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INTRODUCTION |
Signaling via G-protein-coupled receptors
(GPCRs)1 is subject to a
variety of regulatory processes. One of the key regulatory mechanisms
is the control of receptor function by G-protein-coupled receptor
kinases (GRKs). These kinases phosphorylate the agonist-bound state of
GPCRs, and this phosphorylation is often the initial step in homologous
receptor desensitization, the loss of receptor responsiveness upon
repeated or prolonged receptor stimulation (1, 2). To date, six members
of the GRK family are known (3, 4). Of these, GRK2 (previously called
-adrenergic receptor kinase-1) as well as GRK3, GRK5, and GRK6 are
widely expressed throughout the mammalian body whereas GRK1 (also known
as rhodopsin kinase) and GRK4 have been found only in specific tissues.
All GRKs have a similar molecular architecture. A central catalytic
domain is flanked by an N- and C-terminal domain. The function of the
latter is to provide a membrane anchor, which seems to be essential for
receptor phosphorylation (5). The C terminus of GRK1 is farnesylated,
whereas GRK2 and GRK3 possess a pleckstrin homology domain implicated
in phosphatidylinositol bisphosphate and G-protein 
-subunit
binding. GRK4 and GRK6 are palmitoylated, and GRK5 binds phospholipids
through a poorly defined polybasic domain (3, 4). The function of the N
terminus is less clear. It has been shown for GRK1 that antibodies
against an epitope in the N terminus prevent phosphorylation of
light-activated rhodopsin but not of a peptide substrate (6). However,
the epitope against which the antibodies were directed is poorly
conserved in the GRK family. Recent results have indicated that the N
terminus of GRK2 and GRK3 contains an RGS (regulator of G-protein
signaling) domain that specifically binds to and activates
G
q (7, 8). In addition, the extreme N terminus of GRK2
and GRK5 contains a binding site for Ca2+/calmodulin.
Binding of Ca2+/calmodulin to this site causes an
inhibition of GRK activity, and this may represent a mechanism for the
regulation of GRKs (9-11).
In addition to GRKs, other kinases have been found to phosphorylate
GPCRs. The most prominent kinases capable of phosphorylating receptors
are the protein kinases A and C (PKC), which are activated by the
second messengers generated after stimulation of these receptors.
Recent studies have identified cross-talk between PKC and GRKs, which
may provide another level of regulation of GRKs. PKC has been shown to
phosphorylate GRK2 (12, 13) and GRK5 (14). Whereas phosphorylation of
GRK5 by PKC attenuated its activity (14, 15), PKC-phosphorylated GRK2
caused significantly enhanced receptor phosphorylation and
desensitization (12, 13). This enhancement was at least partially
caused by a PKC-mediated stimulation of GRK2 translocation from the
cytosol to the plasma membrane where the substrate receptors are
located (13). The molecular mechanisms of this activation have,
however, not been resolved. Hence, we have investigated the exact
nature of the phosphorylation and regulation of GRK2 by PKC. We report
here that phosphorylation of GRK2 by PKC occurs in the N terminus and that this phosphorylation abolishes the inhibitory effect of
Ca2+/calmodulin on GRK2 activity.
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EXPERIMENTAL PROCEDURES |
Materials--
Crude phosphatidylcholine (type II-S from
soybean) and calmodulin (from bovine testes, activity at least 40,000 units/mg protein) were from Sigma. The GRK2 antiserum has been
described previously (13).
Cell Culture--
Cells were cultured in Dulbecco's modified
Eagle's medium supplemented with 10% fetal calf serum and antibiotics
in a humidified incubator with 7% CO2. HEK293 cells were
transfected with a modified calcium phosphate method (16). COS cells
were transfected using DEAE-dextran (17). Transiently transfected cells
were analyzed ~48 h after transfection.
Mutagenesis--
The construction of the cDNA coding for GST
fused to human GRK2-(552-689) has already been described (13).
cDNAs coding for the N terminus (amino acids 1-185) or the
pleckstrin homology domain (amino acids 552-656) of human GRK2 were
amplified from the full-length cDNA by PCR with Pfu polymerase
(Stratagene) and cloned into the vector pGEX1
T (Amersham Pharmacia
Biotech) for expression as GST fusion proteins (18). Point mutations on
human GRK2 were introduced by the method of Kunkel (19). All constructs were verified by automated sequencing.
Purification of Proteins--
G-protein 
-subunits were
purified from bovine brain according to Sternweis and Robishaw (20).
