©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Phosphorylation and Activation of -Adrenergic Receptor Kinase by Protein Kinase C (*)

(Received for publication, January 30, 1995; and in revised form, May 1, 1995)

Tsu Tshen Chuang Harry LeVine , III (1) Antonio De Blasi (§)

From the Consorzio Mario Negri Sud, Istituto di Ricerche Farmacologiche Mario Negri, Santa Maria Imbaro 66030, Italy and the Department of Neurodegenerative Diseases, Parke-Davis Pharmaceutical Research Division, Warner-Lambert Co., Ann Arbor, Michigan 48105

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The aim of this study was to test the possible modification of beta-adrenergic receptor kinase (betaARK) activity by second messengers and/or their downstream components. Using human mononuclear leukocytes (MNL), we found that calcium ionophores could elevate betaARK activity by about 80% in a protein kinase C (PKC)-dependent manner. This was confirmed by the ability of the PKC activator phorbol 12-myristate 13-acetate (PMA) to produce a similar effect, suggesting a PKC-dependent modulation of betaARK activity. In vitro experiments with purified proteins showed that PKC could directly phosphorylate betaARK1 with an apparent K for betaARK1 of 6 nM. The ability of betaARK1 to phosphorylate rhodopsin was 61% greater when it was phosphorylated by PKC. The level of phosphorylation of betaARK1 immunoprecipitated from MNL and Sf9 cells overexpressing this kinase was enhanced by about 2-3-fold after PMA treatment. Functional significance of PKC-dependent increase in betaARK activity was demonstrated by beta-adrenergic receptor (betaAR) homologous desensitization experiments in MNL. betaAR desensitization, as induced by exposure to 10 µM isoproterenol (5 min at 37 °C), was increased from 42 ± 10% in control to 68 ± 8% in PMA-pretreated MNL. betaARK inhibitor heparin (160 µg/ml) prevented the augmenting effect of PMA on betaAR desensitization. These results show that betaARK activity can be increased through phosphorylation by PKC, thus indicating that betaARK can be preconditioned to modulate the subsequent cellular responsiveness to receptor activation, providing the cell with a mechanism by which specific homologous desensitization can be regulated heterologously.


INTRODUCTION

beta-Adrenergic receptor kinase (betaARK) (^1)is a serine-threonine kinase involved in the process of homologous desensitization of G-protein-coupled receptors(1) , which bind a large array of different molecules, ranging from photons, neurotransmitters, and neuropeptides to autacoid substances, hormones, and immunomodulators acting through different intracellular second messengers. The mechanism of betaARK-mediated homologous desensitization has been most extensively studied on the beta(2)-adrenergic receptors (betaAR). betaARK phosphorylates the agonist-occupied form of the receptor, enabling the binding of its co-factor beta-arrestin (2) to the receptor to result in uncoupling of the receptor from G-proteins and hence effector second messenger systems. betaARK is a member of a multigene family, consisting of six known subtypes, which have also been named G-protein-coupled receptor kinases (GRK 1-6) due to the apparently unique functional association of such kinases with this receptor family(1) . In this scheme rhodopsin kinase corresponds to GRK1, betaARK1 to GRK2, and betaARK2 to GRK3(1) . While the expression of rhodopsin kinase is essentially confined to the retina, where it regulates phototransduction, a wide tissue distribution has been reported for many GRKs, the central nervous system as well as immune cells(3, 4, 5, 6) being sites of relevant expression of most subtypes.

Physical interaction between betaARK and receptor is favored by the binding of the kinase to beta-subunits of heterotrimeric G proteins, and this results in an enhanced receptor phosphorylation(7) . The binding of betaARK to beta-subunits is shown to be mediated by a stretch of amino acids near the C-terminal of the kinase, including sequences in and extending beyond the most C-terminal region of the pleckstrin homology (PH-) domain(8) .

While betaARK modulates receptor-mediated production of second messengers, the likely existence of a feedback loop by which second messengers and/or their downstream components impinge on betaARK to modulate its activity remains an intriguing open question. The present study was aimed to address this point. We found that betaARK activity as well as betaARK-dependent receptor homologous desensitization are enhanced in cells following protein kinase C (PKC) activation. The molecular basis for these effects is provided by in vitro experiments showing that betaARK can be directly phosphorylated by PKC and that this increases the rhodopsin-phosphorylating activity of betaARK.


