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J Biol Chem, Vol. 274, Issue 39, 28003-28010, September 24, 1999

Cyclic AMP-dependent Phosphorylation of Thromboxane A₂ Receptor-associated G₁₃^*

Jeanne M. Manganello, Yasmine Djellas, Catherine Borg, Kostas Antonakis, and Guy C. Le Breton^§

From the Department of Pharmacology, University of Illinois, Chicago, Illinois 60612 and  Vectorologie Moléculaire et Cellulaire, Ecole Nationale Supérieure de Chimie de Paris (Unité Mixte Recherche 133), CNRS, Paris 75231, France

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Although it is well established that cAMP inhibits platelet activation induced by all agonists, the thromboxane A₂ signal transduction pathway was found to be particularly sensitive to such inhibition. Therefore, we examined whether cAMP-dependent kinase mediates phosphorylation of the thromboxane A₂ receptor-G-protein complex. It was found that cAMP induces protein kinase A-dependent [-³²P]ATP labeling of solubilized membrane proteins in the region of G subunits, i.e. 38-45 kDa. Moreover, ligand affinity chromatography purification of thromboxane A₂ receptor-G-protein complexes from these membranes revealed that 38-45-kDa phosphoproteins co-purify with thromboxane A₂ receptors. Immunoprecipitation of the affinity column eluate with a G₁₃ antibody demonstrated that 8-Br-cAMP increased phosphorylation of thromboxane A₂ receptor-associated G₁₃ by 87 ± 27%. In separate experiments, immunopurification of G₁₃ on microtiter wells coated with a different G₁₃ antibody revealed that 8-Br-cAMP increased G₁₃ phosphorylation by 53 ± 19%. Finally, treatment of ³²P-labeled whole platelets with prostacyclin resulted in a 90 ± 14% increase in phosphorylated G₁₃ that was abolished by pretreatment with the adenylate cyclase inhibitor MDL-12. These results provide the first evidence that protein kinase A mediates phosphorylation of G₁₃ both in vitro and in vivo and provides a basis for the preferential inhibition of thromboxane A₂-mediated signaling in platelets by cAMP.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Thromboxane A₂ (TXA₂)¹ is a potent platelet agonist and vasoconstrictor (1, 2) that modulates not only the hemostatic process but also potentiates thrombotic events such as myocardial infarction and stroke (3-7). On this basis, investigators have sought to understand better TXA₂-induced signal transduction processes in an attempt to develop new therapeutic strategies for managing TXA₂ receptor-mediated thrombotic events. However, despite these efforts, significant gaps remain in our understanding of signal transduction, and modulation of signal transduction, through this pathway. At present, it is known that at least one pool of platelet TXA₂ receptors is coupled to phospholipase C through the pertussis toxin-insensitive G-protein G_q (8-14). Thus, TXA₂ receptor activation of phospholipase C leads to inositol triphosphate/diacylglycerol generation and a subsequent mobilization of intraplatelet calcium (8-10). More recently, evidence has been provided that, in addition to G_q, TXA₂ receptors also couple to the G₁₃ subunit (14-17). Although the effector for this putative signaling protein has not yet been established in platelets, a variety of G₁₃-mediated effects in other cell systems (18-25) have been proposed.

Regarding modulation of TXA₂ receptor activity, recent studies have focused on cyclic nucleotide-mediated phosphorylation of specific platelet proteins (26-29). Thus, it is well known that platelet activity is inhibited by elevated cAMP levels, and it is generally accepted that this inhibitory effect is mediated by protein kinase A (PKA)-dependent phosphorylation of different protein substrates. For example, cAMP-induced phosphorylation of certain downstream signaling components such as myosin light chain kinase and thrombolamban causes inhibition of contractile processes and calcium-dependent processes, respectively (30-33). Furthermore, separate studies have established a direct relationship between platelet cAMP levels and the ability of TXA₂ receptors to mobilize intraplatelet calcium (34). This latter phenomenon, in turn, raises the possibility that the TXA₂ signaling components themselves may serve as potential PKA substrates.

