The Amino-terminal Domain of G-protein-coupled Receptor Kinase 2 Is a Regulatory G
Binding Site*
Tanja
Eichmann,
Kristina
Lorenz,
Michaela
Hoffmann,
Jörg
Brockmann,
Cornelius
Krasel,
Martin J.
Lohse, and
Ursula
Quitterer
From the Institut für Pharmakologie und Toxikologie,
Versbacher Strasse 9, D-97078 Würzburg, Germany
Received for publication, May 16, 2002, and in revised form, November 25, 2002
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ABSTRACT |
G-protein-coupled receptor kinase 2 (GRK2) is
activated by free G
subunits. A G
binding site of GRK2 is
localized in the carboxyl-terminal pleckstrin homology domain. This
G
binding site of GRK2 also regulates G
-stimulated
signaling by sequestering free G
subunits. We report here that
truncation of the carboxyl-terminal G
binding site of GRK2 did
not abolish the G
regulatory activity of GRK2 as determined by
the inhibition of a G
-stimulated increase in inositol phosphates
in cells. This finding suggested the presence of a second G
binding site in GRK2. And indeed, the amino terminus of GRK2
(GRK21-185) inhibited a G
-stimulated
inositol phosphate signal in cells, purified
GRK21-185 suppressed the G
-stimulated
phosphorylation of rhodopsin, and GRK21-185 bound directly
to purified G
subunits. The amino-terminal G
regulatory
site does not overlap with the RGS domain of GRK-2 because
GRK21-53 with truncated RGS domain inhibited
G
-mediated signaling with similar potency and efficacy as did
GRK21-185. In addition to the G
regulatory activity,
the amino-terminal G
binding site of GRK2 affects the kinase
activity of GRK2 because antibodies specifically cross-reacting with
the amino terminus of GRK2 suppressed the GRK2-dependent
phosphorylation of rhodopsin. The antibody-mediated inhibition was
released by purified G
subunits, strongly suggesting that G
binding to the amino terminus of GRK2 enhances the kinase activity
toward rhodopsin. Thus, the amino-terminal domain of GRK2 is a
previously unrecognized G
binding site that regulates
GRK2-mediated receptor phosphorylation and inhibits G
-stimulated signaling.
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INTRODUCTION |
Activated G-protein-coupled receptors are switched off by
phosphorylation through G-protein-coupled receptor kinases
(GRKs)1 (1). GRKs are modular
proteins consisting of at least three structural domains with different
functions. The core kinase domain of GRK2 and GRK3, which represents
the
-adrenergic receptor kinase isozymes, is flanked by an
amino-terminal domain, which contains an RGS domain, and a
carboxyl-terminal domain, which contains a pleckstrin homology domain
(PH domain) (2-4). The activation of GRK2 and GRK3 requires the
activation and dissociation of a heterotrimeric G-protein,
i.e. the kinases are activated by free G
subunits (5,
6). A G
binding site of GRK2 and GRK3 is localized in the
carboxyl terminus of the kinase and overlaps the PH domain (7).
Truncation of the PH domain of GRK2 generates a kinase with compromised
regulation by G
subunits (7). The carboxyl-terminal G
binding site of GRK2 also regulates G
-stimulated signaling by
sequestering free G
subunits (8). Analyzing the G
regulatory activity of proteins is a means of identifying G
-binding proteins or localizing G
binding sites of
proteins (9-11). To find out whether the G
regulatory activity
of GRK2 resides entirely in the carboxyl-terminal PH domain, we
analyzed the G
-sequestering activity of wild-type GRK2 and of
carboxyl-terminal-truncated GRK2 mutants. The capacity of those
proteins to inhibit a G
-stimulated increase in inositol
phosphates mediated by activation of phospholipase C
2
was determined (12). We report here that truncation of the carboxyl-terminal G
binding site of GRK2 did not abolish the G
regulatory activity of GRK2. A previously unrecognized G
binding site of GRK2 was identified in the amino terminus of GRK2 that
enhances the kinase activity of GRK2 toward the receptor substrate
rhodopsin and which inhibits G
-stimulated signaling.
