The Amino-terminal Domain of G-protein-coupled Receptor Kinase 2 Is a Regulatory Gbeta gamma Binding Site*

Tanja Eichmann, Kristina Lorenz, Michaela Hoffmann, Jörg Brockmann, Cornelius Krasel, Martin J. Lohse, and Ursula QuittererDagger

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

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

G-protein-coupled receptor kinase 2 (GRK2) is activated by free Gbeta gamma subunits. A Gbeta gamma binding site of GRK2 is localized in the carboxyl-terminal pleckstrin homology domain. This Gbeta gamma binding site of GRK2 also regulates Gbeta gamma -stimulated signaling by sequestering free Gbeta gamma subunits. We report here that truncation of the carboxyl-terminal Gbeta gamma binding site of GRK2 did not abolish the Gbeta gamma regulatory activity of GRK2 as determined by the inhibition of a Gbeta gamma -stimulated increase in inositol phosphates in cells. This finding suggested the presence of a second Gbeta gamma binding site in GRK2. And indeed, the amino terminus of GRK2 (GRK21-185) inhibited a Gbeta gamma -stimulated inositol phosphate signal in cells, purified GRK21-185 suppressed the Gbeta gamma -stimulated phosphorylation of rhodopsin, and GRK21-185 bound directly to purified Gbeta gamma subunits. The amino-terminal Gbeta gamma regulatory site does not overlap with the RGS domain of GRK-2 because GRK21-53 with truncated RGS domain inhibited Gbeta gamma -mediated signaling with similar potency and efficacy as did GRK21-185. In addition to the Gbeta gamma regulatory activity, the amino-terminal Gbeta gamma 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 Gbeta gamma subunits, strongly suggesting that Gbeta gamma binding to the amino terminus of GRK2 enhances the kinase activity toward rhodopsin. Thus, the amino-terminal domain of GRK2 is a previously unrecognized Gbeta gamma binding site that regulates GRK2-mediated receptor phosphorylation and inhibits Gbeta gamma -stimulated signaling.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 beta -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 Gbeta gamma subunits (5, 6). A Gbeta gamma 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 Gbeta gamma subunits (7). The carboxyl-terminal Gbeta gamma binding site of GRK2 also regulates Gbeta gamma -stimulated signaling by sequestering free Gbeta gamma subunits (8). Analyzing the Gbeta gamma regulatory activity of proteins is a means of identifying Gbeta gamma -binding proteins or localizing Gbeta gamma binding sites of proteins (9-11). To find out whether the Gbeta gamma regulatory activity of GRK2 resides entirely in the carboxyl-terminal PH domain, we analyzed the Gbeta gamma -sequestering activity of wild-type GRK2 and of carboxyl-terminal-truncated GRK2 mutants. The capacity of those proteins to inhibit a Gbeta gamma -stimulated increase in inositol phosphates mediated by activation of phospholipase Cbeta 2 was determined (12). We report here that truncation of the carboxyl-terminal Gbeta gamma binding site of GRK2 did not abolish the Gbeta gamma regulatory activity of GRK2. A previously unrecognized Gbeta gamma 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 Gbeta gamma -stimulated signaling.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 Gbeta gamma 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 Cbeta 2, Gbeta 1, and Ggamma 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, Gbeta gamma subunits as indicated, 400 nM rhodopsin, 10 mM MgCl2, 2 mM EDTA, and 50 µM [gamma -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 Gbeta gamma subunits, various concentrations of purified Gbeta gamma 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 Gbeta have been characterized previously (14, 18).

Binding of GRK21-185 to Gbeta gamma -- Purified GST-GRK21-185 (100 nM) coupled to glutathione-Sepharose was incubated with Gbeta gamma 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 Gbeta gamma subunits were eluted with SDS sample buffer, separated by SDS-PAGE, and identified in immunoblot with Gbeta -specific antibodies. As a control, GST-GRK51-200 was used instead of GST-GRK21-185.

