From the 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
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ABSTRACT |
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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 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 Gs 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 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-
Rat 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
[ 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).
-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-
C), which lacked the
C-terminal G
-binding domain and which
was not associated with oocyte membranes, also failed to induce GVBD. Furthermore, injection of rat
-arrestin-2 mRNA also induced
hormone-independent GVBD. Several inhibitors of clathrin-mediated
receptor endocytosis (the clathrin-binding domain of
-arrestin-2,
concanavalin A, and monodansyl cadaverine) significantly reduced the
abilities of GRK3/
-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.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
causes oocyte maturation,
suggesting that endogenous Xenopus Gs
plays a
dominant role in maintaining prophase arrest. On the other hand, we
demonstrated that inhibition of endogenous G protein
subunits
(via injection of G
scavengers) lowers oocyte cAMP
and induces oocyte maturation (11). Furthermore, 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).
-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,
-arrestin. Binding of
-arrestin to the GpCR
prevents the latter from further binding to its target G protein, thus
resulting in GpCR desensitization. In addition,
-arrestin also
serves as an adaptor for clathrin-coated pits. The latter function of
the
-arrestin thus initiates clathrin-mediated endocytosis of the
GpCR. We reasoned that overexpression of GRKs and
-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.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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-
C.
-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
-arrestin-2) and
ligated into pCS2+HA that had previously been digested with
NcoI. The C-terminal clathrin-binding domain of
-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
-arrestin-2. The resultant plasmid encoded HA-
-arrestin-C.
-32P]ATP, 10 µM ATP, 14 µg each of
myelin basic protein and
-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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
View larger version (18K):
[in a new window]
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.
View larger version (28K):
[in a new window]
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 -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 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 G
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 G
(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
G complexes (22). To determine whether
G
binding is required for GRK3 to induce GVBD, we
constructed GRK3-
C in which the C-terminal
G
-binding domain (amino acids 546-688) (23) had been
deleted. Fig. 3A shows that
both HA-GRK3 (lane 1) and HA-GRK3-
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-
C was completely
"cytosolic." Following immunoprecipitation, both HA-GRK3 and
HA-GRK3-
C exhibited in vitro kinase activities, although HA-GRK3-
C was less robust in phosphorylating myelin basic protein and
-casein (Fig. 3B). Despite retaining considerable
in vitro kinase activities, HA-GRK3-
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 G
-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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If GRK3-induced GVBD was mediated via phosphorylation and
desensitization of an endogenous GpCR, it must work together with an
endogenous -arrestin protein (15). We therefore wished to test
whether overexpression of a mammalian
-arrestin could also induce
GVBD. Rat
-arrestin-2 cDNA (20) was PCR-amplified and subcloned
into pCS2-HA vector (18). Injection of HA-
-arrestin-2 mRNA
caused robust GVBD (Fig. 4A).
In contrast, the C-terminal clathrin-binding domain of
-arrestin-2,
HA-
-arrestin-C, was unable to induce GVBD. HA-
-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-
-arrestin-2 co-operate in GVBD
induction, we injected low concentrations (1 ng per oocyte) of GRK3 or
HA-
-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
-arrestin-2 and
GRK3 in GVBD induction, consistent with the involvement of GpCR
desensitization (22).
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If GRK3/-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
-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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In addition to GpCR desensitization, GRKs and -arrestins also
promote GpCR endocytosis (15). In particular,
-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
-arrestin blocks GpCR endocytosis. If
clathrin-mediated GpCR endocytosis played any role in
GRK3/
-arrestin-2-induced GVBD, HA-
-arrestin-C should interfere
with the GVBD induction. Indeed, injection of
-arrestin-C mRNA
significantly reduced, but did not eliminate, the ability of GRK3 or
-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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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 -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-
-cyclodextrin (28), which inhibits
caveolae-mediated endocytosis (29). Methyl-
-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 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
G-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
-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/
-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/
-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 -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
-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. G 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.
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ACKNOWLEDGEMENTS |
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We thank Drs. Robert J. Lefkowitz and Mario Tiberi for various cDNA constructs.
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FOOTNOTES |
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* 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.
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ABBREVIATIONS |
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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..
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