A G Protein-coupled Receptor Kinase Induces Xenopus Oocyte Maturation*

Jing WangDagger § and X. Johné LiuDagger §||

From the Dagger  Ottawa Health Research Institute, Ottawa Hospital Civic Campus, Ottawa, Ontario K1Y 4E9, Canada and the § Department of Biochemistry, Microbiology, and Immunology, and the  Department of Obstetrics and Gynaecology, University of Ottawa, Ottawa, Ontario K1N 6N5, Canada

Received for publication, January 10, 2003, and in revised form, February 17, 2003

    ABSTRACT
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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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-Delta C), which lacked the C-terminal Gbeta gamma -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.

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ABSTRACT
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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 (1). Progesterone, likely through its cytoplasmic receptor xPR (2, 3), regulates intracellular signaling pathways, ultimately leading to the activation of maturation promoting factor or MPF1 (4). 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 (5), likely by inhibiting membrane-bound adenylyl cyclases (6, 7). The importance of cAMP reduction is underlined by the demonstration that both forskolin (8) and isobutylmethylxanthine (9) 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 (10, 11), 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 (12) and later in mice (13), that injection of neutralizing antibodies against mammalian Gsalpha causes oocyte maturation, suggesting that endogenous Xenopus Gsalpha plays a dominant role in maintaining prophase arrest. On the other hand, we demonstrated that inhibition of endogenous G protein beta gamma subunits (via injection of Gbeta gamma scavengers) lowers oocyte cAMP and induces oocyte maturation (11). Furthermore, overexpression of Gbeta gamma 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).

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 beta -adrenergic receptors and many other GpCRs (15). A G protein-coupled receptor kinase (GRK) phosphorylates activated, usually agonist-occupied, GpCR, creating a binding site for a regulatory protein, beta -arrestin. Binding of beta -arrestin to the GpCR prevents the latter from further binding to its target G protein, thus resulting in GpCR desensitization. In addition, beta -arrestin also serves as an adaptor for clathrin-coated pits. The latter function of the beta -arrestin thus initiates clathrin-mediated endocytosis of the GpCR. We reasoned that overexpression of GRKs and beta -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.

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cDNA Constructs-- The nucleotide sequence encoding full-length rat GRK3 (16) (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 with StuI and ligated into StuI-digested pCS2+ (17) or pCS2+HA (18). 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 (19) and confirmed by DNA sequencing.

A C-terminally truncated GRK, GRK3-Delta C (containing amino acids 1-544 (16)), 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 with StuI. These manipulations resulted in HA-GRK3-Delta C.

Rat beta -arrestin-2 (20) (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 with NcoI (limited digestion was necessary due to the presence of an NcoI within the coding sequence of beta -arrestin-2) and ligated into pCS2+HA that had previously been digested with NcoI. The C-terminal clathrin-binding domain of beta -arrestin-2 (amino acids 319-410) (21) 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 beta -arrestin-2. The resultant plasmid encoded HA-beta -arrestin-C.

In Vitro GRK3 Kinase Assays-- 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 mM NaCl, 1% Triton X-100, 1 µg/ml leupeptin, 100 µM phenylmethylsulfonyl 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 [gamma -32P]ATP, 10 µM ATP, 14 µg each of myelin basic protein and beta -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 (2, 11, 18).

    RESULTS AND DISCUSSION
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To test whether mammalian GRKs can induce frog oocyte maturation, we subcloned rat GRK3 into pCS2+ (17) 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. 2A). Typically, GRK3-induced GVBD lagged several hours behind progesterone-induced GVBD (Fig. 2A).


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Fig. 1.   Schematic representation of plasmid constructs used in this study. Details are presented under "Experimental Procedures." The N-terminal HA tag is not represented here. The boundaries of the identified domains are marked by single-letter codes of amino acids and their positions. PH, pleckstrin homology.


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Fig. 2.   Kinase 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 H1 as an in vitro substrate) (40). D, oocytes injected with water or mRNA for HA-GRK3 or HA-GRK3-K200R were incubated overnight in OR2. Extracts were prepared and subjected to immunoprecipitation with anti-HA monoclonal antibodies. The immune complexes were subjected to in vitro kinase assays using both beta -casein and myelin basic protein (MBP) as substrates (41) (upper panel). An aliquot of the immune complexes were also subjected to immune blotting with anti-HA antibodies (lower panel). Shown are representatives of four independent experiments.