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EXPERIMENTAL PROCEDURES |
Cell Culture and Cell Transfection--
Human embryonic kidney
cells (HEK-293) were cultured and transfected as described previously
(13) with plasmids encoding human GRK2 or the indicated truncation
mutants of GRK2 or GRK5. The mutants were generated by polymerase chain
reaction and were sequenced entirely to confirm the identity of the mutants.
Determination of Cellular Inositol Phosphate Levels--
Total
inositol phosphate levels of HEK-293 cells were determined as described
(13). For determination of the G
regulatory activity of wild-type
GRK2 and of the different truncation mutants, cells were co-transfected
with plasmids encoding the indicated GRK2 mutants and phospholipase
C
2, G
1, and G
2 (11).
Protein Purification of GRK2--
Human GRK2 was expressed in
Sf9 cells using a recombinant baculovirus. GRK2 was purified by
SP- Sepharose and heparin-Sepharose according to established
protocols (14, 15). The amino-terminal domain of GRK2
(GRK21-185), GRK21-53,
GRK254-185, and GRK51-200 were expressed
in Escherichia coli as glutathione S-transferase (GST) fusion proteins and purified by affinity chromatography on
glutathione-Sepharose 4B according to the manufacturer's protocol (Amersham Biosciences).
Phosphorylation of Rhodopsin by GRK2--
The kinase activity of
GRK2 was assessed by phosphorylation of the receptor substrate
rhodopsin in a total volume of 50 µl of buffer (20 mM
HEPES, pH 7.4) containing 20 nM GRK2, G
subunits as
indicated, 400 nM rhodopsin, 10 mM
MgCl2, 2 mM EDTA, and 50 µM
[
-32P]ATP. Phosphorylation was initiated by light and
proceeded for 20 min at room temperature. After SDS-PAGE, receptor
phosphorylation was assessed by autoradiography. Rhodopsin-enriched
membranes were prepared from dark-adapted bovine retinae by sucrose
gradient centrifugation (14).
To determine the activation of GRK2 by G
subunits, various
concentrations of purified G
subunits from bovine brain were incubated in the phosphorylation mixture (16). To assess the effect of
antibodies specifically cross-reacting with the amino terminus or with
the carboxyl terminus of GRK2, polyclonal anti-GRK2 antibodies were
immunoselected by affinity chromatography on GRK21-185
or on GRK2561-689, respectively, covalently coupled to
Affi-Gel-10 (13). The purified antibodies were incubated in the
phosphorylation mixture as indicated. Specificity and cross-reactivity
of the antibodies with GRK21-185 or with
GRK2561-689 were analyzed in immunoblot.
Immunoblot Detection of Proteins--
Proteins were separated on
SDS-containing polyacrylamide gels, transferred to polyvinylidene
difluoride membranes, and identified in immunoblot similarly as
described (17). Antibodies specific for GRK2 or for G
have been
characterized previously (14, 18).
Binding of GRK21-185 to G
--
Purified
GST-GRK21-185 (100 nM) coupled to
glutathione-Sepharose was incubated with G
subunits (3 nM) in a total volume of 500 µl of buffer (100 mM NaCl, 20 mM HEPES, pH 7.4). After extensive washing with the same buffer, bound G
subunits were eluted with SDS sample buffer, separated by SDS-PAGE, and identified in immunoblot with G
-specific antibodies. As a control, GST-GRK51-200
was used instead of GST-GRK21-185.
ADP-ribosylation of G
o--
The G
-mediated
enhancement of the pertussis toxin-catalyzed ADP-ribosylation of
G
o was performed with 8 nM G
o
and 12 nM G
in the absence or presence of increasing
concentrations (10 nM-3 µM) of
GST-GRK21-185, GST-GRK21-53, or
GST-GRK254-185 similarly as described previously (19).
