we considered of importance to identify and characterize the Gbetagamma regulated AC isoform(s) that are expressed in the Xenopus oocyte. Through biochemical studies, we found that stage VI plasma membrane oocyte AC activity showed attributes of an AC2 isoform. Furthermore, exogenous Gbetagamma was capable to activate oocyte AC only in the presence of the activated form of Galphas (Galphas-GTPgammaS), which is in agreement with the Ggammabeta conditional activation reported for the mammalian AC2 and AC4 isotypes. In order to study the functional role of AC in oocyte maturation we cloned from a Xenopus oocyte cDNA library a gene encoding an AC with high identity to AC7 (xAC7). Based on this sequence, we constructed a minigene encoding the AC-Gbetagamma interacting region (xAC7pep) to block, within the oocyte, this interaction. We found that microinjection of the xAC7pep potentiated progesterone-induced maturation, as did the AC2 minigene. From these results we can conclude that a Gbetagamma-activated AC is playing an important role in Xenopus oocyte meiotic arrest in a Galphas-GTP dependent manner.