We have previously shown that the non-catalytic C terminus of bovine GRK2 (71% identical to its counterpart in rat GRK3), when engineered with a geranylgeranylation site for membrane attachment, caused GVBD by scavenging endogenous G protein beta gamma complexes (11). The geranylgeranylation site was necessary since the wild type C terminus was ineffective (11). As GRK3 (or GRK2) does not contain a geranylgeranylation site (or any other sites for posttranslational modifications for membrane attachment), it was unlikely that GRK3 functioned as efficient Gbeta gamma scavengers to induce GVBD. Nevertheless, to rule out this possibility, we substituted the catalytically essential lysine residue (Lys220) (16) with arginine (K220R). In contrast to wild type GRK3, GRK3-K220R was completely ineffective in GVBD induction (Fig. 2B). To facilitate biochemical analyses of GRK3 and GRK3-K220R, we engineered an N-terminal HA tag (18) to each of them, generating HA-GRK3 and HA-GRK3-K220R respectively. HA-GRK3 was slightly less efficient than untagged GRK3 in GVBD induction, whereas HA-GRK3-K220R was, as expected, incapable of inducing GVBD (Fig. 2B). 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. Clearly, HA-GRK3 or GRK3, but not their kinase-dead counterparts, induced MOS synthesis and activated both MAP kinase and MPF (Fig. 2C). We carried out immune kinase assays to determine the kinase activities of HA-GRK3 and HA-GRK3-K220R. Anti-HA antibodies pulled down similar amounts of the two proteins in mRNA-injected oocytes (Fig. 2D, lower panel). However, only HA-GRK3 immunoprecipitates contained significantly greater kinase activities when compared with control immunoprecipitates (derived from extracts of water-injected oocytes) (Fig. 2D, upper panel). These results clearly indicate that the kinase activity of GRK3 is essential for GVBD induction and suggest that GRK3 induced GVBD via a mechanism different from scavenging oocyte Gbeta gamma (11).

In intact cells, the abilities of GRKs to phosphorylate their substrates, agonist-occupied GpCRs, are dependent on their membrane association (22). Whereas some GRKs (GRK1, GRK4, and GRK6) are intrinsically membrane-bound because they contain posttranslational lipid attachments, GRK3 and GRK2 need to bind membrane-bound Gbeta gamma complexes (22). To determine whether Gbeta gamma binding is required for GRK3 to induce GVBD, we constructed GRK3-Delta C in which the C-terminal Gbeta gamma -binding domain (amino acids 546-688) (23) had been deleted. Fig. 3A shows that both HA-GRK3 (lane 1) and HA-GRK3-Delta C (lane 2) were expressed efficiently in mRNA-injected oocytes. However, only wild type HA-GRK3 was partially associated with oocyte membranes (Fig. 3A, lane 5), whereas HA-GRK3-Delta C was completely "cytosolic." Following immunoprecipitation, both HA-GRK3 and HA-GRK3-Delta C exhibited in vitro kinase activities, although HA-GRK3-Delta C was less robust in phosphorylating myelin basic protein and beta -casein (Fig. 3B). Despite retaining considerable in vitro kinase activities, HA-GRK3-Delta C was not able to induce any GVBD, nor was it capable of activating any of the protein kinases in frog oocytes (Fig. 3C). These results indicate the essential role of the C-terminal Gbeta gamma -binding domain of GRK in membrane association and in GVBD induction and, therefore, lend further support that GRK3 induced GVBD via phosphorylating a membrane-bound GpCR.