MATERIALS AND METHODS

Human mononuclear leukocytes (MNL) preparation and treatments, cytosolic betaARK preparation as well as bovine rod outer segment (ROS) phosphorylation assay, and quantification of phosphate incorporation were as described(4) . Phorbol 12-myristate 13-acetate (PMA) and ionomycin were dissolved in Me(2)SO (0.01% Me(2)SO final concentration). Intracellular and extracellular calcium was chelated by incubation with 1 mM EGTA and 10 nM ionomycin for 45 min, resulting in 80% reduction in the subsequent ionomycin-induced calcium accumulation (not shown). DEAE-Sephacel chromatography was carried out as described in (9) .

For immunoprecipitation and immunoblots, an affinity-purified rabbit polyclonal anti-betaARK1 antibody raised against GST-human betaARK1 C terminus (222-amino acid) fusion protein was used. For immunoprecipitation, 1 µg of antibody was used according to (10) . The immune complexes were precipitated by protein A/Sepharose CL-4B conjugate and washed three times with a buffer containing 10 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.5% Nonidet P-40, then SDS sample buffer added, boiled for 5 min at 100 °C, and analyzed by 10% polyacrylamide gel electrophoresis. For immunoblots, 144 ng/ml antibody was used according to (11) . Blots were developed with donkey anti-rabbit IgG coupled to alkaline phosphatase according to the manufacturer's instructions.

PKC was partially purified from bovine brain as described(12) , except that PKC was eluted from the DEAE-Sephacel column with a NaCl gradient of 0-200 mM to improve the enzyme purity. The fractions containing peak PKC activity for Histone IIIS (116 pmol phosphate/min/mg total eluted proteins) were used. This gave an about 3-fold purification and the activity of PKC, which in this preparation is estimated to represent 2% of total protein(12, 13) , can also be expressed as 116 pmol phosphate/min/20 µg PKC. Histone IIIS phosphorylation activity of 1 nmol of phosphate incorporation/min at 30 °C was defined as 1 unit of PKC. Pure PKC (13) was a gift of Dr. P. J. Parker.

Most of the in vitro experiments were performed using a recombinant human betaARK1 overexpressed in Sf9 insect cells infected with a multiplicity of infection of 4 and purified as described(14) . betaARK1 obtained using this procedure migrated on the polyacrylamide gel as a doublet with apparent molecular mass of about 80 kDa, as seen by Coomassie Blue staining and immunoblot. More recently, using a different source of Sf9 cells infected with a multiplicity of infection of 8 we obtained new preparations in which purified betaARK1 migrated on polyacrylamide gel as a single band (80 kDa), as identified by immunoblotting. The results obtained with the former preparation were confirmed using this new preparation of purified betaARK1. In the PKC phosphorylation assay, the phosphorylation mixture (48 µl) contained 30 mM Tris-HCl, pH 7.5, 7.5 mM MgCl(2), 1 mM CaCl(2), 100 µg/ml phosphatidylserine, 1 µM PMA, 50 µM [-P]ATP (2-8 µCi/reaction), with 200 microunits of PKC, and/or 10.4 nM of betaARK1 was usually incubated at 30 °C for 1 h. The activity of betaARK1 was similar before and after this incubation (4.3 ± 0.3 and 4.5 ± 0.9 nmol/min/mg respectively, n = 3). Since PKC undergoes autophosphorylation and migrates on the gel near betaARK, at the end of the phosphorylation reaction PKC was pelleted together with phosphatidylserine vesicles (70,000 g for 20 min at 4 °C), and hence separated from betaARK1. The resulting supernatant was electrophoresed on 8% polyacrylamide gel and autoradiography of dried gels was for 12-18 h. Calphostin C was used as described(15) .

For P labeling in living cells, MNL prepared as described above were washed three times with phosphate-free Dulbecco's modified Eagle's medium (DMEM) supplemented with L-arginine, L-cysteine, D-glucose, L-glutamine, L-inositol, L-leucine, and L-methionine according to the manufacturer's recipe, as well as 25 mM of HEPES. MNL were then incubated in the same medium. Sf9 cells were collected at 60 h after infection and washed with phosphate-free Grace's medium (supplemented with 7.5 mM HEPES) three times before being incubated in the same medium. Both cell types were incubated for 3 h in the presence of 1 mCi/ml [P]orthophosphate, at 37 °C for MNL, and at 27 °C for Sf9 cells. They were then treated with PMA (1 µM) or vehicle for 20 min. Cells were then washed three times with ice-cold PBS before being lysed in cell lysis buffer and the cytosol prepared as described(4) , and betaARK1 was immunoprecipitated as described above.