In this connection, it is known that phosphorylation of receptors by second messenger kinases can modulate their function (35-38). This phenomenon has been well studied in the -adrenergic receptor system, in which PKA-induced receptor phosphorylation has been linked to heterologous receptor desensitization (39, 40). On the other hand, the physiological consequence of PKA-mediated phosphorylation of TXA₂ receptors is unclear. Thus, even though it was found that TXA₂ receptor protein can be phosphorylated by PKA in receptor expression systems, the authors of this study (27) concluded that PKA had no functional significance in mediating agonist-induced TXA₂ receptor desensitization. In contrast to cAMP, however, other investigators (29) have recently reported that cGMP-dependent phosphorylation of TXA₂ receptors by protein kinase G (PKG) inhibits agonist-induced GTPase activity in platelet membranes. This finding may in part explain the inhibitory effects of cGMP on platelet function.

Finally, phosphorylation of G-proteins has also been shown to constitute a mechanism for signal modulation through seven transmembrane receptors. For example, protein kinase C (PKC) has been shown to induce phosphorylation of at least three G subunits, i.e. G_i, G_z, and G₁₂ (41-47). Results regarding PKC-mediated phosphorylation of G₁₃, however, are not definitive. Thus, whereas Offermanns et al. (47) found that G₁₃ can be phosphorylated by PKC in platelets as well as in a reconstituted system, Kozasa and Gilman (46) reported that the purified G₁₃ subunit was not a PKC substrate. In contrast to PKC, there have been fewer reports of G-protein phosphorylation induced by cyclic nucleotides. In this regard, PKA- and PKG-mediated phosphorylation of G subunits has only been described to occur in limited cell types and has not yet been demonstrated to occur in platelets (43, 47-51).

In summary, various mechanisms have been proposed for cyclic nucleotide-induced modulation of signaling either at the TXA₂ receptor level itself or at more distant effector sites. In order to provide additional information concerning this potentially important regulatory process, the present study investigated the ability of cAMP to induce phosphorylation of TXA₂ receptor-associated G-proteins. Since other investigators (43) have reported that platelet G_q is not a PKA substrate, we examined the ability of PKA to phosphorylate TXA₂ receptor-coupled G₁₃. In these studies, it was found in both platelet membranes and intact platelets that cAMP stimulates phosphorylation of G₁₃. These results provide the first evidence for PKA-mediated phosphorylation of an endogenous G-protein  subunit in human platelets and identify one such  subunit as being TXA₂ receptor-associated G₁₃. This ability of cAMP to cause G₁₃ phosphorylation may, in part, explain our separate finding that TXA₂-mediated signal transduction is preferentially sensitive to inhibition by prostacyclin (PGI₂).

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- Outdated platelet units were obtained from Heartland Blood Center (Aurora, IL) and Life Source Blood Services (Chicago, IL); BM13.177 was generously supplied by Dr. K. Stegmeier, Roche Molecular Biochemicals; the SQ intermediate (ethyl-(1S)-(1,2-(Z),3,4)-7-((3-aminomethyl)-7-oxabicyclo[2.2.1]hept-2-yl)-5-heptenoate) for the synthesis of SQ31,491 was provided by the Squibb Institute for Medical Research (NJ). Cyclic AMP, 8-Br-cAMP, ADP, IP₂₀, CHAPS, A23187, indomethacin, isobutylmethylxanthine (IBMX), apyrase, normal rabbit IgG, bovine serum albumin, and protein A-Sepharose CL-4B were purchased from Sigma; asolectin was from the American Lecithin Co. (Atlanta, GA); MDL-12 and okadaic acid were obtained from Calbiochem; [-³²P]ATP and [³²P]H₃PO₄ were purchased from ICN Biochemical, Inc. (Irvine, CA). The thrombin receptor-activating protein (TRAP) refers to the first six amino acids of the new amino terminus revealed after thrombin cleavage, i.e. SFLLRN (TRAP^42-47) and was purchased from Research Genetics, (Huntsville, AL). U46619 and PGI₂ were purchased from Cayman Chemicals (Ann Arbor, MI); tissue solubilizer-1 was obtained from Research Products International, Corp. (Mount Prospect, IL); 4-chloro-1-naphthol (horseradish peroxidase color development reagent) was from Bio-Rad; biotinylated goat anti-rabbit IgG (H + L) and the Vectastain ABC kits were purchased from Vector Laboratories (Burlingame, CA). The G₁₃-N IgG rabbit polyclonal antibody was purchased from Santa Cruz Laboratories (Santa Cruz, CA). The anti-peptide G-13 antibody, directed against an internal G₁₃ sequence, (Table I) was raised in rabbits according to standard protocols, and the IgG fraction was purified by protein A-Sepharose chromatography as described (17).