GST fusion proteins were expressed in Escherichia coli BL21
and purified by affinity chromatography as described previously (13).
Human and bovine GRK2 were expressed in baculovirus-infected Sf9
cells and purified according to Kim et al. (21). PKC
isozymes were purified from baculovirus-infected Sf9 cells as
described previously (22). Protein concentrations were determined
according to Bradford (23).
Determination of PKC and GRK2 Activity--
PKC activity was
determined using a soluble peptide substrate,
[Ser25]PKC
-(19-31) as described previously (22). GRK2
activity was usually measured by using urea-treated rod outer segments
(50 pmol of rhodopsin) as the substrate in the presence of 50 nM purified G-protein 
-subunits as described
previously (24). Incubation was carried out at 30 °C for 8 min.
32P incorporation into rhodopsin was determined by
SDS-polyacrylamide gel electrophoresis of the reaction mixture,
excision of the rhodopsin band, and Cerenkov counting or quantification
with a phosphorimager. To assess phosphorylation of soluble substrates
by GRKs, 16 pmol of a GST fusion protein of the C terminus of the
parathyroid hormone receptor were incubated with GRK2 for 30 min at
30 °C. For investigation of the effects of calmodulin and/or PKC on
GRK2 activity, GRK2 was preincubated with 70 units of calmodulin and/or
25 pmol of PKC for 30 min at 30 °C in the dark before adding rod
outer segments. PKC was activated with 10 µM PMA.
Phosphorylation of Recombinant Proteins by
PKC--
Approximately 3 pmol of purified PKC were incubated with the
indicated amounts of recombinant proteins (GRK2 or GRK2 fragments) in
the presence of 4 µg of crude phosphatidylcholine vesicles in 20 mM Tris/HCl, pH 7.2, 2 mM EDTA, 8 mM MgCl2, 50 µM
CaCl2, 1 µM PMA, 100 µM
[
-32P]ATP (~500 cpm/pmol) for 45 min at 30 °C.
Proteins were resolved by SDS-polyacrylamide gel electrophoresis and
visualized by autoradiography.
Mass Spectrometry--
200 ng of purified GRK2 were
phosphorylated with 10 ng of recombinant PKC
in the presence of 20 µM [
-32P]ATP (1 Ci/mmol). The mix was
separated by SDS-polyacrylamide gel electrophoresis, and the
phosphorylated band was identified using autoradiography. It was then
excised from the gel and digested with endopeptidase LysC, and the
peptide mixture was separated by reverse-phase HPLC coupled to a
radiodetector and an API 100 quadrupole mass spectrometer as described
previously (25).
Two-dimensional Peptide Mapping--
GRK2 was phosphorylated and
digested as above. Labeled peptides were analyzed by two-dimensional
phosphopeptide mapping as described (26). The experiment was also
performed with a synthetic peptide comprising residues 7-34 of GRK2
with identical results.
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RESULTS |
PKC Isoform Specificity for GRK2 and GRK5--
It has been
previously shown that crude preparations of PKC purified from rat or
bovine brain are capable of phosphorylating GRK2 and GRK5 (12-14). To
examine which of the PKC isoforms were responsible for phosphorylation
of GRKs, we analyzed PKC preparations from baculovirus-infected
Sf9 cells for their ability to phosphorylate GRK2 and GRK5. The
activity of these fractions was calibrated with histone H1 as a
substrate. Equal amounts of the various PKC isoforms were then used to
phosphorylate purified GRK2 and GRK5 (Fig.
1). Whereas GRK5 was quite a good
substrate for all PKC isoforms tested except for PKC
, GRK2 was only
significantly phosphorylated by PKCs
,
, and
. The
radioactivity incorporated into GRKs in the absence of PKC (Fig. 1,
A and B, right lanes) is caused by
autophosphorylation. Because GRK2 is selectively phosphorylated by only
certain PKC isoforms, whereas, with the exception of PKC
, GRK5
serves as substrate for all PKCs, these data suggest that the two types
of phosphorylation might be different. Because of their higher activity
toward GRK2, all subsequent experiments were done with recombinant
PKC
or PKC
.

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Fig. 1.
PKC isoenzyme specificity for GRKs 2 and
5. The activities of different PKC isoenzymes were standardized
using the peptide substrate [Ser25]PKC -(19-31).
Equivalent amounts were then used to phosphorylate 10 pmol of purified
GRK2 (A) or GRK5 (B). Shown are the results from
a representative experiment, which was repeated three times with
similar results. Phosphorylation in the absence of GRK is a result of
autokinase activity.