For desensitization experiments MNL suspended in DMEM were untreated (control) or exposed to 1 µM PMA for 15 min at 37 °C and then incubated in the absence (undesensitized cells) or presence (desensitized cells) of 10 µM isoproterenol for 5 min at 37 °C to induce desensitization, followed by extensive washing at 4 °C. For cAMP accumulation experiments (16) , MNL were exposed to 10 µM isoproterenol for a further 5 min, and intracellular cAMP accumulated was measured. For experiments with heparin, MNL were permeabilized by streptolysin-O (0.8 unit/ml) for 5 min with or without heparin (160 µg/ml; Sigma H-3125) after PMA pretreatment and prior to induction of desensitization by isoproterenol, and membrane adenylyl cyclase assay was carried out as in (17) . Titration with heparin showed that 160 µg/ml heparin was the minimal concentration for inhibition of homologous desensitization. Desensitization was measured as the difference in isoproterenol-stimulated cAMP production in cells or by membranes that were undesensitized and desensitized.

DMEM and phosphate-free DMEM were obtained from Life Technologies, Inc., RPMI 1640 medium from Bioproducts; glutamine and penicillin/streptomycin from Seromed; Tris, SDS, bromphenol blue, acrylamide, and bisacrylamide from Bio-Rad; EDTA, urea, and magnesium chloride from Merck; DEAE-Sephacel from Pharmacia; Me(2)SO from Research Industries Corporations; cAMP assay kit, [-P]ATP and [P]orthophosphate from Amersham; ionomycin and H7 (1-5(isoquinoline sulfonyl)-2-methylpiperazine) from Calbiochem; streptolysin-O from Wellcome; donkey anti-rabbit IgG coupled to alkaline phosphatase from Pierce. All the other materials were obtained from Sigma.


RESULTS

MNL have been shown to express high levels of betaARK mRNA and activity(4, 5) , thereby providing an excellent model for studying intracellular regulation of this kinase. We found that exposure of MNL to calcium ionophores ionomycin and A23187 induced a 70-80% increase in kinase activity (Fig. 1A) as measured by a ROS phosphorylation assay which in these cells mostly measure the GRK subtype betaARK1(9) . The maximal effect was reached between 10 and 30 min of treatment with 1 µM ionomycin and was completely prevented by calcium chelation (Fig. 1A). Since calcium/calmodulin do not directly affect betaARK activity(18) , the observed effect of calcium ionophores appeared to be mediated by component(s) downstream of calcium. Possible candidates include PKC and calcium/calmodulin-dependent kinases(19, 20) . We found that coincubation of ionomycin with two PKC inhibitors, H7 and staurosporin, reverted the effect of ionomycin on betaARK activity (Fig. 1B), suggesting that PKC mediates at least part of the effect of ionomycin. This was confirmed by the finding that the PKC agonist PMA was able to increase cytosolic betaARK activity to similar extents as by ionomycin (Fig. 1C). Characterization of the cytosolic rhodopsin phosphorylating activity obtained from MNL treated with ionomycin or PMA met the criteria for it being mediated by betaARK, i.e. the activity was completely inhibited by heparin and was agonist (light)-dependent (Fig. 2A). Rhodopsin is also a substrate of PKC (21) , but the contribution of PKC under our assay conditions is excluded by the following pieces of evidence. First, rhodopsin phosphorylation by PKC requires the addition of PKC activators Ca, diacylglycerol, and phosphatidylserine(21) , which are absent in our phosphorylation assay. Second, PKC phosphorylation of rhodopsin is light independent (21) whereas light (agonist) is strictly required to activate betaARK (see Fig. 2A). Third, the augmented betaARK activity by ionomycin was still observed after the samples had passed through DEAE-Sephacel gel, which retains PKC and protein kinase A (18) thereby removing potentially interfering kinases from the phosphorylation assay (not shown). Finally, the PKC inhibitor calphostin C did not interfere with the phosphorylation of rhodopsin (Fig. 2B). Activation of protein kinase A by exposing MNL to 1 mM dibutyryl cAMP did not affect the level of betaARK activity (not shown). Taken together these findings on living MNL point to a direct or indirect modulation of betaARK activity mediated by PKC.