Platelet Membrane Preparation and Solubilization-- Typically, 12 units of outdated human platelet concentrate were pooled and incubated with 3 mM aspirin for 45 min. Solubilized platelet membranes were then prepared as described (52) and typically resulted in a final protein concentration of 4.5 mg/ml. Platelet membranes were prepared using a modification of this protocol. Specifically, platelet concentrate was centrifuged at 160 × g for 15 min to remove red blood cells. The platelet-rich plasma was supplemented with 40 nM PGI₂, and platelets were pelleted by centrifugation at 1600 × g for 20 min. The platelet pellet was suspended in buffer A (25 mM Tris-HCl, 5 mM MgCl₂, pH 7.4) plus PGI₂ (40 nM) and was re-centrifuged at 1600 × g for 20 min. The resulting platelet pellet was resuspended in buffer A, was sonicated on ice (45-s bursts with 15-s rests) for 4 min, and was centrifuged at 1600 × g for 5 min. The supernatant was placed into centrifuge tubes, and the pellet was again resuspended, sonicated, and centrifuged. The supernatants were then combined and centrifuged at 100,000 × g for 30 min. The resulting membrane pellet was resuspended in buffer A to yield a final protein concentration of 2-4 mg/ml.

Polyacrylamide Gel Electrophoresis-- SDS-PAGE was performed according to the method of Laemmli (53), and the gels were silver-stained as described by Morrissey (54), dried, and subjected to autoradiography. Silver-stained gels and autoradiographic films were analyzed by using a Protein Databases, Inc. densitometer (Huntington Station, NY).

Phosphorylation of Platelet Membranes and Solubilized Platelet Membranes-- Solubilized platelet membranes were phosphorylated (2-4 mg of protein) in a reaction mixture containing 1 µM cAMP, 1 mM 8-Br-cAMP or vehicle (deionized water), 100-150 µM [-³²P]ATP (4500 Ci/mmol), 60 µM CaCl₂, and incubation buffer (45 mM histidine HCl, 50 mM KH₂PO₄, 20 mM NaF, 120 mM KCl, pH 7.4) in a total volume of 1600-2300 µl. In some experiments, NaF was 40 mM, and 2 mM sodium orthovanadate was added as an additional phosphatase inhibitor. In other experiments, the specific PKA inhibitor, IP₂₀ (55), was added (3.6 µM). The reaction mixture was allowed to incubate for 30 min at 25 °C, after which time the phosphorylated proteins were either added to Laemmli sample buffer (62.5 mM Tris-HCl, 3% SDS, 10% glycerol, pH 6.8) and resolved by SDS-PAGE, purified by ligand affinity chromatography as described under "Ligand Affinity Purification of TXA₂ Receptor-G-Protein Complexes," or added to G-13 antibody-coated microtiter plates as described under "Immunological Techniques."