ADP-ribosylation of Galpha o-- The Gbeta gamma -mediated enhancement of the pertussis toxin-catalyzed ADP-ribosylation of Galpha o was performed with 8 nM Galpha o and 12 nM Gbeta gamma 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).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Gbeta gamma Regulatory Activity of a GRK2 Mutant with Truncated Carboxyl-terminal PH Domain-- The carboxyl-terminal PH domain of GRK2 is essential for the Gbeta gamma -dependent phosphorylation of receptor substrates by GRK2 (7). Furthermore, PH domains can regulate Gbeta gamma -stimulated signaling by sequestering free Gbeta gamma subunits (9). To analyze whether the PH domain of GRK2 is entirely responsible for the Gbeta gamma regulatory activity of GRK2, the PH domain of GRK2 was truncated, and the Gbeta gamma regulatory activity of the truncated GRK2 mutant (GRK21-558CVLL) was analyzed in intact cells by determining the inhibition of a Gbeta gamma -stimulated increase in inositol phosphates mediated by activation of phospholipase Cbeta 2 (11, 12). To exclude that a cytosolic localization of the truncated GRK2 mutant prevented the interaction with membrane-anchored Gbeta gamma 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 Gbeta gamma regulatory activity (Fig. 1B). Wild-type GRK2 and GRK21-558CVLL inhibited the Gbeta gamma -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 Gbeta gamma -stimulated signal (Fig. 1B, column 3). Expression levels of Gbeta gamma and of phospholipase Cbeta 2 were similar in the different experiments (not shown). Together, these data demonstrate that the PH domain of GRK2 inhibits Gbeta gamma -stimulated signaling in cells but that the Gbeta gamma 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 Cbeta 2, Gbeta 1, Ggamma 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).

Gbeta gamma Regulatory Activity of GRK21-485-- To identify the additional domain involved in the Gbeta gamma regulatory activity of GRK2, GRK21-558CVLL was further truncated. In GRK21-485 the entire carboxyl-terminal Gbeta gamma 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 Gbeta gamma -stimulated increase in inositol phosphates (Fig. 2B, column 2 versus column c). The inhibition of the Gbeta gamma -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 Gbeta gamma binding domain of GRK2, GRK2495-689, was also expressed (Fig. 2A, lane 4). GRK2495-689 inhibited the Gbeta gamma -stimulated signal similarly to GRK21-485, confirming the Gbeta gamma regulatory capacity of the carboxyl-terminal Gbeta gamma binding site of GRK2 (Fig. 2B, column 4). Together, these findings demonstrate that truncation of the carboxyl-terminal Gbeta gamma binding site of GRK2 does not abolish the Gbeta gamma regulatory activity of this kinase, suggesting that GRK2 contains a previously unrecognized Gbeta gamma binding site in addition to the carboxyl-terminal site.


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Fig. 2.   Gbeta gamma 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 Cbeta 2, Gbeta 1, Ggamma 2 (column c). Coexpression of GRK2 (column 1), GRK21-485 (column 2), GRK21-485CVLL (column 3), or of GRK2495-689 (column 4) decreased the Gbeta gamma -stimulated signal. Lower panel, topology model of GRK2. Data ± S.E. are the means (n = 8).

The Amino-terminal Domain of GRK2 Regulates Gbeta gamma -stimulated Signaling-- In search for the second Gbeta gamma binding site of GRK2, the Gbeta gamma 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 Gbeta gamma -stimulated increase in inositol phosphates by ~40% similarly to GRK21-485 (Fig. 3A, column 1 versus c and cf. Fig. 2B). Again the Gbeta gamma 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 Gbeta gamma subunits (20). GRK51-200 or GRK51-200CVLL did not significantly affect the Gbeta gamma -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 Gbeta gamma -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 Gbeta gamma regulatory site, which is apparently absent in GRK5.


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Fig. 3.   The amino-terminal domain of GRK2 regulates Gbeta gamma -stimulated signaling in cells. A, expression of GRK21-185 or of GRK21-185CVLL in HEK-293 cells suppressed the Gbeta gamma -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 Cbeta 2, Gbeta 1, and Ggamma 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 Gbeta gamma -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).