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Fig. 3.   GRK3-Delta C was defective in membrane association and for GVBD induction. A, oocytes injected with mRNA for HA-GRK3 (lanes 1, 3, and 5) or HA-GRK3-Delta C (lanes 2, 4, and 6) were incubated overnight in OR2. Oocytes were lysed in detergent-free homogenization buffer (11) (250 µl for 15 oocyte). Following low speed (900 × g) clarification, the extracts were subjected to centrifugation for 1 h at 100,000 × g. Unfractionated extracts (total, representing one oocyte), the supernatant (cytosol, representing one oocyte), or pellets (membranes, representing 1.5 oocytes) were analyzed by immunoblotting with anti-HA antibodies. Shown is a representative of three independent experiments. B, extracts from the variously injected oocytes were subjected to immune kinase assays as described in Fig. 2D. The upper panel shows a representative immunoprecipitation, as probed by anti-HA immunoblotting. The middle panel shows the corresponding in vitro kinase assays, using both beta -casein and myelin basic protein (MBP) as substrates. The lower panel shows the relative kinase activities of each immunoprecipitate, as determined by phosphorimaging scanning (total phosphorylation of both substrates). C, oocytes injected with water or mRNA for GRK3 or GRK3-Delta C were incubated overnight in OR2. GVBD were scored and expressed as % of total injected oocytes (means with S.D. of four independent experiments). The lower panels show representative analyses for MOS accumulation and MPF assays.

If GRK3-induced GVBD was mediated via phosphorylation and desensitization of an endogenous GpCR, it must work together with an endogenous beta -arrestin protein (15). We therefore wished to test whether overexpression of a mammalian beta -arrestin could also induce GVBD. Rat beta -arrestin-2 cDNA (20) was PCR-amplified and subcloned into pCS2-HA vector (18). Injection of HA-beta -arrestin-2 mRNA caused robust GVBD (Fig. 4A). In contrast, the C-terminal clathrin-binding domain of beta -arrestin-2, HA-beta -arrestin-C, was unable to induce GVBD. HA-beta -arrestin-2-induced GVBD followed a time course very similar to that of GRK3-induced GVBD and both lagged behind progesterone-induced GVBD (Fig. 4B). To determine whether GRK3 and HA-beta -arrestin-2 co-operate in GVBD induction, we injected low concentrations (1 ng per oocyte) of GRK3 or HA-beta -arrestin-2 mRNA alone or in combination. As shown in Fig. 4C, injection of either mRNA alone caused little GVBD, whereas co-injection of both caused significant percentages of GVBD. These results clearly indicate synergism between beta -arrestin-2 and GRK3 in GVBD induction, consistent with the involvement of GpCR desensitization (22).


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Fig. 4.   beta -arrestin-2 induced GVBD. A, groups of 20 or more oocytes were injected with water or mRNA for HA-beta -arrestin-2 or HA-beta -arrestin-C. Following overnight incubation in OR2, GVBD were scored and expressed as % of total injected oocytes. Shown are means with S.D. of three to four independent experiments. B, groups of 50 or more oocytes were incubated in OR2 (control) or OR2 containing progesterone (Pg, 1 µM) or injected with mRNA for GRK3 or HA-beta -arrestin-2. GVBD were scored at the indicated time following the addition of progesterone or the injection of mRNA. Shown is a representative of three independent experiments. C, groups of 30 or more oocytes were injected with water or the indicated mRNA (1 ng per oocyte). Following overnight incubation in OR2, oocytes were scored for GVBD. Shown are means with S.D. of three independent experiments. The sign ** denotes p < 0.05 in pair-wise Student's t test.

If GRK3/beta -arrestin-2 induced GVBD via the desensitization of a cAMP-raising GpCR, we expected that the GVBD induction would be blocked by forskolin. Indeed, incubation of GRK3- or beta -arrestin-2-injected oocytes with forskolin completely blocked GVBD induction (Fig. 5). Upon removal of forskolin (by simply transferring oocyte to fresh OR2) and further incubation, significant percentages of the mRNA-injected oocytes eventually underwent GVBD (Fig. 5).


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Fig. 5.   Forskolin blocked GRK3/beta -arrestin-2-induced GVBD. Oocytes were injected with water or the indicated mRNA. mRNA-injected oocytes were immediately split into two groups. One group was placed in OR2, and the other in OR2 containing forskolin (50 µM). Following overnight incubation, oocytes were scored for GVBD. Shown are means with S.D. of at least three independent experiments. In two experiments, after GVBD scoring, forskolin-treated oocytes were transferred to fresh OR2 and were further incubated for 7-8 h before being scored again for GVBD (indicated by +/- forskolin).