In separate experiments, platelet membranes (1-2 mg of protein) were phosphorylated in a reaction mixture containing 1 µM cAMP, 86 µM [-³²P]ATP (4500 Ci/mmol), 1 mM EGTA, and incubation buffer in a volume of 2.25 ml. Following incubation for 30 min at 25 °C, the membranes were centrifuged for 15 min at 100,0 × g. The pellet was washed twice with buffer A and was solubilized in 2 ml of buffer B (10 mM CHAPS, 50 mM Tris, 5 mM MgCl_2, pH 7.4) that was supplemented with 20 mM NaF. The solubilized membranes were then centrifuged at 100,000 × g for 30 min. The resulting supernatant was subjected to ligand affinity chromatography in order to purify the TXA₂ receptor-G-protein complex, as described below.

Ligand Affinity Purification of TXA₂ Receptor-G-Protein Complexes-- Platelet TXA₂ receptor-G-protein complexes were purified as described previously (52). Briefly, phosphorylated, solubilized platelet membranes were supplemented with 500 mM KCl, 0.5 mg/ml asolectin, 20% glycerol, 0.2 mM EGTA, and 10 mM CHAPS and were incubated overnight with the ligand affinity column matrix, which utilizes the TXA₂ receptor antagonist SQ31,491 coupled to Affi-Gel 102. Unbound proteins were washed away with buffer D (20 mM Tris base, 10 mM CHAPS, 20% glycerol, 500 mM KCl, 0.2 mM EGTA, 0.5 mg/ml asolectin, pH 7.4) that was supplemented with 20 mM NaF and 2 mM sodium orthovanadate. The TXA₂ receptor-G-protein complexes were eluted from the column using the TXA₂ antagonist 50 mM BM13.177 in buffer D at a flow rate of 1 ml/8 min. The first 1-ml elution fraction was collected, and the column was clamped for 30 min. Subsequently, two 1-ml elution fractions were collected, and the fractions were pooled, concentrated, and subjected to SDS-PAGE. To determine whether the same amount of receptor was eluted from each column, the pooled elution fraction was subjected to SDS-PAGE, and the gels were silver-stained. Densitometric analysis of the stained receptor bands verified that the same amount of receptor protein was eluted in each set of experiments. In separate experiments, the pooled elution fraction was used for immunoprecipitation studies using the G₁₃-N IgG as described under "Immunological Techniques."

Incubation of Platelets with [³²P]H₃PO₄-- Human platelet-rich plasma (PRP) was prepared from acid/citrate/dextrose anticoagulated blood as described (34). Platelets were then ³²P-labeled by a modification of the method of Carlson et al. (41). Briefly, PRP was incubated with 3 mM aspirin for 45 min at room temperature to inhibit cyclooxygenase. Platelets were collected from PRP by centrifugation at 1000 × g for 10 min and were washed once in a solution of modified Tyrode's buffer (20 mM HEPES, 138 mM NaCl, 2.9 mM KCl, 1 mM MgCl₂, 1 mM glucose, 5 mM EDTA, 0.35% bovine serum albumin, 60 µg/ml apyrase) to which 50 units/ml heparin was added. Platelets were pelleted by centrifugation at 1000 × g for 10 min, resuspended in modified Tyrode's buffer (without heparin) to a concentration of 2 × 10⁹ cells/ml, and were incubated with 0.5 mCi/ml [³²P]H₃PO₄ (9000 Ci/mmol) for 60 min at room temperature. Following incubation with 3.3 mM NaH₃PO₄ for 15 min, portions of the platelet suspension (e.g. 2 × 10⁹ cells) were incubated with 500 µM MDL-12 or vehicle (10 mM HEPES, 150 mM NaCl, pH 7.4) for 15 min and were subsequently incubated with 1 mM IBMX and 1 µM PGI₂ or vehicle (ethanol and 0.05 M Tris buffer, pH 9, respectively) for 5 min. Samples were immediately cooled in an ice slurry, and the platelets were pelleted by centrifugation at 7000 × g for 3 min and were washed twice in ice-cold buffer A that was supplemented with 2 µM okadaic acid. Platelets were solubilized in buffer B containing 2 µM okadaic acid and were sonicated (30-s bursts with 15-s rests) for 4 min and homogenized (10 strokes with a Teflon homogenizer). The solubilized mixture was centrifuged at 100,000 × g for 30 min, and the sonication and centrifugation steps were repeated. The solubilized proteins were subjected to immunoprecipitation as described below. Precipitated ³²P counts were normalized for the amount of immunoprecipitated G₁₃ by immunoblotting the precipitated protein fraction with the G₁₃-N antibody and analyzing the labeled protein by densitometry.

