A G Protein-coupled Receptor Kinase Induces XenopusOocyte Maturation

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 β-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-ΔC), which lacked the C-terminal Gβγ-binding domain and which was not associated with oocyte membranes, also failed to induce GVBD. Furthermore, injection of rat β-arrestin-2 mRNA also induced hormone-independent GVBD. Several inhibitors of clathrin-mediated receptor endocytosis (the clathrin-binding domain of β-arrestin-2, concanavalin A, and monodansyl cadaverine) significantly reduced the abilities of GRK3/β-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. 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 β-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-ΔC), which lacked the C-terminal Gβγ-binding domain and which was not associated with oocyte membranes, also failed to induce GVBD. Furthermore, injection of rat β-arrestin-2 mRNA also induced hormone-independent GVBD. Several inhibitors of clathrin-mediated receptor endocytosis (the clathrin-binding domain of β-arrestin-2, concanavalin A, and monodansyl cadaverine) significantly reduced the abilities of GRK3/β-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. maturation promoting factor G protein-coupled receptor germinal vesicle breakdown G protein-coupled receptor kinase hemagglutinin mitogen-activated protein concanavalin A monodansyl cadaverine. Fully grown Xenopus laevis oocytes are arrested at the prophase of meiosis I. Reinitiating of meiosis, or oocyte maturation, is triggered by the ovarian hormone progesterone (1Masui Y. J. Exp. Zool. 1967; 166: 365-376Crossref PubMed Scopus (212) Google Scholar). Progesterone, likely through its cytoplasmic receptor xPR (2Bayaa M. Booth R.A. Sheng Y. Liu X.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12607-12612Crossref PubMed Scopus (186) Google Scholar, 3Tian J. Kim S. Hellig E. Ruderman J.V. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14358-14363Crossref PubMed Scopus (150) Google Scholar), regulates intracellular signaling pathways, ultimately leading to the activation of maturation promoting factor or MPF1 (4Masui Y. Markert C.L. J. Exp. Zool. 1971; 177: 129-146Crossref PubMed Scopus (1149) Google Scholar). One important signaling pathway regulated by progesterone appears to be intracellular cAMP. It is generally agreed that progesterone induces a modest (20%) reduction of cAMP (5Smith L.D. Development. 1989; 107: 685-699PubMed Google Scholar), likely by inhibiting membrane-bound adenylyl cyclases (6Sadler S.E. Maller J.L. J. Biol. Chem. 1981; 256: 6368-6373Abstract Full Text PDF PubMed Google Scholar, 7Finidori-Lepicard J. Schorderet-Slatkine S. Hanoune J. Baulieu E.E. Nature. 1981; 292: 255-257Crossref PubMed Scopus (178) Google Scholar). The importance of cAMP reduction is underlined by the demonstration that both forskolin (8Schorderet-Slatkine S. Baulieu E.-E. Endocrinology. 1982; 111: 1385-1387Crossref PubMed Scopus (46) Google Scholar) and isobutylmethylxanthine (9Sadler S.E. Maller J.L. J. Biol. Chem. 1987; 262: 10644-10650Abstract Full Text PDF PubMed Google Scholar) block progesterone-induced oocyte maturation. However, the classical Gi inhibitor Bordetella pertussis toxin does not prevent progesterone from inhibiting adenylyl cyclase or inducing oocyte maturation (10Sadler S.E. Maller J.L. Cooper D.M. Mol. Pharmacol. 1984; 26: 526-531PubMed Google Scholar, 11Sheng Y. Tiberi M. Booth R.A. Ma C. Liu X.J. Curr. Biol. 2001; 11: 405-416Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar), suggesting that progesterone action is not mediated by the activation of classical Gi proteins. This puzzle appears to be resolved by the finding that inhibition of endogenous adenylyl cyclase-activating G proteins causes spontaneous oocyte maturation. Jaffe and colleagues demonstrated, first in the frog (12Gallo C.J. Hand A.R. Jones T.L.Z. Jaffe L.A. J. Cell Biol. 1995; 130: 275-284Crossref PubMed Scopus (86) Google Scholar) and later in mice (13Mehlmann L.M. Jones T.L. Jaffe L.A. Science. 