Figure 1: MNL cytosolic betaARK activity is increased in a PKC-dependent manner. A, rhodopsin phosphorylation by cytosolic betaARK obtained from freshly prepared MNL untreated (control, C) or exposed for 20 min to calcium ionophores A23187 1 µM, ionomycin 1 µM (I), and ionomycin after calcium chelation (I w/o Ca). The arrow indicates bands of phosphorylated rhodopsin (opsin) as revealed by autoradiography after polyacrylamide gel electrophoresis. B, the effect of ionomycin (I) was prevented by the presence of two PKC inhibitors H7 20 µM (I + H7), and staurosporin 0.5 µM (I + S). C, a similar increase of betaARK activity was induced by ionomycin 1 µM (I) and PMA 1 µM. The histogram on the right summarizes the results from six independent experiments (means ± S.E.). *, significantly different from control (p < 0.05; analysis of variance). All experiments were repeated at least twice.




Figure 2: Characterization of MNL cytosolic rhodopsin phosphorylating activity. A, rhodopsin phosphorylation by cytosolic preparations obtained from MNL untreated (Control), treated with ionomycin (Iono, 1 µM) or PMA (1 µM) was carried out in the presence and absence of light (agonist) or the betaARK inhibitor heparin (10 µg/ml). B, lack of effect of calphostin C on rhodopsin phosphorylation by MNL cytosolic preparations. MNL were untreated (Control) or treated for 20 min with 1 µM PMA and their respective cytosolic fractions prepared. Samples containing 50 µg of cytosolic proteins were then tested for their rhodopsin phosphorylating activities, and the ROS phosphorylation assay was performed in the absence or presence of calphostin C (1 µM). All experiments were repeated at least twice.



As betaARK contains in its amino acid sequence a number of phosphorylation site motifs for PKC(4) , it represents a possible substrate for this kinase. Direct phosphorylation by PKC may then lead to an increase of betaARK activity. To investigate this hypothesis, a partially pure PKC was used to phosphorylate betaARK1 purified from the baculovirus/Sf9 cell expression system. betaARK1, which appeared on the polyacrylamide gel as a doublet with apparent molecular mass of about 80 kDa, was indeed phosphorylated by the PKC preparation, and this phosphorylation was nearly completely inhibited by the selective PKC inhibitors staurosporin and calphostin C (Fig. 3A), and was prevented in the absence of the PKC activators PMA, calcium and phosphatidylserine (not shown). Phosphate incorporation was increased by 5-fold from the autophosphorylation level of 0.09 ± 0.01 to 0.49 ± 0.03 mol phosphate/mol of betaARK1 in the presence of PKC (n = 4, p < 0.001), and the time course of this reaction is depicted in Fig. 3, B and C. Immunoprecipitation using a specific anti-betaARK1 antibody after the PKC phosphorylation reaction specifically showed that betaARK1 was phosphorylated by PKC (Fig. 4A). By immunoblotting, it was shown that betaARK1 remained essentially in the supernatant after fractionation by centrifugation (see ``Materials and Methods''). This also shows that equal amounts of betaARK1 were present in the control and PKC-phosphorylated samples (Fig. 4B). Autoradiography of the same nitrocellulose membrane used for immunoblot confirmed the phosphorylation of betaARK1 by PKC (Fig. 4B).


Figure 3: In vitro phosphorylation of betaARK1 by partially purified PKC. A, samples containing PKC only (lane 1), PKC + betaARK1 (lane 2), betaARK1 only (lane 3), PKC + betaARK1 + 0.1 µM staurosporin (lane 4), and PKC + betaARK1 + 1 µM calphostin C (lane 5) were incubated under phosphorylation conditions for 1 h. The arrow indicates the bands corresponding to betaARK1 as revealed by autoradiography after polyacrylamide gel electrophoresis. B, time course of betaARK1 phosphorylation by PKC. Samples containing PKC only (lanes 1), PKC + betaARK1 (lanes 2), and betaARK1 only (lanes 3) were incubated for the indicated times. C, graph showing average values of time-dependent PKC-induced phosphate incorporation from two experiments, calculated from radioactivity of excised bands of phosphorylated betaARK1 minus autophosphorylated betaARK1 and PKC background. All experiments were repeated at least twice.