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RESULTS |
G
Regulatory Activity of a GRK2 Mutant with Truncated
Carboxyl-terminal PH Domain--
The carboxyl-terminal PH domain of
GRK2 is essential for the G
-dependent phosphorylation
of receptor substrates by GRK2 (7). Furthermore, PH domains can
regulate G
-stimulated signaling by sequestering free G
subunits (9). To analyze whether the PH domain of GRK2 is entirely
responsible for the G
regulatory activity of GRK2, the PH domain
of GRK2 was truncated, and the G
regulatory activity of the
truncated GRK2 mutant (GRK21-558CVLL) was analyzed in
intact cells by determining the inhibition of a G
-stimulated
increase in inositol phosphates mediated by activation of phospholipase
C
2 (11, 12). To exclude that a cytosolic localization of
the truncated GRK2 mutant prevented the interaction with
membrane-anchored G
subunits in cells, a membrane-anchoring CAAX motif was introduced in
GRK21-558CVLL similarly as described (7). Wild-type
GRK2, the carboxyl-terminal-truncated mutant
GRK21-558CVLL, and the carboxyl terminus of GRK2
containing the PH domain, GRK2561-689, were expressed in
HEK-293 cells (Fig. 1A,
lanes 1-3) and analyzed for their G
regulatory
activity (Fig. 1B). Wild-type GRK2 and GRK21-558CVLL inhibited the G
-stimulated increase in
inositol phosphates (Fig. 1B, columns 1 and
2 versus c). GRK2561-689
comprising the PH domain of GRK2 also significantly decreased the
G
-stimulated signal (Fig. 1B, column 3).
Expression levels of G
and of phospholipase C
2
were similar in the different experiments (not shown). Together, these
data demonstrate that the PH domain of GRK2 inhibits G
-stimulated
signaling in cells but that the G
regulatory activity of GRK2 is
not entirely mediated by the carboxyl-terminal PH domain.

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Fig. 1.
Truncation of the PH domain of GRK2.
A, expression of GRK2 (lane 1),
GRK21-558CVLL (lane 2), or
GRK2561-689 (lane 3) in HEK-293 cells as
determined in immunoblot (IB) with GRK2-specific antibodies.
B, inositol phosphate levels of cells expressing
phospholipase C 2, G 1, G 2
(column c, 8-10-fold stimulation of basal), and of cells
coexpressing GRK2 (column 1), GRK21-558CVLL
(column 2), or GRK2561-689 (column
3). A topology model of GRK2 depicts the localization of the
different GRK2 proteins. Data ± S.E. are the means
(n = 8).
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G
Regulatory Activity of GRK21-485--
To
identify the additional domain involved in the G
regulatory
activity of GRK2, GRK21-558CVLL was further truncated. In
GRK21-485 the entire carboxyl-terminal G
binding
site of GRK2 was truncated (7). Wild-type GRK2 and
GRK21-485 were expressed in HEK-293 cells (Fig.
2A, lanes 1 and
2). Full-length GRK2 (Fig. 2B, column
1) and the truncated mutant GRK21-485 significantly
inhibited the G
-stimulated increase in inositol phosphates (Fig.
2B, column 2 versus column c). The inhibition of
the G
-stimulated signal was not dependent on the introduction of
a membrane-anchoring CAAX motif in
GRK21-485CVLL (Fig. 2, A and B,
lane 3, column 3). The carboxyl-terminal G
binding domain of GRK2, GRK2495-689, was also expressed
(Fig. 2A, lane 4). GRK2495-689
inhibited the G
-stimulated signal similarly to
GRK21-485, confirming the G
regulatory capacity of
the carboxyl-terminal G
binding site of GRK2 (Fig. 2B,
column 4). Together, these findings demonstrate that
truncation of the carboxyl-terminal G
binding site of GRK2 does
not abolish the G
regulatory activity of this kinase, suggesting
that GRK2 contains a previously unrecognized G
binding site in
addition to the carboxyl-terminal site.

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Fig. 2.
G
regulatory activity of
GRK21-485.