2002; 297: 1343-1345Crossref PubMed Scopus (196) Google Scholar), that injection of neutralizing antibodies against mammalian Gsα causes oocyte maturation, suggesting that endogenous Xenopus Gsα plays a dominant role in maintaining prophase arrest. On the other hand, we demonstrated that inhibition of endogenous G protein βγ subunits (via injection of Gβγ scavengers) lowers oocyte cAMP and induces oocyte maturation (11Sheng Y. Tiberi M. Booth R.A. Ma C. Liu X.J. Curr. Biol. 2001; 11: 405-416Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Furthermore, overexpression of Gβγ subunits increases oocyte cAMP (11Sheng Y. Tiberi M. Booth R.A. Ma C. Liu X.J. Curr. Biol. 2001; 11: 405-416Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar) and inhibits progesterone-induced oocyte maturation (11Sheng Y. Tiberi M. Booth R.A. Ma C. Liu X.J. Curr. Biol. 2001; 11: 405-416Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 14Lutz L.B. Kim B. Jahani D. Hammes S.R. J. Biol. Chem. 2000; 275: 41512-41520Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). 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 (11Sheng Y. Tiberi M. Booth R.A. Ma C. Liu X.J. Curr. Biol. 2001; 11: 405-416Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). In this study, we wished to explore a well characterized aspect of GpCR signaling, receptor desensitization, to validate the existence and function of the putative GpCR in maintaining prophase arrest in frog oocytes. A general mechanism governing GpCR desensitization has emerged from work carried out on β-adrenergic receptors and many other GpCRs (15Claing A. Laporte S.A. Caron M.G. Lefkowitz R.J. Prog. Neurobiol. 2002; 66: 61-79Crossref PubMed Scopus (451) Google Scholar). A G protein-coupled receptor kinase (GRK) phosphorylates activated, usually agonist-occupied, GpCR, creating a binding site for a regulatory protein, β-arrestin. Binding of β-arrestin to the GpCR prevents the latter from further binding to its target G protein, thus resulting in GpCR desensitization. In addition, β-arrestin also serves as an adaptor for clathrin-coated pits. The latter function of the β-arrestin thus initiates clathrin-mediated endocytosis of the GpCR. We reasoned that overexpression of GRKs and β-arrestin in frog oocytes may disrupt the tonic GpCR signaling in prophase oocytes causing GpCR desensitization and endocytosis. Such interventions may therefore cause spontaneous oocyte maturation. The nucleotide sequence encoding full-length rat GRK3 (16Arriza J.L. Dawson T.M. Simerly R.B. Martin L.J. Caron M.G. Snyder S.H. Lefkowitz R.J. J. Neurosci. 1992; 12: 4045-4055Crossref PubMed Google Scholar) (a gift from Dr. Robert Lefkowitz) was PCR-amplified using the following primers: forward primer, 5′-TAT AGG CCT GCC ATG GCG GAC CTG GAG G-3′; reverse primer, 5′-TAT AGG CCT CAG AGG CCG CTG CTA TTT CTG-3′. The amplified DNA was digested withStuI and ligated into StuI-digested pCS2+ (17Turner D.L. Weintraub H. Genes Dev. 1994; 8: 1434-1447Crossref PubMed Scopus (951) Google Scholar) or pCS2+HA (18Booth R.A. Cummings C. Tiberi M. Liu X.J. J. Biol. Chem. 2002; 277: 6719-6725Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). The resultant plasmids encoded, respectively, untagged or hemagglutinin (HA)-tagged GRK3. The kinase-dead mutant of GRK3 (GRK3-K220R) was generated by the two-step PCR procedure (19Vallette F. Mege E. Reiss A. Adesnik M. Nucleic Acids Res. 1989; 17: 723-733Crossref PubMed Scopus (175) Google Scholar) and confirmed by DNA sequencing. A C-terminally truncated GRK, GRK3-ΔC (containing amino acids 1–544 (16Arriza J.L. Dawson T.M. Simerly R.B. Martin L.J. Caron M.G. Snyder S.H. Lefkowitz R.J. J. Neurosci. 