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Identification of the PKC Phosphorylation Site on GRK2--
We
had shown previously that GRK2 could be phosphorylated by rat brain PKC
preparations to a stoichiometry of about one, suggesting a single
phosphorylation site on GRK2. To verify the existence of a single
phosphorylation site, we performed two-dimensional phosphopeptide
mapping of GRK2. GRK2 was phosphorylated in the presence of
[
-32P]ATP with PKC
, digested with endopeptidase
LysC, and the peptides resolved by two-dimensional electrophoresis.
Only a single radiolabeled spot derived from GRK2 was identified,
indicating the presence of a single phosphorylation site (Fig.
2).

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Fig. 2.
Two-dimensional phosphopeptide mapping of
PKC -phosphorylated GRK2. GRK2 was
phosphorylated in vitro by PKC and cleaved with LysC. The
resulting peptide mixture was separated by two-dimensional
phosphopeptide mapping, and the phosphopeptides were visualized by
autoradiography. A, PKC-phosphorylated GRK2; B,
autophosphorylated GRK2; C, mixture of PKC-phosphorylated
and autophosphorylated GRK2.
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To map this phosphorylation site, we performed HPLC-MS analysis of the
LysC-digested peptides. The peptide containing the transferred
phosphate was detected online with a radiodetector, and its mass was
directly analyzed by electrospray-MS (Fig.
3). The major peak of radioactivity was
identified as (AT)PAARASK based on the mass of the peptide and
the fragmentation spectrum. This region corresponds to residues 22-30
of human GRK2 and identifies Ser29 as the single
phosphorylation site for PKC in GRK2.

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Fig. 3.
HPLC-MS analysis of PKC-phosphorylated
GRK2. 200 ng of purified GRK2 were phosphorylated in
vitro with PKC and 20 µM
[ -32P]ATP. The labeled protein was digested with LysC,
and the resulting peptides analyzed by HPLC coupled to a radiodetector
and an API 100 quadrupole mass spectrometer. The upper panel
shows the total ion current and the middle panel represents
the detection profile of the radiocounter. The delay between
radiodetection and MS was ~4.6 min as indicated by the two
lines. The lower panel shows the mass spectrum of the
peak at 32.1 min. Despite weak signals, sequence information of the
peptide with m/z = 872.4 could be generated
by front-end fragmentation. The peak at 58.6 min (middle
panel) contains partially digested protein.
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To confirm this finding, a synthetic peptide comprising residues 7-34
of human GRK2 was synthesized, phosphorylated with PKC
, digested
with endopeptidase LysC, and subjected to two-dimensional peptide
mapping as described above. The resulting phosphopeptide pattern was
identical to the one obtained with full-length GRK2, as shown in
separate or overlay analyses (Fig. 4).
These data corroborate the correct identification of the
phosphorylation site.

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Fig. 4.
Comparison of two-dimensional phosphopeptide
maps of PKC -phosphorylated GRK2 and a
PKC -phosphorylated peptide comprising residues
22-30. A, GRK2. B, peptide ATPAARASK.
C, GRK2 + peptide.
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Ser29 Is the PKC Phosphorylation Site in Vitro and in
Vivo--
To verify that Ser29 is indeed the site
phosphorylated by PKC, we constructed a mutant of human GRK2 in which
this residue was replaced with alanine (S29A). This mutant was
expressed in Sf9 cells using the baculovirus system and purified
in a way identical to the wild-type protein. Purified S29A GRK2 was
able to phosphorylate rhodopsin in a manner similar to the wild-type
protein although its specific activity was lower (data not shown). As
expected, PKC
was unable to phosphorylate the S29A GRK2, in contrast
to wild-type GRK2 (Fig. 5). These data
confirm that Ser29 is indeed the only phosphorylation site
for PKC in GRK2.

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Fig. 5.
Phosphorylation of purified wild-type GRK2
(WT) or mutant GRK2 (S29A) with
PKC. 50 pmol of GRK2 were phosphorylated with 125 pmol of PKC in a
volume of 40 µl for 30 min at 30 °C, and proteins were separated
by SDS-polyacrylamide gel electrophoresis. PKC and GRK2 have an
identical apparent molecular weight on SDS-polyacrylamide gels.
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Phosphorylation of Ser29 in GRK2 Abolishes the
Inhibitory Effect of Calmodulin--
We and others have shown
previously that phosphorylation of GRK2 with PKC purified from bovine
or rat brain leads to increased GRK2 activity on rhodopsin (12, 13).