A, immunoblot (IB) of GRK2 (lane 1),
GRK21-485 (lane 2), GRK21-485CVLL
(lane 3), or of GRK2495-689 (lane 4)
expressed in HEK-293 cells. B, inositol phosphate levels of
cells expressing phospholipase C 2, G 1,
G 2 (column c). Coexpression of GRK2
(column 1), GRK21-485 (column 2),
GRK21-485CVLL (column 3), or of
GRK2495-689 (column 4) decreased the
G -stimulated signal. Lower panel, topology model of
GRK2. Data ± S.E. are the means (n = 8).
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The Amino-terminal Domain of GRK2 Regulates G
-stimulated
Signaling--
In search for the second G
binding site of GRK2,
the G
regulatory effect of the amino-terminal domain of GRK2 was
analyzed. GRK21-185 was expressed in HEK-293 cells (Fig.
3A, upper panel,
lane 1). GRK21-185 inhibited the
G
-stimulated increase in inositol phosphates by ~40% similarly
to GRK21-485 (Fig. 3A, column 1 versus
c and cf. Fig. 2B). Again the G
regulatory activity was independent of a CAAX
membrane-anchoring motif (Fig. 3A, lane 2,
column 2). As a control, the amino-terminal domain of GRK5
was expressed (Fig. 3B, upper panel, lanes
1 and 2) because GRK5 has been reported to
phosphorylate receptor substrates independently of the addition of
G
subunits (20). GRK51-200 or
GRK51-200CVLL did not significantly affect the
G
-stimulated increase in inositol phosphates under the applied
experimental conditions (Fig. 3B, columns 1 and
2 versus c). In contrast, the amino terminus of GRK2 lacking
the receptor interacting site (21), GRK215-185, inhibited
the G
-stimulated signal similarly as did GRK21-185
(Fig. 3B, column 3, versus Fig.
3A). The expression levels of GRK51-200, of
GRK51-200CVLL, and of GRK215-185 were similar
when determined in immunoblot with antibodies specific for GRK5 or for
GRK2 (Fig. 3B, upper panel, lanes
1-3). Thus, the amino-terminal domain of GRK2 contains a G
regulatory site, which is apparently absent in GRK5.

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Fig. 3.
The amino-terminal domain of GRK2 regulates
G -stimulated signaling in
cells. A, expression of GRK21-185 or of
GRK21-185CVLL in HEK-293 cells suppressed the
G -stimulated increase in inositol phosphate generation.
Upper panel, immunoblot (IB) detection of
GRK21-185 (lane 1) and of
GRK21-185CVLL (lane 2) by anti-GRK2 antibodies.
Lower panel, inositol phosphate levels of cells expressing
phospholipase C 2, G 1, and
G 2 (column c) and of cells coexpressing
GRK21-185 (column 1) or GRK21-185
CVLL (column 2). B, immunoblot of cells
expressing GRK51-200 (lane 1) or
GRK51-200CVLL (lane 2) as detected by anti-GRK5
antibodies and of cells expressing GRK215-185 (lane
3) detected by anti-GRK2 antibodies (upper panel). The
anti-GRK5 and anti-GRK2 antibodies were standardized with purified GRK5
or GRK2, respectively, to produce a signal of equal intensity in
immunoblot with equimolar amounts of GRK protein. Lower
panel, GRK215-185 with truncated receptor interacting
site (column 3) inhibited the G -stimulated increase in
inositol phosphates (column c), whereas
GRK51-200 (column 1) or
GRK51-200CVLL (column 2) had no significant
effect. Data ± S.E. are the means (n = 8).
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GRK21-185 Binds G
Subunits in
Vitro--
Because GRK21-185 regulated G
-stimulated
signaling in intact cells, we asked whether purified
GRK21-185 interacted with G
subunits directly.
GRK21-185 was purified as a GST fusion protein and tested
for the inhibition of G
-stimulated rhodopsin phosphorylation.
Purified GST-GRK21-185 inhibited the GRK2-mediated
phosphorylation of rhodopsin stimulated by 30 nM G
(IC50: 340 ± 30 nM, Fig.
4A). GST as a control did not
affect the phosphorylation of rhodopsin at concentrations <10
µM (not shown). These findings demonstrate that the
amino-terminal domain of GRK2 regulates a G
-stimulated signal in
cells and in vitro.