1992; 12: 4045-4055Crossref PubMed Google Scholar)), was PCR-amplified using the same forward primer as above and the following reverse primer, 5′-AGG CCT TCA TTT ATT TTT AGC CTT CTT CCT GGC-3′. The amplified DNA was digested with StuI and then ligated into pCS2+HA that had been previously digested withStuI. These manipulations resulted in HA-GRK3-ΔC. Rat β-arrestin-2 (20Attramadal H. Arriza J.L. Aoki C. Dawson T.M. Codina J. Kwatra M.M. Snyder S.H. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1992; 267: 17882-17890Abstract Full Text PDF PubMed Google Scholar) (a gift from Dr. Robert Lefkowitz) was PCR-amplified using the following primers: forward primer, 5′-TAT CCA TGG GTG AAA AAC CCG GGA CC-3′; reverse primer, 5′-TAT CCA TGG CAG AAC TGG TCA TCA CAG TC-3′. The amplified DNA was digested withNcoI (limited digestion was necessary due to the presence of an NcoI within the coding sequence of β-arrestin-2) and ligated into pCS2+HA that had previously been digested withNcoI. The C-terminal clathrin-binding domain of β-arrestin-2 (amino acids 319–410) (21Krupnick J.G. Santini F. Gagnon A.W. Keen J.H. Benovic J.L. J. Biol. Chem. 1997; 272: 32507-32512Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar) was PCR-amplified using the same reverse primer (above) and the following forward primer, 5′-TAT CCA TGG GAA TCC TAG TAT CCT AC-3′. The amplified cDNA was digested and ligated into pCS2+HA, as described above for full-length β-arrestin-2. The resultant plasmid encoded HA-β-arrestin-C. Oocytes were injected with mRNA (10 ng per oocyte, unless otherwise indicated) encoding HA-GRK3 or HA-GRK3-K220R. Following an overnight incubation, 20 oocytes were lysed in ice-cold phosphate-buffered saline lysis buffer (10 mm sodium phosphate buffer, pH 7.5, 150 mmNaCl, 1% Triton X-100, 1 μg/ml leupeptin, 100 μmphenylmethylsulfonyl fluoride; 10 μl/oocyte) by forcing through a pipette tip. The lysates were clarified by centrifugation and then were immune-precipitated with anti-HA antibodies. Immune complexes were resuspended in kinase buffer (50 mm Hepes, pH 7.3, 10 mm MgCl2, 2 mm MnCl2, 1 mm dithiothreitol, 0.05% Triton X-100; 15 μl per sample). Kinase reaction was initiated with the addition of 5 μCi of [γ-32P]ATP, 10 μm ATP, 14 μg each of myelin basic protein and β-casein. The kinase reaction was carried out at room temperature for 30 min and was stopped with the addition of equal volume of 2× SDS-sample buffer. Proteins were separated on a 15% SDS-PAGE, dried, and visualized by autoradiography. Other procedures employed in this study have been described in our previous publications (2Bayaa M. Booth R.A. Sheng Y. Liu X.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12607-12612Crossref PubMed Scopus (186) Google Scholar, 11Sheng Y. Tiberi M. Booth R.A. Ma C. Liu X.J. Curr. Biol. 2001; 11: 405-416Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 18Booth R.A. Cummings C. Tiberi M. Liu X.J. J. Biol. Chem. 2002; 277: 6719-6725Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). To test whether mammalian GRKs can induce frog oocyte maturation, we subcloned rat GRK3 into pCS2+ (17Turner D.L. Weintraub H. Genes Dev. 1994; 8: 1434-1447Crossref PubMed Scopus (951) Google Scholar) for in vitro mRNA synthesis. (All cDNA constructs are depicted in Fig.1.) Injection of GRK3 mRNA indeed caused efficient GVBD, with maturation spots indistinguishable from that induced by progesterone (Fig.2 A). Typically, GRK3-induced GVBD lagged several hours behind progesterone-induced GVBD (Fig.2 A).Figure 2Kinase activity of GRK3 was essential