Immunological Techniques-- For immunoprecipitation experiments, 1 ml of the pooled ligand affinity column eluate was incubated with 75 µl of either G₁₃-N IgG (200 µg/ml) or preimmune IgG (200 µg/ml) and was mixed overnight at 4 °C. Protein-A Sepharose beads were then added (55 µl of a 10% (w/v) suspension). After mixing overnight at 4 °C, the beads were isolated by centrifugation and were washed three times with 400 µl of buffer containing 10 mM CHAPS in phosphate-buffered saline, pH 7.4. The final pellet was boiled for 5 min in the presence of Laemmli sample buffer (which was devoid of bromphenol blue) plus buffer B, and the mixture was centrifuged to pellet the beads. The resulting supernatant (60 µl) was added to scintillation mixture, and the ³²P-labeled proteins were quantified using a liquid scintillation spectrometer (Beckman LS 6800). Specific phosphorylation was calculated as the difference between counts obtained with G₁₃-N IgG and preimmune IgG. For the whole cell experiments, G₁₃ was precipitated using 150 µl of G₁₃-N IgG and 55 µl of the protein A-Sepharose bead suspension.

For immunoaffinity purification of G₁₃ on microtiter plates, Immulon 2 microtiter wells were coated overnight at 4 °C with 50 µl of G-13 IgG (100 µg/ml) or preimmune IgG (100 µg/ml). The wells were then blocked with 3% bovine serum albumin in phosphate-buffered saline for 1 h at 25 °C and were washed once with 300 µl of phosphate-buffered saline. Solubilized platelet membranes that were phosphorylated in the presence or absence of 1 mM 8-Br-cAMP (50 µl), as described previously, were incubated on the antibody-coated microtiter wells overnight at 4 °C. The wells were washed three times with 300 µl of buffer B, and the labeled protein was eluted from the wells using 50 µl of tissue solubilizer. The eluted, ³²P-labeled proteins were added to scintillation mixture and were quantified by liquid scintillation counting. Specific phosphorylation was calculated as the difference between counts obtained with G-13 IgG and preimmune IgG.

Immunoblot Analysis-- Proteins were transferred electrophoretically from SDS gels onto nitrocellulose membranes according to the method of Towbin et al. (56), and membranes were blocked with 3% gelatin in Tris-buffered saline. The membranes were incubated overnight at room temperature with the indicated dilution of G₁₃-N IgG or preimmune IgG. Membranes were then washed with 0.1% Tween in Tris-buffered saline and were treated with biotinylated goat anti-rabbit IgG (H + L) as the secondary antibody. Immunoreactive proteins were conjugated with horseradish peroxidase-labeled avidin and were detected using 4-chloro-1-naphthol as the substrate (0.5 mg/ml).

Platelet Aggregation-- Blood was drawn into acid/citrate/dextrose anticoagulant, and PRP was prepared as described above. Platelets were treated for 1 min with indomethacin (10 µM) in order to prevent endogenous TXA₂ production. Aggregation was stimulated with either U46619, ADP, TRAP, or A23187 at a concentration that yielded approximately 85% of the maximal aggregation response (sub-maximal dose), and the aggregation response was measured by the turbidimetric method (57) using a model 400 Lumi-aggregometer (Chronolog Corp., Havertown, PA). The inhibitory effect of cAMP was measured by adding PGI₂ (0.25-1 nM) to the PRP 1 min prior to addition of agonist. The concentration of PGI₂ was titrated to a dose that substantially inhibited U46619-induced aggregation (at the sub-maximal concentration), and the effect of that same dose of PGI₂ was measured on aggregation induced by the sub-maximal concentrations of ADP, TRAP, and A23187. The dose-dependent inhibitory effect of PGI₂ was determined in indomethacin-treated platelets by incubating PRP for 1 min with various concentrations of PGI₂, as indicated, and subsequently stimulating aggregation with the sub-maximal dose of each agonist.