In addition to GpCR desensitization, GRKs and beta -arrestins also promote GpCR endocytosis (15). In particular, beta -arrestins serve to target GpCR to clathrin-coated pits (15). Krupnick et al. (21) have previously demonstrated that the overexpression of the clathrin-binding domain of beta -arrestin blocks GpCR endocytosis. If clathrin-mediated GpCR endocytosis played any role in GRK3/beta -arrestin-2-induced GVBD, HA-beta -arrestin-C should interfere with the GVBD induction. Indeed, injection of beta -arrestin-C mRNA significantly reduced, but did not eliminate, the ability of GRK3 or beta -arrestin-2 to induce GVBD (Fig. 6A). As a control, we injected equal amounts of an mRNA (pCS2+MT (17)) that encoded a similarly sized polypeptide.


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Fig. 6.   Inhibitors of clathrin-pits, but not that of caveoli, reduced GRK3-induced GVBD. A, groups of 90 or more oocytes were injected with a control mRNA (pCS2+MT, coding for six copies of the 13-amino acid Myc tag (17)) or mRNA for HA-beta -arrestin-C. Each of the two groups of oocytes was split and immediately injected with mRNA for either GRK3 or HA-beta -arrestin-2. Oocytes were incubated in OR2 for 6-8 h (when either GRK3- or HA-beta -arrestin-2-injected oocytes had reached maximum GVBD response). All groups were scored for GVBD at the same time. Shown are means with S.D. of three independent experiments. B, oocytes were injected with mRNA for GRK3 or v-Ras and were immediately transferred to OR2 with (+) or without concanavalin A (250 µg/ml). Oocytes were incubated for 6-8 h (when GRK3- or v-Ras injected oocytes had reached maximum GVBD responses). Therefore, the GRK3 groups (lanes 1 and 2) were always scored for GVBD and lysed at the same time. So were the v-Ras groups (lanes 3 and 4). Shown are means with S.D. of three to four independent experiments. The lower panels show representative immunoblots with anti-xMAP kinase or anti-phosphor MAP kinase. C, oocytes were injected with mRNA for GRK3 or v-Ras and were immediately transferred to OR2 or OR2 containing the indicated inhibitors monodansyl cadaverine (MDC, 300 µM) or methyl-beta -cyclondextrin (Mbeta CD, 1%). GVBD scoring and oocyte lysis were performed as described in B. Shown are means with S.D. of three independent experiments. The lower panels show representative immunoblots with anti-xMAP kinase or anti-phosphor MAP kinase.

To further examine the involvement of clathrin-mediated endocytic pathway in GRK3-induced GVBD, we tested two widely used chemical inhibitors. The plant lectin concanavalin A (Con A) binds cell-surface glycoproteins and interferes with the formation of clathrin-coated pits (24). Treatment of cells with Con A blocks beta -adrenergic receptor endocytosis without affecting G protein activation and activation of the second messenger cAMP (25). Indeed, treatment of oocytes with Con A significantly reduced, but did not eliminate, the ability of GRK3 to induce GVBD (Fig. 6B, lane 2). Another inhibitor for clathrin-pits is the polyamine monodansyl cadaverine (MDC). Unlike Con A, which acts extracellularly, MDC acts intracellularly to block clathrin polymerization (26). As shown in Fig. 6C, MDC similarly reduced GRK3-induced GVBD. To rule out general toxicity or other nonspecific effects of the inhibitors in oocytes, we tested the effects of these inhibitors on v-Ras-induced GVBD. Although the mechanism by which v-Ras induces GVBD remains controversial (27), it is unlikely that it would require receptor endocytosis. Indeed, neither Con A (Fig. 6B) nor MDC (Fig. 6C) had any inhibitory effect on v-Ras-induced GVBD. To further strengthen the argument that Con A and MDC inhibited GRK-induced GVBD through blockade of clathrin-mediated GpCR endocytosis, we tested the cholesterol-binding agent methyl-beta -cyclodextrin (28), which inhibits caveolae-mediated endocytosis (29). Methyl-beta -cyclodextrin had no effect on GRK3- or v-Ras-induced GVBD (Fig. 6C), suggesting that caveoli are not involved in GRK3-induced GpCR endocytosis.