for GVBD induction. A, 50 or more oocytes were either injected with water (control) or GRK3 mRNA or incubated with progesterone (Pg, 1 μm). At the indicated time following the injection (or the addition of progesterone), GVBD were scored and expressed as % of total treated oocytes. Although different batches of oocytes varied in their GVBD response time, GRK3-induced GVBD always lagged several hours behind Pg-induced GVBD. Typical images of oocytes in each group were shown. B, oocytes injected with the indicated mRNAs were incubated overnight in OR2. GVBD were scored and expressed as % of total injected oocytes. Shown are means with S.D. of four to six independent experiments. Shown above the bars are actual numbers of GVBD-positive oocytes over those of treated oocytes. The sign * denotes p < 0.001 in pair-wise Student's t test. C, following GVBD scoring (as in B), oocytes were lysed and the resultant extracts were analyzed for MOS accumulation, Xenopus MAP kinase (xMAPK) phosphorylation, and MPF assays (using histone as an in vitro A.R. J. 12: PubMed Scopus Google Scholar). oocytes injected with water or mRNA for HA-GRK3 or were incubated overnight in OR2. were and to with anti-HA antibodies. The complexes were to in vitro kinase assays using both and myelin basic protein as J.L. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1989; Full Text PDF PubMed Google Scholar) of the complexes were also to with anti-HA antibodies Shown are of four independent We have previously that the of to its in rat with a site for caused GVBD by endogenous G protein βγ complexes (11Sheng Y. Tiberi M. Booth R.A. Ma C. Liu X.J. Curr. Biol. 2001; 11: 405-416Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). The site was necessary the was (11Sheng Y. Tiberi M. Booth R.A. Ma C. Liu X.J. Curr. Biol. 2001; 11: 405-416Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). GRK3 (or does not a site (or other for for was that GRK3 as efficient Gβγ to induce GVBD. to out this we the essential (16Arriza J.L. Dawson T.M. Simerly R.B. Martin L.J. Caron M.G. Snyder S.H. Lefkowitz R.J. J. Neurosci. 1992; 12: 4045-4055Crossref PubMed Google Scholar) with In to was completely in GVBD induction 2 To of GRK3 and we an (18Booth R.A. Cummings C. Tiberi M. Liu X.J. J. Biol. Chem. 2002; 277: 6719-6725Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar) to each of HA-GRK3 and HA-GRK3 was efficient untagged GRK3 in GVBD as of inducing GVBD 2 To that GRK3-induced GVBD was by activation of the protein MAP and we analyzed in extracts from oocytes. HA-GRK3 or not their kinase-dead induced MOS and activated both MAP kinase and MPF (Fig.2 We carried out kinase assays to the kinase of HA-GRK3 and HA-GRK3-K220R. antibodies of the proteins in oocytes 2 However, HA-GRK3 significantly kinase with from extracts of 2 These results that the kinase activity of GRK3 is essential for GVBD induction and suggest that GRK3 induced GVBD a mechanism different from oocyte Y. Tiberi M. Booth R.A. Ma C. Liu X.J. Curr. Biol. 2001; 11: 405-416Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). In the abilities of GRKs to their are on their Lefkowitz R.J. PubMed Scopus Google Scholar). GRKs and are membrane-bound GRK3 and to membrane-bound Gβγ complexes Lefkowitz R.J. PubMed Scopus Google Scholar). To whether Gβγ binding is for GRK3 to induce GVBD, we GRK3-ΔC in which the C-terminal Gβγ-binding domain (amino acids J. L.M. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1994; PubMed Scopus Google Scholar) had been A that both HA-GRK3 and were expressed in oocytes. However, HA-GRK3 was associated with oocyte A, was completely Following both HA-GRK3 and in vitro kinase was in myelin basic protein and vitro kinase was not to induce GVBD, was of of the protein in frog oocytes These results the essential role of the C-terminal Gβγ-binding domain of in and in GVBD induction further support that GRK3 induced GVBD a membrane-bound GpCR. GRK3-induced GVBD was mediated and desensitization of an endogenous GpCR, work together with an endogenous β-arrestin protein (15Claing A. Laporte S.A. Caron M.G. Lefkowitz R.J. Prog. Neurobiol. 2002; 66: 61-79Crossref PubMed Scopus (451) Google Scholar). We therefore wished to test whether overexpression of a mammalian β-arrestin also induce GVBD. Rat β-arrestin-2 cDNA (20Attramadal H. Arriza J.L. Aoki C. Dawson T.M. Codina J. Kwatra M.M. Snyder S.H. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1992; 267: 17882-17890Abstract Full Text PDF PubMed Google Scholar) was PCR-amplified and subcloned into (18Booth R.A. Cummings C. Tiberi M. Liu X.J. J. Biol. Chem. 2002; 277: 6719-6725Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Injection of mRNA caused GVBD A). In the C-terminal clathrin-binding domain of β-arrestin-2, was to induce GVBD. GVBD a time to that of GRK3-induced GVBD and both lagged behind progesterone-induced GVBD To whether GRK3 and in GVBD we injected ng per of GRK3 or mRNA or in in C, injection of either mRNA caused GVBD, of both caused of GVBD. These results β-arrestin-2 and GRK3 in GVBD with the of GpCR desensitization Lefkowitz R.J. PubMed Scopus Google Scholar). GRK3/β-arrestin-2 induced GVBD the desensitization of a GpCR, we that the GVBD induction be by of or oocytes with forskolin completely GVBD induction of forskolin oocyte to and further incubation, of the oocytes GVBD In addition to GpCR desensitization, GRKs and also GpCR endocytosis (15Claing A. Laporte S.A. Caron M.G. Lefkowitz R.J. Prog. Neurobiol. 2002; 66: 61-79Crossref PubMed Scopus (451) Google Scholar). In to target GpCR to clathrin-coated (15Claing A. Laporte S.A. Caron M.G. Lefkowitz R.J. Prog. Neurobiol. 2002; 66: 61-79Crossref PubMed Scopus (451) Google Scholar). J.G. Santini F. Gagnon A.W. Keen J.H. Benovic J.L. J. Biol. Chem. 1997; 272: 32507-32512Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar) have previously demonstrated that the overexpression of the clathrin-binding domain of β-arrestin GpCR endocytosis. clathrin-mediated GpCR endocytosis role in GVBD, with the GVBD induction. injection of mRNA significantly not the of GRK3 or β-arrestin-2 to induce GVBD A). a we injected equal of an mRNA (17Turner D.L. Weintraub H. Genes Dev. 1994; 8: 1434-1447Crossref PubMed Scopus (951) Google that encoded a To further the of clathrin-mediated pathway in GRK3-induced GVBD, we The concanavalin A and with the of clathrin-coated J.L. Biol. 1982; PubMed Google Scholar). of with A β-adrenergic receptor endocytosis G protein activation and activation of the cAMP J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of oocytes with A significantly not the of GRK3 to induce GVBD B, Another inhibitor for is the monodansyl A, which to block H. 1981; PubMed Scopus Google Scholar). in C, reduced GRK3-induced GVBD. To out general or other of the inhibitors in we the of these inhibitors on GVBD. Although the mechanism by which induces GVBD A. J. C. 2002; PubMed Scopus Google Scholar), is that receptor endocytosis. A had on GVBD. To further the that A and GVBD through of clathrin-mediated GpCR we the J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), which inhibits endocytosis J. Cell Biol. PubMed Scopus Google Scholar). had on or GVBD suggesting that are not in GRK3-induced GpCR endocytosis. studies have indicated that G proteins in maintaining meiosis arrest in both (11Sheng Y. Tiberi M. Booth R.A. Ma C. Liu X.J. Curr. Biol. 2001; 11: 405-416Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, C.J. Hand A.R. Jones T.L.Z. Jaffe L.A. J. Cell Biol. 1995; 130: 275-284Crossref PubMed Scopus (86) Google Scholar, 14Lutz L.B. Kim B. Jahani D. Hammes S.R. J. Biol. Chem. 2000; 275: 41512-41520Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar) and (13Mehlmann L.M. Jones T.L. Jaffe L.A. Science. 