Figure 4: Identification of phosphorylated betaARK1. A, immunoprecipitation of betaARK1 after phosphorylation by PKC. Samples containing PKC only (lane 1), PKC + betaARK1 (lane 2), betaARK1 only (lane 3) were incubated under phosphorylation conditions for 1 h followed by immunoprecipitation using an anti-betaARK1 antibody. The arrow indicates the bands corresponding to betaARK1 as revealed by autoradiography after polyacrylamide gel electrophoresis of the immunoprecipitated material. B, samples containing PKC only (lanes 1), PKC + betaARK1 (lanes 2), and betaARK1 only (lanes 3) were incubated under phosphorylation conditions for 1 h followed by centrifugation at 70,000 g for 20 min. The resulting supernatant and pellet were electrophoresed on 8% polyacrylamide gel and transferred to nitrocellulose paper. betaARK1 (arrows) was then detected by immunoblotting (upper panel) and autoradiography (lower panel) from the same blot. All experiments were repeated at least twice.



For kinetic and functional studies, PKC purified from bovine brain to >90% homogeneity was used to avoid interfering proteins in the phosphorylation assay (Fig. 5). Pure PKC was able to phosphorylate betaARK1 to a phosphate incorporation level of 0.74 ± 0.07 mol/mol of betaARK1 (n = 3, p < 0.05 versus autophosphorylation, Fig. 5A). PKC showed a high affinity for betaARK1, with an apparent K of 6 nM, as calculated from a double-reciprocal plot (Fig. 5B). According to the experiments in intact cells (Fig. 1), phosphorylation of betaARK1 by PKC should lead to increased rhodopsin phosphorylating activity of the kinase. This was the case as the ability of betaARK1 to phosphorylate rhodopsin was 61% greater when it was phosphorylated by PKC compared to betaARK1 that was not exposed to PKC (see Fig. 5C).


Figure 5: Increased betaARK1 activity upon phosphorylation by PKC purified from bovine brain. 400 microunits of PKC and 2.6 nM of betaARK1 (unless otherwise stated) were used in PKC phosphorylation reactions. A, phosphorylation of betaARK1 by pure PKC. Samples containing PKC only (lane 1), PKC + betaARK1 (lane 2), and betaARK1 only (lane 3) were incubated under phosphorylation conditions for 1 h. The arrow indicates the bands corresponding to betaARK1 as revealed by autoradiography after polyacrylamide gel electrophoresis. Numbers on the left are molecular mass markers in kDa. B, assessment of the apparent K of PKC for betaARK1 by double-reciprocal plot in which the concentration of betaARK1 was varied. Each point is the average from two experiments. C, assessment of rhodopsin phosphorylating activity of betaARK1 after phosphorylation by PKC. Rhodopsin (250 nM) was phosphorylated in the presence of 21 µg/ml purified brain beta-subunits (35) by 20 µl of supernatant from samples containing PKC only (lane 1), PKC + betaARK1 (lane 2), and betaARK1 only (lane 3), which were previously incubated for 1 h at 30 °C to allow phosphorylation of betaARK1 by PKC. The arrow indicates bands of phosphorylated rhodopsin (opsin) as revealed by autoradiography after polyacrylamide gel electrophoresis. Results from three independent experiments (means ± S.E.) are shown in the histogram on the right. *, significantly different from betaARK1 activity not exposed to PKC (p < 0.005, n = 3).



The effect of PKC on kinetic parameters of betaARK phosphorylating activity were determined in experiments in which different concentrations of rhodopsin were used as substrate. Using a double-reciprocal plot, we found a 31% decrease in K and a 10% increase in V(max) values (average of two experiments, data not shown), and these changes can account for the increased phosphorylating activity of betaARK induced by PKC.