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Fig. 4.
GRK21-185
binds to G subunits
in vitro. A, increasing concentrations
of purified GST-GRK21-185 (lane 1, 75 nM; lane 2, 150 nM; lane
3, 300 nM; lane 4, 600 nM;
lane 5, 1.25 µM; lane 6, 2.5 µM) suppressed the GRK2-mediated phosphorylation of
rhodopsin (Rh) stimulated by 30 nM G
(lane c, 100%). B, interaction of purified
G subunits with GST-GRK21-185 coupled to
glutathione-Sepharose (lane 2, column 2). As a
control, GST-GRK51-200 was coupled instead of
GST-GRK21-185 (lane 1, column 1).
Bound G subunits were eluted and detected in immunoblot
(IB) with anti-G antibodies. In lane 3, 100%
of the G -load (total) was detected in the immunoblot. Data ± S.E. are the means (n = 3).
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To further confirm that the amino-terminal domain of GRK2 interacts
directly with G
subunits, binding of
GST- GRK21-185 to G
subunits was measured.
GST-GRK21-185 or GST-GRK51-200 as a control
were coupled to glutathione-Sepharose and incubated with purified
G
subunits. Bound G
subunits were eluted and detected in
immunoblot with G
-specific antibodies (Fig. 4B). Although
under the experimental conditions G
subunits did not bind in
significant amounts to GST-GRK51-200, which was used as a
control (Fig. 4B, lane 1, column 1),
the amino-terminal domain of GRK2, GST-GRK21-185,
interacted specifically with G
subunits (Fig. 4B,
lane 2, column 2), i.e. nearly 100%
of the loaded G
subunits were bound by the
GST-GRK21-185-Sepharose (Fig. 4B, lane
3). Together these findings demonstrate that
GRK21-185 binds directly to G
subunits. Thus,
GRK2 contains a second G
binding site in the amino terminus in
addition to the carboxyl-terminal PH domain.
The Amino-terminal G
Binding Domain of GRK2 Regulates Kinase
Activity--
The carboxyl-terminal G
binding domain of GRK2 is
essential for the G
-dependent stimulation of the
kinase activity toward receptor substrates (7). Does the amino-terminal
G
binding domain also affect the kinase activity of GRK2? To
determine the effect of the amino terminus on the kinase activity, the
amino-terminal G
binding domain of GRK2 was targeted with
immunoselected antibodies. The purified polyclonal antibodies used for
this experiment specifically cross-reacted with GRK21-185
but did not interact with the carboxyl-terminal G
binding domain of GRK2 as determined in immunoblot (not shown). The presence of 100 nM antibodies to the amino terminus of GRK2 suppressed the
G
-stimulated (10-40 nM) phosphorylation of rhodopsin
by 20 nM GRK2 (Fig. 5,
B, lanes 1-3, versus A,
lanes 1-3), suggesting that the amino terminus of GRK2 is
involved in phosphorylating rhodopsin.

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Fig. 5.
The amino-terminal
G binding domain of GRK2
regulates the kinase activity. A, phosphorylation of
rhodopsin (Rh) by 20 nM GRK2 in the presence of
increasing concentrations of G subunits (lane 1, 13 nM; lane 2, 20 nM; lane
3, 40 nM; lane 4, 110 nM;
lane 5, 300 nM; lane 6, control, 1 µM; lane 7, 3 µM). B,
effect of 100 nM immunoselected antibodies specifically
cross-reacting with the amino terminus of GRK2 on the GRK2-mediated
phosphorylation of rhodopsin determined as in A in the
presence of increasing concentrations of purified G subunits
(lanes 1-7). Data ± S.E. are the means
(n = 3).
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To analyze whether the antibodies interfered with the binding of
G
subunits to the amino terminus of GRK2, the concentration of
the purified G
subunits was increased (Fig. 5, A and
B, lanes 4-7). G
stimulated the
phosphorylation of rhodopsin by GRK2 in the absence of antibodies
(EC50 = 38 ± 7 nM, Fig. 5A).