2002; 297: 1343-1345Crossref PubMed Scopus (196) Google Scholar) oocytes. However, the of the G proteins or whether these G proteins are regulated by classical GpCRs receptors with protein kinase have been in G proteins (18Booth R.A. Cummings C. Tiberi M. Liu X.J. J. Biol. Chem. 2002; 277: 6719-6725Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, L.M. Lefkowitz R.J. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, H. F. M.G. Mol. Biol. 2001; 12: PubMed Scopus Google Scholar), with binding of the receptors to the subunits of the G proteins S. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, H. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). GRKs are protein that activated as to or GpCRs Lefkowitz R.J. PubMed Scopus Google Scholar). GRK3 was to induce GVBD therefore the first and support for the that prophase oocytes an GpCR and that this GpCR is responsible for maintaining meiosis arrest (11Sheng Y. Tiberi M. Booth R.A. Ma C. Liu X.J. Curr. Biol. 2001; 11: 405-416Abstract Full Text Full Text PDF PubMed Scopus (62) Google L.M. Jones T.L. Jaffe L.A. Science. 2002; 297: 1343-1345Crossref PubMed Scopus (196) Google Scholar). studies have indicated that GRKs are to proteins. R.A. Y. J. S. S. Caron M.G. Lefkowitz R.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) and (a of molecular proteins of A. Benovic J.L. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar) can be by and GRK3. However, the of these Another of is the regulatory which is by a of not by other or other GRKs R.A. Lefkowitz R.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). We that GRK3-induced GVBD involves of a GpCR, of these GRK3-induced GVBD of the kinase mediated by the C-terminal Gβγ-binding with the of a membrane-bound GpCR. GRK3-induced GVBD was by the of inhibition of cAMP signaling both GRK3 and β-arrestin-2 are GVBD and with the of GpCR desensitization. Furthermore, several inhibitors of clathrin-mediated endocytosis significantly reduced the abilities of GRK3/β-arrestin-2 to induce GVBD, a role of GpCR endocytosis in GVBD induction. However, as inhibition was with these inhibitors appears that clathrin-mediated endocytosis may not be for GVBD induction. In other GpCR desensitization caused by GRK3/β-arrestin-2 may be to cause GVBD. In these support the existence of an activated GpCR that is responsible for maintaining prophase arrest. GRK3 and β-arrestin-2 are known to cause desensitization of GpCR in mammalian (15Claing A. Laporte S.A. Caron M.G. Lefkowitz R.J. Prog. Neurobiol. 2002; 66: 61-79Crossref PubMed Scopus (451) Google Scholar), the not by suggest GpCR as the oocyte However, the β-arrestin-2 constructs provide a to this putative oocyte meiosis inhibitor (11Sheng Y. Tiberi M. Booth R.A. Ma C. Liu X.J. Curr. Biol. 2001; 11: 405-416Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). The results the of whether progesterone induces GVBD by endogenous GpCR We have to whether progesterone can HA-GRK3 by kinase These have not the of GRK3 Gβγ binding in and be following lysis and However, we have and J. to mammalian and the other to from a cDNA D.L. Full Text PDF PubMed Scopus Google Scholar) by PCR using on B. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). work be to the role of these endogenous GRKs in progesterone-induced oocyte maturation. We Robert J. Lefkowitz and Tiberi for cDNA

Fetched live from OpenAlex and de-inverted. Abstracts are not stored in this database: the inverted indexes are 8.6 GB of the frame’s 9.3 GB of text, and the host has 13 GB free.