Surprisingly, when we tried to reproduce these findings with PKC
or
PKC
purified from Sf9 cells, we obtained no stimulation.
Instead, we observed a slight nonsignificant reduction of GRK2 activity
after PKC phosphorylation (Fig. 6,
left).

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Fig. 6.
Activity of bovine GRK2 in the presence of
PKC and/or calmodulin. GRK2 was preincubated with 25 pmol of PKC
and/or 70 units of calmodulin for 30 min at 30 °C in the dark.
Rhodopsin was added, and the reaction was continued for another 8 min
under light. Proteins were separated by SDS-polyacrylamide gel
electrophoresis, and 32P incorporation into rhodopsin was
measured by excising the rhodopsin band from the gel and Cerenkov
counting. Data are the mean ± S.E. from three experiments.
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This discrepancy between our data suggests that the brain PKC
preparations might have contained additional proteins that influence the activity of GRK2. One such protein might be calmodulin. Calmodulin has been shown to attenuate GRK2 activity, and the calmodulin binding
site encompasses Ser29 of GRK2 (11). To investigate whether
calmodulin could in fact be the postulated "missing protein," we
examined the influence of calmodulin on GRK2 activity.
Calmodulin inhibited the activity of bovine GRK2 by more than 75% as
measured by the GRK2-mediated phosphorylation of light-activated rhodopsin (Fig. 6, right). This inhibition was completely
reversed when active PKC was present in the assay (Fig. 6,
right). These data show that PKC activates GRK2 by relieving
the Ca2+/calmodulin-mediated inhibition.
Mutation of Ser29 Abolishes PKC-mediated Activation of
GRK2 in Intact Cells--
Finally we wanted to verify that
Ser29 represents the functionally relevant PKC
phosphorylation site in intact cells. To achieve this, HEK293 cells
were transiently transfected with wild-type or S29A mutant GRK2. When
the cells were stimulated with PMA, cells transfected with wild-type
GRK2 showed a roughly 2-fold increase in cytoplasmic GRK activity (Fig.
7, left). In the case of the
S29A mutant, this stimulation of GRK activity by PMA was almost
completely abolished (Fig. 7, right).

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Fig. 7.
Activation of GRK2 by PMA in intact
cells. HEK293 cells transfected with 0.5 µg of wild-type GRK2
(WT, left) or S29A (right) plasmid
were incubated with 1 µM PMA or vehicle for 20 min. Cells
were then lysed, cytosol was prepared, and GRK2 activity was measured
using rhodopsin as the substrate as described under "Experimental
Procedures." Shown are the mean ± S.E. from three experiments.
The inset shows a representative experiment. The amounts of
transfected GRK2 were roughly equivalent as determined by Western
blotting (data not shown).
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The residual effects of PMA seen under these conditions are probably
caused by endogenous wild-type GRK2. These results indicate that
PKC-mediated phosphorylation of GRK2 at Ser29 does indeed
cause its activation in intact cells by relieving a constitutive
inhibition through calmodulin or a related protein.
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DISCUSSION |
The function and activity of G-protein-coupled receptors is
controlled by several classes of proteins via a multitude of
mechanisms. A general feature of these mechanisms is that they are
interrelated and that they appear to be controlled at several different
levels. A particularly intriguing example of this complex control is
the inhibition of receptor activity by GRKs, which is itself subject to
control by PKC. Whereas GRK5 activity toward receptors was decreased
following phosphorylation by PKC (14), GRK2 activity toward small
soluble receptor substrates was also decreased by PKC, but its activity
versus membrane-bound receptors was increased (12, 13).
These data suggested that PKC might activate GRK2 by increasing its
association with membrane-bound receptors (13).
PKC phosphorylation studies utilizing a protein comprised of the C
terminus of GRK2 fused to GST had initially suggested the presence of a
C-terminal phosphorylation site in GRK2 (13). In the present study,
using intact GRK2 and purified recombinant PKC isozymes, we have mapped
the PKC phosphorylation site in GRK2 unequivocally to serine 29 in the
N terminus, and we can exclude phosphorylation of the GRK2 C terminus
by PKC. We have further shown that PKC phosphorylation of GRK2 does not
directly stimulate the activity of the isolated GRK2 in
vitro.