The presence of 100 nM antibodies specifically
cross-reacting with the amino terminus of GRK2 increased the
EC50 value of G
in stimulating GRK2-mediated
rhodopsin phosphorylation more than 20-fold (Fig. 5, B
versus A), but the G
subunits were capable of reversing the antibody-mediated inhibition of the GRK2-induced rhodopsin phosphorylation (Fig. 5B). A higher concentration
of the antibodies (250 nM) further increased the
EC50 value of G
in stimulating GRK2 (not shown). As a
control, unrelated antibodies not cross-reactive with GRK2 did not
affect the phosphorylation of rhodopsin by GRK2 (not shown). Together
these findings provide strong evidence that the antibodies compete with
G
subunits for binding to the amino terminus of GRK2, thereby
preventing the stimulatory interaction of G
with the amino
terminus. The interaction of G
with the amino-terminal G
binding domain of GRK2 may thus contribute to the
G
-dependence of GRK2.
Differentiation of the Amino- and Carboxyl-terminal G
Binding
Sites of GRK2--
To differentiate between the amino- and
carboxyl-terminal G
binding sites of GRK2, the effect of
domain-specific antibodies to the carboxyl terminus was assessed in the
rhodopsin phosphorylation assay. Antibodies specifically cross-reacting
with the carboxyl-terminal domain, GRK2561-689 (anti-C),
inhibited the stimulatory effect of G
at concentrations ranging
from 20 nM to 1 µM (Fig.
6A). Interestingly, these
antibodies did not alter the GRK2-mediated rhodopsin phosphorylation in
the presence of less than 20 nM G
as did the
antibodies cross-reacting with the amino terminus (Fig. 6A
versus Fig. 5B). Considering that these low
G
concentrations in the rhodopsin phosphorylation assay are
achieved without the addition of purified G
because the G
subunits come from the rhodopsin-enriched membranes as determined in
immunoblot (not shown), this finding is in good agreement with previous
observations; a GRK2 mutant lacking the carboxyl-terminal G
binding site is capable of phosphorylating rhodopsin but lacks the
G
-enhancing effect exerted by the addition of purified G
subunits (7). Together these data indicate that the amino- and
carboxyl-terminal G
binding sites of GRK2 are functionally
different.

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Fig. 6.
Differentiation of the amino- and
carboxyl-terminal G binding
sites of GRK2. A, effect of 100 nM
immunoselected antibodies specifically cross-reacting with the carboxyl
terminus of GRK2 (+anti-C) on the GRK2-mediated
phosphorylation of rhodopsin (Rh) in the presence of
increasing concentrations of G subunits as indicated. As a
control, the rhodopsin phosphorylation was determined in the absence of
antibodies ( anti-C). B, increasing
concentrations of G -specific antibodies (anti-G ) used
as G scavenger inhibited the rhodopsin phosphorylation by GRK2
( anti-C), and 100 nM antibodies specifically
cross-reacting with the carboxyl terminus of GRK2 did not alter this
inhibition (+anti-C). C, shielding of the amino
terminus of GRK2 with increasing concentrations of site-directed
antibodies (anti-N) inhibited the rhodopsin phosphorylation
by GRK2 in the presence of 10 nM G
( anti-C). Again, 100 nM antibodies
specifically cross-reacting with the carboxyl terminus of GRK2 did not
alter this inhibition (+anti-C). The rhodopsin
phosphorylation is expressed as % of control (i.e. the
phosphorylation mediated by GRK2 in the presence of 10 nM
G but in the absence of antibodies). Data ± S.E. are the
means of three independent experiments (upper panels), and
the bottom panels show autoradiograms of representative
experiments.
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The GRK2 activity that was not blocked by the
carboxyl-terminal-specific antibodies was still
G
-dependent because increasing concentrations of
G
-specific antibodies used as G
scavenger inhibited the
residual rhodopsin phosphorylation entirely (Fig. 6B).
Similar results were obtained with several other G
-binding proteins such as G
o or the Raf kinase (not shown).