The betaARK1 purified from Sf9/baculovirus expression system used for the above experiments migrated on the gel as a doublet, indicating that a fraction of protein generated by this expression protocol was not properly processed. This can explain the relatively low phosphorylation activity toward rhodopsin of this betaARK1 preparation compared to those reported by others(22, 23) . More recently, using slightly different protocol of infection and a different batch of cells (see ``Materials and Methods''), we obtained new preparations in which betaARK1 migrated on polyacrylamide gel as a single band (80 kDa) and had a much higher rhodopsin phosphorylating activity (4-6 nmol phosphate/min/mg in the absence of G/beta) compared to the previous batch. This activity is within the range of the values previously reported(22, 23) . The identification of this band was confirmed by immunoblotting. Phosphorylation and activation of betaARK by PKC was confirmed in experiments done with this new preparation of betaARK1. PKC was able to phosphorylate this betaARK1 (Fig. 6A), and this resulted in an increased ability of betaARK1 to phosphorylate rhodopsin (from 3.6 to 7.0 nmol/min/mg without or with phosphorylation by PKC, respectively, Fig. 6B).


Figure 6: Phosphorylation and activation of betaARK1, obtained by an alternative expression protocol, by PKC. A, a new betaARK1 preparation from Sf9 cells in which the kinase appeared as a single band on the gel was used (see ``Materials and Methods'' for details). Samples containing partially purified PKC only (lane 1), PKC + betaARK1 (lane 2), and betaARK1 only (lane 3) were incubated under phosphorylation conditions for 1 h. The arrow indicates the band corresponding to betaARK1 as revealed by autoradiography after polyacrylamide gel electrophoresis. B, assessment of rhodopsin phosphorylating activity of betaARK1 after phosphorylation by PKC. Rhodopsin phosphorylation was carried out as described under ``Materials and Methods,'' using 20 µl of supernatant from samples containing PKC only (lane 1), PKC + betaARK1 (lane 2), and betaARK1 only (lane 3), which were previously incubated for 1 h at 30 °C to allow phosphorylation of betaARK1 by PKC. The arrow indicates bands of phosphorylated rhodopsin (opsin) as revealed by autoradiography after polyacrylamide gel electrophoresis. These experiments are representatives of two.



To investigate whether PKC can phosphorylate betaARK in living cells, the phosphorylation state of betaARK1 was examined in MNL and Sf9 cells preloaded with labeled [P]orthophosphate and then treated with PMA. First, MNL were used since PKC activation in these cells caused increases in betaARK activity (see Fig. 1). We found that the level of phosphorylation of betaARK1 immunoprecipitated from MNL treated with PMA was enhanced by about 3-fold compared to that from control MNL (Fig. 7). Immunoblot performed on parallel samples confirmed that equal amounts of betaARK1 were immunoprecipitated (Fig. 7). Sf9 cells overexpressing betaARK1 were also used for the same experiments to provide an improved signal of phosphorylated betaARK1 and to obtain adequate amount of immunoprecipitated betaARK1 for functional assays(24) . We found that the PKC-induced betaARK1 phosphate incorporation level was increased by approximately 2-fold compared to the autophosphorylation level. The rhodopsin phosphorylating activity of betaARK1 immunoprecipitated from PMA-treated Sf9 was 30% higher than that from untreated cells (not shown).


Figure 7: Phosphorylation of betaARK1 by PKC in living cells. MNL and Sf9 cells overexpressing betaARK1, prelabeled for 3 h in medium containing 1 mCi/ml [P]orthophosphate, were treated with PMA (1 µM) or vehicle for 20 min. betaARK1 was then immunoprecipitated from their cytosolic preparations and analyzed by polyacrylamide gel electrophoresis. On the left, immunoblot (imm) and autoradiogram (auto) of parallel samples of immunoprecipitated betaARK1 from MNL are shown. On the right is the autoradiogram of immunoprecipitated betaARK1 from Sf9 cells. The arrow indicates bands of betaARK1. The results shown are representatives of two similar experiments.



