Abstract
Several recent studies have suggested that resumption of oocyte meiosis, indicated by germinal vesicle breakdown or GVBD, involves inhibition of endogenous heterotrimeric G proteins in both frogs and mice. These studies imply that a heterotrimeric G protein(s), and hence its upstream activator (a G protein-coupled receptor or GpCR), is activated in prophase oocytes and is responsible for maintaining meiosis arrest. To test the existence and function of this putative GpCR, we utilized a mammalian G-protein-coupled receptor kinase (GRK3) and beta-arrestin-2, which together are known to cause GpCR desensitization. Injection of mRNA for rat GRK3 caused hormone-independent GVBD. The kinase activity of GRK3 was essential for GVBD induction as its kinase-dead mutant (GRK3-K220R) was completely ineffective. Another GRK3 mutant (GRK3-DeltaC), which lacked the C-terminal G(betagamma)-binding domain and which was not associated with oocyte membranes, also failed to induce GVBD. Furthermore, injection of rat beta-arrestin-2 mRNA also induced hormone-independent GVBD. Several inhibitors of clathrin-mediated receptor endocytosis (the clathrin-binding domain of beta-arrestin-2, concanavalin A, and monodansyl cadaverine) significantly reduced the abilities of GRK3/beta-arrestin-2 to induce GVBD. These results support the central role of a yet-unidentified GpCR in maintaining prophase arrest in frog oocytes and provide a potential means for its molecular identification.
Highlights
Grown Xenopus laevis oocytes are arrested at the prophase of meiosis I
We demonstrated that inhibition of endogenous G protein ␥ subunits lowers oocyte cAMP and induces oocyte maturation [11]
Overexpression of G␥ subunits increases oocyte cAMP [11] and inhibits progesterone-induced oocyte maturation [11, 14]. Together these studies suggest the existence of an activated G protein(s) in prophase oocytes that maintains high levels of cAMP and prophase arrest. We postulated that this G protein(s) is activated by an endogenous G protein-coupled receptor (GpCR) that is activated in prophase oocytes [11]
Summary
Grown Xenopus laevis oocytes are arrested at the prophase of meiosis I. (All cDNA constructs are depicted in Fig. 1.) Injection of GRK3 mRNA caused efficient GVBD, with maturation spots indistinguishable from that induced by progesterone (Fig. 2A). To confirm that GRK3-induced GVBD was accompanied by activation of the various maturation-specific protein kinases (MOS, MAP kinase, and MPF), we analyzed them in extracts derived from mRNA-injected oocytes.
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