Because the carboxyl-terminal-specific antibodies did not interfere
with the G
scavenger (Fig. 6B), these findings reveal
again the second G
binding site in GRK2, which is distinct from
the carboxyl-terminal site (Fig. 6B). For comparison,
antibodies to the amino terminus of GRK2 inhibited the GRK2-mediated
rhodopsin phosphorylation under similar conditions in a
concentration-dependent manner (Fig. 6C). As
controls, the antibodies to the amino terminus of GRK2 did not bind
G
(not shown), and carboxyl-terminal antibodies did not interfere
with the inhibition exerted by the amino-terminal-specific antibodies
(Fig. 6C). Thus, the second G
binding site in the amino terminus of GRK2 is functionally different from the
carboxyl-terminal site and is involved in rhodopsin phosphorylation at
low concentrations of G
(<20 nM).
The RGS Domain of GRK2 Does Not Interfere with G
Binding--
The amino terminus of GRK2 contains a previously
identified RGS domain (Ref. 2 and 3 and Fig.
7D). Does the RGS domain overlap with the
amino-terminal G
binding domain? Two different GST fusion
proteins were prepared, GST-GRK21-53 and
GST-GRK254-185, encompassing the RGS domain (Fig.
7D). Although GRK21-53 inhibited the
G
-stimulated phosphorylation of rhodopsin by GRK2 similarly to
GRK21-185 (Fig. 7A, upper panel
versus Fig. 4A), the RGS domain,
GRK254-185, had no significant effect when applied at
similar concentrations (Fig. 7A, lower panel).
This finding strongly suggests that the RGS domain and the
amino-terminal G
regulatory site of GRK2 do not overlap. In
addition to the G
binding site, GRK21-53 contains
other regulatory sites such as a receptor interacting site (21) or a
calmodulin binding site (22). To exclude the possibility that
GRK21-53 interfered with the kinase activity of GRK2 in a
G
-independent manner, we analyzed the G
regulatory effects
of this protein in another G
-dependent assay, the
G
-mediated enhancement of the pertussis toxin-catalyzed
ADP-ribosylation of G
o (19). GRK21-53
inhibited the enhancing effect of G
on the ADP-ribosylation of
G
o (Fig. 7B) with similar potency and
efficacy as did GRK21-185 (IC50, 290 ± 20 and 360 ± 30 nM of GRK21-53 and
GRK21-185, respectively). In contrast, the RGS domain,
GRK254-185, had no significant effect at concentrations
<1 µM (Fig. 7B). Thus, GRK21-53
encompasses the functionally important portion of the
amino-terminal G
regulatory site of GRK2, whereas the RGS domain
of GRK2, GRK254-185, did not interfere significantly with
G
binding. The in vitro findings were confirmed in
cells. Although GRK21-53 inhibited G
-stimulated
signaling similarly to GRK21-185, the RGS domain
GRK254-185 did not affect the G
-stimulated increase
in inositol phosphates mediated by PLC-
2 (Fig.
7C, first through fourth columns). By contrast, the RGS domain-containing GRK21-185 and
GRK254-185 efficiently inhibited a
G
q-stimulated inositol phosphate signal mediated by
PLC-
1 that was not affected by GRK21-53
(Fig. 7C, fifth through eighth columns).

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Fig. 7.
The RGS domain of GRK2 does not inhibit
G -stimulated effects.
A, increasing concentrations of GST-GRK21-53
(lane c, 0 nM; lane 1, 14 nM; lane 2, 42 nM; lane
3, 140 nM; lane 4, 420 nM;
lane 5, 1.4 µM; lane 6, 4.2 µM) inhibited the G -stimulated phosphorylation of
rhodopsin by GRK2 (upper panel), whereas the RGS domain
GST-GRK254-185 when applied at similar concentrations had
no significant effect (lower panel). The experiment shown is
representative of three independent experiments each with similar
results. B, GST-GRK21-53 and
GST-GRK21-185 inhibited the G -mediated enhancement
of the pertussis toxin-catalyzed ADP-ribosylation of G o
(100%) with similar potency and efficacy, whereas
GST-GRK254-185 was ineffective at concentrations <1
µM. Data are the means (±S.E., n = 6).