GRK21-185 Binds Gbeta gamma Subunits in Vitro-- Because GRK21-185 regulated Gbeta gamma -stimulated signaling in intact cells, we asked whether purified GRK21-185 interacted with Gbeta gamma subunits directly. GRK21-185 was purified as a GST fusion protein and tested for the inhibition of Gbeta gamma -stimulated rhodopsin phosphorylation. Purified GST-GRK21-185 inhibited the GRK2-mediated phosphorylation of rhodopsin stimulated by 30 nM Gbeta gamma (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 Gbeta gamma -stimulated signal in cells and in vitro.


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Fig. 4.   GRK21-185 binds to Gbeta gamma 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 Gbeta gamma (lane c, 100%). B, interaction of purified Gbeta gamma 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 Gbeta gamma subunits were eluted and detected in immunoblot (IB) with anti-Gbeta antibodies. In lane 3, 100% of the Gbeta -load (total) was detected in the immunoblot. Data ± S.E. are the means (n = 3).

To further confirm that the amino-terminal domain of GRK2 interacts directly with Gbeta gamma subunits, binding of GST- GRK21-185 to Gbeta gamma subunits was measured. GST-GRK21-185 or GST-GRK51-200 as a control were coupled to glutathione-Sepharose and incubated with purified Gbeta gamma subunits. Bound Gbeta gamma subunits were eluted and detected in immunoblot with Gbeta -specific antibodies (Fig. 4B). Although under the experimental conditions Gbeta gamma 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 Gbeta gamma subunits (Fig. 4B, lane 2, column 2), i.e. nearly 100% of the loaded Gbeta gamma subunits were bound by the GST-GRK21-185-Sepharose (Fig. 4B, lane 3). Together these findings demonstrate that GRK21-185 binds directly to Gbeta gamma subunits. Thus, GRK2 contains a second Gbeta gamma binding site in the amino terminus in addition to the carboxyl-terminal PH domain.

The Amino-terminal Gbeta gamma Binding Domain of GRK2 Regulates Kinase Activity-- The carboxyl-terminal Gbeta gamma binding domain of GRK2 is essential for the Gbeta gamma -dependent stimulation of the kinase activity toward receptor substrates (7). Does the amino-terminal Gbeta gamma binding domain also affect the kinase activity of GRK2? To determine the effect of the amino terminus on the kinase activity, the amino-terminal Gbeta gamma 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 Gbeta gamma binding domain of GRK2 as determined in immunoblot (not shown). The presence of 100 nM antibodies to the amino terminus of GRK2 suppressed the Gbeta gamma -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 Gbeta gamma binding domain of GRK2 regulates the kinase activity. A, phosphorylation of rhodopsin (Rh) by 20 nM GRK2 in the presence of increasing concentrations of Gbeta gamma 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 Gbeta gamma subunits (lanes 1-7). Data ± S.E. are the means (n = 3).

To analyze whether the antibodies interfered with the binding of Gbeta gamma subunits to the amino terminus of GRK2, the concentration of the purified Gbeta gamma subunits was increased (Fig. 5, A and B, lanes 4-7). Gbeta gamma 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 Gbeta gamma in stimulating GRK2-mediated rhodopsin phosphorylation more than 20-fold (Fig. 5, B versus A), but the Gbeta gamma 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 Gbeta gamma 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 Gbeta gamma subunits for binding to the amino terminus of GRK2, thereby preventing the stimulatory interaction of Gbeta gamma with the amino terminus. The interaction of Gbeta gamma with the amino-terminal Gbeta gamma binding domain of GRK2 may thus contribute to the Gbeta gamma -dependence of GRK2.

Differentiation of the Amino- and Carboxyl-terminal Gbeta gamma Binding Sites of GRK2-- To differentiate between the amino- and carboxyl-terminal Gbeta gamma 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 Gbeta gamma 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 Gbeta gamma as did the antibodies cross-reacting with the amino terminus (Fig. 6A versus Fig. 5B). Considering that these low Gbeta gamma concentrations in the rhodopsin phosphorylation assay are achieved without the addition of purified Gbeta gamma because the Gbeta gamma 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 Gbeta gamma binding site is capable of phosphorylating rhodopsin but lacks the Gbeta gamma -enhancing effect exerted by the addition of purified Gbeta gamma subunits (7). Together these data indicate that the amino- and carboxyl-terminal Gbeta gamma binding sites of GRK2 are functionally different.