On the other hand, our previous experiments clearly indicated that
phosphorylation of GRK2 with PKC preparations obtained from bovine
brain caused an ~2.5-fold increase in the incorporation of phosphate
into rhodopsin (13). Similarly, Chuang et al. (12) also
observed activation of GRK2 after phosphorylation with a PKC
preparation purified from bovine brain.
As shown here, we were unable to reproduce these results using PKC
isozymes purified from baculovirus-infected Sf9 cells. These
discrepancies raised the possibility that the GRK2 activation observed
after phosphorylation with PKC might be the result of additional
protein(s) present in the brain PKC preparations, but not in the PKCs
purified from Sf9 cells. One candidate for such a protein was
calmodulin, which has previously been shown to inhibit GRK2 activity
(10, 11). Indeed, purified recombinant GRK2 was inhibited by
calmodulin, and this inhibition could be fully reversed when GRK2 was
phosphorylated by PKC. We and others have previously reported that PKC
activation of GRK2 also occurs in intact cells (12, 13). Here, we show
that removal of the PKC phosphorylation site on GRK2 also abolishes
GRK2 activation through PKC. This indicates that a pool of GRK2 in the
cell may be tonically inhibited by forming a complex with calmodulin or
a related protein from which it is released through PKC phosphorylation.
Whereas our experiments offer a mechanistic explanation for the
phenomenon of GRK2 activation by PKC, they also raise several questions. First, how does calmodulin inhibit GRK2, and how is this
inhibition altered following phosphorylation of GRK2 by PKC? It is
conceivable that calmodulin competes with the agonist-activated receptor for GRK2. This suggestion is supported by findings obtained with GRK1 (rhodopsin kinase) and rhodopsin. An antiserum directed against amino acids 17-34 of GRK1 was able to inhibit phosphorylation of light-activated rhodopsin but not of a peptide substrate (6). Interestingly, this region of GRK1 is also able to bind rhodopsin, as
is the corresponding region of GRK5 (11). Furthermore, N-terminal truncation of GRKs 2 and 5 impairs their ability to phosphorylate rhodopsin (27). These data indicate that the N terminus of GRKs might
be involved in binding to the receptor substrates. The sequence homology of the calmodulin-binding sites in GRK2 and GRK5 is, however,
quite low.
The proximal C terminus of GRK2 (residues 457-546 of 689) has been
implicated in receptor binding as well (28). This suggests that both C-
and N-terminal regions of GRK are involved in receptor association. The
C terminus has been shown to interact with light-activated rhodopsin by
itself, suggesting that it may be sensitive to the conformational
changes occurring in G-protein-coupled receptors upon activation (28).
In contrast, truncation of the N terminus does not abolish the ability
of GRKs to be activated by agonist-occupied receptors (27). It
therefore appears that the N terminus is not involved in sensing the
activated state of the receptor but may contribute some weak
interaction that is required for efficient substrate recognition.
Evidently, additional experiments are necessary to prove this hypothesis.
Another question is how phosphorylation of GRK2 by PKC affects its
regulation by Ca2+/calmodulin. Such inhibition might be
because of a reduction in direct binding affinity (measured to be
around 40 nM, see Ref. 11), but it might also be more
complex and involve other regions of the GRKs. (A second calmodulin
binding domain has been mapped to residues 466-689 of GRK2, Ref. 11.)
Such a more complex interaction is suggested by the fact that
phosphorylation of GRK2 by PKC (as well as of GRK5) has a direct
inhibitory effect on the activity of the catalytic core region of GRKs
if, instead of a membrane-bound receptor, a soluble substrate was
phosphorylated. This was either a peptide (13, 14) or the soluble C
terminus of the parathyroid hormone receptor (data not shown).
It is remarkable that GRK2 seems to possess a special versatility for
regulating the activity of Gq-coupled receptors. It is
inhibited by Ca2+/calmodulin and thereby regulated by
Gq-mediated alterations in intracellular free
Ca2+-levels. This inhibition is released through PKC
activity, which can also be controlled via Gq. In addition,
GRK2 contains an RGS domain, which leads to fairly selective inhibition
of Gq-mediated signal transduction pathways (7, 8). The
reasons for this specialization of GRK2 are at the moment completely
unclear, in particular because GRK2 was initially thought to be
particularly active toward the Gs-coupled
-adrenergic receptors.
Taken together, our data delineate a previously unknown mechanism of
regulation of G-protein-coupled receptors, which is exerted by an
interplay of PKC and Ca2+/calmodulin in regulating the
activity of GRK2. These observations are a further step in the complex
machinery regulating the function of these receptors.