C, inositol phosphate levels of HEK-293 cells coexpressing
either G and PLC- 2 (columns 1-4) or
G q and PLC- 1 (columns 5-8)
and the indicated GRK2 protein. Data ± S.E. are the means
(n = 6). D, topology model of GRK2 with the
amino- and carboxyl-terminal G binding sites, depicted in
white.
|
|
 |
DISCUSSION |
The kinase activity of GRK2 and GRK3 toward receptor substrates is
strongly enhanced by G
subunits. The G
dependence links the
kinase activity of these GRKs to the activation of a heterotrimeric G-protein. A carboxyl-terminal PH domain in GRK2 and GRK3 is
essentially involved in the G
dependence of GRKs (7). Here we
present strong evidence that the amino-terminal domain of GRK2 contains a second G
binding site that contributes to the regulation of GRK2 by low concentrations of G
subunits; (i) the amino terminus of GRK2 inhibited G
-stimulated signaling in cells and in
vitro, (ii) GRK21-185 interacted directly with
purified G
subunits, (iii) targeting of the amino-terminal
G
binding domain of GRK2 by site-directed antibodies suppressed
the GRK2-mediated phosphorylation of rhodopsin, and (iv) this
inhibition was released by an excess of free G
subunits.
The amino terminus of GRK2 contains several important structural
elements, an RGS-domain affecting G
q-stimulated
signaling (2, 3), a calmodulin binding site (22), which is regulated by
protein kinase C phosphorylation (23), and a receptor interacting site
(21). A receptor interacting site and a calmodulin binding site were
also localized in the carboxyl-terminal domain of GRK2 (22, 24). These
functional similarities of the amino- and the carboxyl-terminal domains
of GRK2 are complemented by the localization of a previously
unrecognized G
binding site in the amino terminus of GRK2. The
novel amino-terminal G
binding site is involved in the G
dependence of GRK2 in addition to the carboxyl terminus. A topological
model of GRK2 appears to consist of a core kinase domain flanked by two
structurally and functionally different G
binding domains. With
two G
binding sites, GRK2 activity is tightly controlled by
G
subunits over a wide concentration range. Thereby G
subunits translate the intensity of a G-protein-stimulated signal into
GRK2 activity to switch off the signal-generating receptor.
Apart from the functional importance of the newly identified G
binding site in the amino terminus of GRK2, this domain may constitute
a novel target allowing the selective inhibition of GRK2-mediated
receptor phosphorylation by pharmacological tools. Site-directed
antibodies to the kinase amino terminus suppressed the phosphorylation
of rhodopsin by GRK2. Because such an inhibition was released by the
addition of an excess of G
subunits, blockade of GRK2 activity by
pharmacological compounds binding to the amino terminus of GRK2 would
be reversed upon excessive G-protein activation, i.e.
G
release. The proposed mechanism could allow the design of
fine-tuning GRK inhibitors, which would amplify low threshold signals
and maintain desensitization of excessive stimuli. Additional experiments will have to identify such compounds to validate the proposed principle under physiological conditions.
 |
ACKNOWLEDGEMENTS |
We thank C. Dees for purification of
rhodopsin, GRK2, G
, and G
o and M. Fischer for
insect cell culture.
 |
FOOTNOTES |
*
This work was supported by the Deutsche
Forschungsgemeinschaft.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.
To whom correspondence should be addressed. Tel.:
49-931-201-48982; Fax: 49-931-201-48539; E-mail:
toph029@rzbox.uni-wuerzburg.de.
Published, JBC Papers in Press, December 16, 2002, DOI 10.1074/jbc.M204795200
 |
ABBREVIATIONS |
The abbreviations used are:
GRK, G-protein-coupled receptor kinase 2;
HEK-293 cells, human embryonic
kidney cells;
PH domain, pleckstrin homology domain;
GST, glutathione
S-transferase.
 |
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