Recent studies have clearly indicated that G proteins play critical roles in maintaining meiosis arrest in both amphibian (11, 12, 14) and mouse (13) oocytes. However, the identity of the G proteins or whether these G proteins are regulated by classical GpCRs remains unclear. For example, receptors with intrinsic protein tyrosine kinase activities have been implicated in regulating G proteins (18, 30-32), with direct binding of the receptors to the alpha  subunits of the G proteins (33, 34). GRKs are highly specialized protein kinases that preferentially phosphorylate activated (agonist-occupied), as opposed to inactive or antagonist-occupied, GpCRs (22). That GRK3 was able to induce GVBD therefore represents the first and compelling experimental support for the notion that prophase oocytes contain an active GpCR and that this GpCR is responsible for maintaining meiosis arrest (11, 13).

Recent studies have indicated that GRKs are able to phosphorylate non-GpCR proteins. Tubulin (35) and synuleins (a class of low molecular weight proteins of unknown biological functions) (36) can be phosphorylated by GRK2 and GRK3. However, the physiological significance of these phosphorylation events remains unknown. Another example of non-GpCR substrate is the Na(+)/H(+) exchanger regulatory factor, which is specifically phosphorylated by a splicing variant of GRK6 (GRK6A) but not by other GRK6 variants or other GRKs (37).

We believe that GRK3-induced GVBD involves phosphorylation of a GpCR, rather than any of these non-GpCR substrates. First, GRK3-induced GVBD required membrane association of the kinase mediated by the C-terminal Gbeta gamma -binding domain, consistent with the involvement of a membrane-bound GpCR. Second, GRK3-induced GVBD was blocked by forskolin, indicating the involvement of inhibition of cAMP signaling pathway. Third, both GRK3 and beta -arrestin-2 are potent GVBD inducers and act synergistically, consistent with the involvement of GpCR desensitization. Furthermore, several inhibitors of clathrin-mediated endocytosis significantly reduced the abilities of GRK3/beta -arrestin-2 to induce GVBD, supporting a role of GpCR endocytosis in GVBD induction. However, as only partial inhibition was observed with these inhibitors (Fig. 6, A-C), it appears that clathrin-mediated endocytosis may not be absolutely required for GVBD induction. In other words, GpCR desensitization caused by GRK3/beta -arrestin-2 may be sufficient to cause GVBD. In any event, these data strongly support the existence of an activated GpCR that is responsible for maintaining prophase arrest.

As GRK3 and beta -arrestin-2 are known to cause desensitization of multiple GpCR in mammalian cells (15), the data presented here do not by themselves suggest any specific GpCR as the oocyte target. However, the beta -arrestin-2 constructs should provide a valuable tool to identify this putative oocyte meiosis inhibitor (11).

The results presented here raise the interesting question of whether progesterone induces GVBD by activating endogenous GRK-mediated GpCR desensitization/endocytosis. We have attempted to determine whether progesterone can modulate HA-GRK3 activities by immune kinase assays. These attempts have not yielded consistent results, perhaps because the "active" state of GRK3 (e.g. Gbeta gamma binding and/or membrane association) only exists in intact cells and cannot be maintained following cell lysis and immunoprecipitation. However, we have isolated two partial cDNAs,2 one most similar to mammalian GRK2 and the other to GRK3, from a Xenopus oocyte cDNA library (38) by PCR amplification using degenerate primers based on conserved GRK sequences (39). Further work will be required to clarify the possible role of these endogenous GRKs in regulating progesterone-induced oocyte maturation.

    ACKNOWLEDGEMENTS

We thank Drs. Robert J. Lefkowitz and Mario Tiberi for various cDNA constructs.

    FOOTNOTES

* This work was supported by operating grants (to X. J. L.) from the National Cancer Institute of Canada and National Science and Engineering Research Council of Canada.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

|| Recipient of Premier's Research Excellent Awards. To whom correspondence should be addressed: Ottawa Health Research Institute, 725 Parkdale Ave., Ottawa, K1Y 4E9, Canada. Tel.: 613-798-5555 (ext. 17752); Fax: 613- 761-5411 or 613-761-5365; E-mail: jliu@ohri.ca.

Published, JBC Papers in Press, February 24, 2003, DOI 10.1074/jbc.M300320200

2 J. Wang and X. J. Liu, unpublished data.

    ABBREVIATIONS

The abbreviations used are: MPF, maturation promoting factor; GpCR, G protein-coupled receptor; GVBD, germinal vesicle breakdown; GRK, G protein-coupled receptor kinase; HA, hemagglutinin; MAP, mitogen-activated protein; Con A, concanavalin A; MDC, monodansyl cadaverine..

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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