To test the physiological significance of PKC-mediated augmentation in cytosolic betaARK activity, we examined the effect of PMA pretreatment on betaAR homologous desensitization in MNL. In control cells, betaAR homologous desensitization resulted in 42 ± 10% reduction in receptor responsiveness as measured by isoproterenol-induced cAMP accumulation, while in PMA-treated MNL, the level of desensitization was increased up to 68 ± 8% (Fig. 8, left panel). cAMP accumulation values (pmol/mg of proteins) were 375 ± 32 and 220 ± 47 for undesensitized and desensitized control MNL, respectively, and 464 ± 9 and 148 ± 39 for undesensitized and desensitized PMA-treated MNL, respectively. Forskolin-stimulated cAMP accumulation was unaffected by isoproterenol pretreatment in both control and PMA-pretreated cells (not shown), indicating that homologous (i.e. agonist-specific) desensitization is selectively augmented by PMA. To confirm that the PMA-induced increase in homologous desensitization was mediated by betaARK, experiments were carried out using the betaARK inhibitor heparin on permeabilized cells(25) . In the presence of heparin (Fig. 8, right panel), homologous desensitization, as assessed by receptor-stimulated adenylyl cyclase activity, was decreased to almost the same level in cells with or without PMA pretreatment. Isoproterenol-stimulated adenylyl cyclase activity values (pmol cAMP/mg/min) were, in the absence of heparin, 48.8 ± 1.3 and 30.3 ± 0.8 for undesensitized and desensitized control MNL, respectively, and 47.9 ± 0.4 and 23.5 ± 0.5 for undesensitized and desensitized PMA-treated MNL, respectively; in the presence of heparin, 36.2 ± 1.5 and 34.1 ± 0.3 for undesensitized and desensitized control MNL, respectively, and 42.1 ± 0.3 and 39.9 ± 1.5 for undesensitized and desensitized PMA-treated MNL, respectively.


Figure 8: PMA pretreatment increased betaARK-mediated beta-adrenergic receptor homologous desensitization in MNL. The level of desensitization (as induced by 10 µM isoproterenol for 5 min at 37 °C) was compared in MNL untreated (considered as 100%) or exposed to 1 µM PMA for 15 min. In undesensitized and desensitized MNL, receptor responsiveness was assessed by either agonist-stimulated cAMP accumulation (left panel) or adenylyl cyclase activity in membrane preparation (right panel). Shown are means ± S.E.; *, significantly different from relative control (for the left panel, p < 0.01, n = 5; for the right panel, p < 0.05, n = 4). The experiments on the right panel, in which heparin was used as betaARK inhibitor, were performed on permeabilized MNL to allow heparin to enter the cells.




DISCUSSION

In the present study we show that betaARK can be phosphorylated by PKC and that this results in enhanced potency of homologous desensitization. Different types of converging evidence support this conclusion: (i) cytosolic betaARK activity was increased after activation of PKC in MNL; (ii) betaARK1 was directly phosphorylated by PKC; (iii) rhodopsin phosphorylating activity of betaARK1 was increased after phosphorylation by purified PKC; (iv) betaARK1 was phosphorylated in living cells after PMA treatment; (v) the potency of betaAR homologous desensitization was enhanced in PMA-treated MNL, and this effect was prevented by the betaARK inhibitor heparin.

Direct phosphorylation of pure betaARK1 by PKC was demonstrated using purified PKC in an in vitro assay. Pharmacological proof that the phosphorylation of betaARK1 was due to PKC was provided by its inhibition by staurosporin and, importantly, by calphostin C, a highly specific PKC inhibitor which acts on the regulatory domain of PKC, with negligible activity on other protein kinases(15, 26) . Immunoprecipitation and immunoblotting with anti-betaARK1 antibodies further identified phosphorylated betaARK1. Finally, using a near homogeneous preparation of PKC, phosphorylation of betaARK1 was confirmed.

Phosphorylation of betaARK1 by PKC resulted in an increased betaARK activity toward rhodopsin. The magnitude of this increase in betaARK activity was similar to that observed on betaARK from living MNL in which PKC was activated either by ionomycin or PMA (see Fig. 1C). Significantly, PKC displays a high affinity toward betaARK1, suggesting that the interaction between these two kinases is biologically relevant.

Phosphorylation of betaARK1 by PKC was observed on living cells using two different cellular models. MNL provided a physiological model since the quantitative and qualitative parameters of betaARK1 and PKC were defined physiologically by the cells. Sf9 cells with overexpressed betaARK1 provided a less physiological model especially with regard to the PKC/betaARK1 stoichiometry. This may be the reason why the magnitude of increase in phosphate incorporation into betaARK1 in PMA-treated cells was lower in Sf9 cells than in MNL. It did nevertheless provide a convenient means for visualization of betaARK1 phosphorylation(24) . In addition, the high levels of betaARK1 in these cells allowed us to examine specifically the activity of betaARK1 immunoprecipitated. Activity of betaARK1 immunoprecipitated from Sf9 cells treated with PMA was increased compared to that from control cells. These results strongly indicate that the phosphorylation of betaARK1 by PKC is physiologically relevant.