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Fig. 6.   Differentiation of the amino- and carboxyl-terminal Gbeta gamma 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 Gbeta gamma subunits as indicated. As a control, the rhodopsin phosphorylation was determined in the absence of antibodies (-anti-C). B, increasing concentrations of Gbeta -specific antibodies (anti-Gbeta ) used as Gbeta gamma 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 Gbeta gamma (-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 Gbeta gamma 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.

The GRK2 activity that was not blocked by the carboxyl-terminal-specific antibodies was still Gbeta gamma -dependent because increasing concentrations of Gbeta -specific antibodies used as Gbeta gamma scavenger inhibited the residual rhodopsin phosphorylation entirely (Fig. 6B). Similar results were obtained with several other Gbeta gamma -binding proteins such as Galpha o or the Raf kinase (not shown). Because the carboxyl-terminal-specific antibodies did not interfere with the Gbeta gamma scavenger (Fig. 6B), these findings reveal again the second Gbeta gamma 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 Gbeta gamma (not shown), and carboxyl-terminal antibodies did not interfere with the inhibition exerted by the amino-terminal-specific antibodies (Fig. 6C). Thus, the second Gbeta gamma 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 Gbeta gamma (<20 nM).

The RGS Domain of GRK2 Does Not Interfere with Gbeta gamma 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 Gbeta gamma 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 Gbeta gamma -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 Gbeta gamma regulatory site of GRK2 do not overlap. In addition to the Gbeta gamma 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 Gbeta gamma -independent manner, we analyzed the Gbeta gamma regulatory effects of this protein in another Gbeta gamma -dependent assay, the Gbeta gamma -mediated enhancement of the pertussis toxin-catalyzed ADP-ribosylation of Galpha o (19). GRK21-53 inhibited the enhancing effect of Gbeta gamma on the ADP-ribosylation of Galpha 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 Gbeta gamma regulatory site of GRK2, whereas the RGS domain of GRK2, GRK254-185, did not interfere significantly with Gbeta gamma binding. The in vitro findings were confirmed in cells. Although GRK21-53 inhibited Gbeta gamma -stimulated signaling similarly to GRK21-185, the RGS domain GRK254-185 did not affect the Gbeta gamma -stimulated increase in inositol phosphates mediated by PLC-beta 2 (Fig. 7C, first through fourth columns). By contrast, the RGS domain-containing GRK21-185 and GRK254-185 efficiently inhibited a Galpha q-stimulated inositol phosphate signal mediated by PLC-beta 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 Gbeta gamma -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 Gbeta gamma -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 Gbeta gamma -mediated enhancement of the pertussis toxin-catalyzed ADP-ribosylation of Galpha 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 Gbeta gamma and PLC-beta 2 (columns 1-4) or Galpha q and PLC-beta 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 Gbeta gamma binding sites, depicted in white.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The kinase activity of GRK2 and GRK3 toward receptor substrates is strongly enhanced by Gbeta gamma subunits. The Gbeta gamma 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 Gbeta gamma dependence of GRKs (7). Here we present strong evidence that the amino-terminal domain of GRK2 contains a second Gbeta gamma binding site that contributes to the regulation of GRK2 by low concentrations of Gbeta gamma subunits; (i) the amino terminus of GRK2 inhibited Gbeta gamma -stimulated signaling in cells and in vitro, (ii) GRK21-185 interacted directly with purified Gbeta gamma subunits, (iii) targeting of the amino-terminal Gbeta gamma 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 Gbeta gamma subunits.

The amino terminus of GRK2 contains several important structural elements, an RGS-domain affecting Galpha 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 Gbeta gamma binding site in the amino terminus of GRK2. The novel amino-terminal Gbeta gamma binding site is involved in the Gbeta gamma 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 Gbeta gamma binding domains. With two Gbeta gamma binding sites, GRK2 activity is tightly controlled by Gbeta gamma subunits over a wide concentration range. Thereby Gbeta gamma 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 Gbeta gamma 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 Gbeta gamma 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. Gbeta gamma 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, Gbeta gamma , and Galpha 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.

Dagger 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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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