To confirm the effect of PKC activation on betaARK with a functional approach on cells, the potency of isoproterenol-induced rapid homologous desensitization following PMA pretreatment was examined. In the present desensitization experiments, 5 min were allowed for 10 µM of isoproterenol to induce homologous desensitization to ensure that the desensitization was mediated mainly by betaARK(17, 27) . PMA pretreatment potentiated the degree of desensitization from 42 to 68%, which is the functional effect expected for the increased betaARK activity observed in these cells (see Fig. 1). Similar results were obtained by Chambaut-Guerin and Thomopoulos (28) in murine macrophage J774 cells. Additionally, as in their study, we also observed that PMA increased betaAR responsiveness, as measured by isoproterenol-induced cAMP accumulation. This is not surprising as it is known that PKC may affect the betaAR-G(s)-adenylate cyclase axis to alter the receptor-mediated response(29) . Using heparin to inhibit betaARK in permeabilized cells(25) , the increase in homologous desensitization caused by PMA pretreatment was prevented, thus confirming that betaARK was responsible for this effect.

Four subtypes of GRKs are expressed in MNL, namely betaARK1, betaARK2, GRK5, and GRK6(9) . The present results in vitro as well as in living cells strongly suggest a major role of betaARK1 in the PKC-induced increase in homologous desensitization. However, the possible role of other subtype(s) in this process remains to be investigated.

The mechanism mediating betaARK-PKC interaction is not known, although other studies suggest the involvement of PH-domain(30, 31) . The PH-domain is composed of approximately 100 loosely conserved amino acids, so far found to be present in a large nubmer of intracellular signaling proteins, including betaARK1 and 2(30) . Recently, two kinases have been shown to bind directly to PKC through their respective PH-domains(31, 32) . Although no consensus amino acid sequence or structure within these two PH-domains critical for binding PKC were identified, mutation of Arg-28 of the Bruton tyrosine kinase, which causes the genetic disease agammaglobulinemia, reduced the capacity of its PH-domain in binding PKC(31) . Arg-28 of Bruton tyrosine kinase was therefore suggested to participate in PKC binding. Interestingly, this amino acid is conserved in most of the PH-domains identified so far, including that of betaARK1 (Arg-579)(30) . This would support direct binding of PKC to betaARK1, as is suggested by our observation of direct phosphorylation of betaARK1 by PKC.

Our results suggest a novel level of signal transduction cross-talk by showing that the activity of betaARK can be increased by direct PKC phosphorylation. This indicates that the efficacy of homologous desensitization, which regulates the responsiveness of many receptors, can be modulated heterologously within the cell. Other members of the GRK family may also be modified by PKC and/or other intracellular enzymes and such a mechanism may underlie phenomena like ``receptor class desensitization''(33) . In view of the fact that PKC is a key enzyme involved in signal transduction and cell proliferation(19) , and that a number of G-protein-coupled receptors have been shown to be protooncogenes(34) , the present finding may have significantly wider implications.


FOOTNOTES

*
This work was supported in part by a grant from P. F. Ingegneria Genetica, CNR, and by Telethon Grant E. 62 (Italy). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Consorzio Mario Negri Sud, via Nazionale, 66030 S. Maria Imbaro, Italy. Tel.: 39-872-570.351; Fax: 39-872-578.240; deblasi{at}cmns.mnegri.it.

^1
The abbreviations used are: betaARK, beta-adrenergic receptor kinase; betaAR, beta-adrenergic receptor; DMEM, Dulbecco's modified Eagle's medium; GRK, G-protein coupled receptor kinase; H7, (1-5(isoquinoline sulfonyl)-2-methylpiperazine); MNL, mononuclear leukocytes; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; ROS, rod outer segment; PH-domain, pleckstrin homology domain; G/beta, beta-subunits of heterotrimeric G-protein.


ACKNOWLEDGEMENTS

We are grateful to Dr. P. J. Parker of the Imperial Cancer Research Funds for the generous gift of pure PKC. We thank E. Pompili for advice in immunoprecipitation, M. Sallese and S. Pirocchi for Sf9 betaARK1, M. Molino for calcium measurements, S. Sozzani for advice in PKC experiments, D. Talone for excellent technical assistance, and R. Bertazzi and S. Menna for expert assistance in preparation of the figures.


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