Statistical Analysis-- Data were analyzed according to the Student's two-tailed paired t test or one-way analysis of variance, as indicated, using GraphPad PRISM statistical software (San Diego, CA). Statistical significance is defined as p < 0.05 or p < 0.01, as indicated.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

cAMP-dependent Phosphorylation of Solubilized Platelet Membranes-- In order to study the possibility that cAMP-dependent kinase can induce phosphorylation of G-protein  subunits, we examined whether cAMP induces phosphorylation of a platelet membrane protein(s) at the molecular weight region in which G proteins have been detected immunologically (58-60). In these experiments, platelet membranes were solubilized in CHAPS and were phosphorylated by [-³²P]ATP in the presence or absence of cAMP (1 µM). After a 30-min incubation, the reaction was terminated by adding samples to Laemmli buffer, and the phosphorylated proteins were separated by SDS-PAGE and were visualized by silver staining. Detection of the ³²P-labeled proteins by autoradiography revealed that cAMP induced substantial phosphorylation at several molecular masses, notably at 22 and 38 45 kDa (Fig. 1). The PKA dependence of this phosphorylation was next examined using the specific PKA inhibitor IP₂₀ to inhibit endogenous PKA activity. In these experiments, solubilized platelet membranes were incubated with [-³²P]ATP in the presence of cAMP (1 µM), cAMP (1 µM) plus IP₂₀ (3.6 µM), or vehicle. After a 30-min incubation, the phosphorylated proteins were resolved by SDS-PAGE. It can be seen from the autoradiogram (Fig. 2) that the increase in phosphorylation induced by cAMP in both the 22-kDa and the 38-45-kDa regions (lane 2) was inhibited to nearly control levels (lane 1) by IP₂₀ (lane 3). These results, therefore, establish that a significant amount of the observed phosphorylation is PKA-specific. Although the 22-kDa protein is thought to be PKA-phosphorylated thrombolamban (32, 33), the identity of the 38-45-kDa proteins is currently unknown. However, since G-protein  subunits are known to migrate to this molecular weight region upon electrophoresis, the next series of experiments investigated whether the 38-45-kDa phosphoproteins may represent PKA-phosphorylated G-proteins.

View larger version (35K): [in this window] [in a new window]   Fig. 1.   Phosphorylation of platelet membranes by cAMP. Solubilized platelet membranes were incubated with [-32P]ATP in the presence or absence of 1 µM cAMP as described under "Experimental Procedures." The phosphorylated proteins were separated by SDS-PAGE and were silver-stained. The autoradiogram shown is representative of five separate experiments. Lane 1, vehicle; lane 2, 1 µM cAMP.

View larger version (38K): [in this window] [in a new window]   Fig. 2.   Specificity of cAMP-induced phosphorylation of 38-45-kDa proteins. Solubilized platelet membranes were incubated with [-32P]ATP in the presence of vehicle (lane 1), 1 µM cAMP (lane 2), or 1 µM cAMP plus 3.6 µM IP20 (lane 3) as described under "Experimental Procedures." The phosphorylated proteins were resolved by SDS-PAGE and were visualized by silver staining. The autoradiogram shown is representative of three separate experiments.

Purification of TXA₂ Receptor-G-protein Complexes-- Previous studies in our laboratory have demonstrated that ligand affinity chromatography purification of TXA₂ receptors from solubilized platelet membranes results in co-purification of TXA₂ receptors with their associated G-proteins (13, 17, 52). Based on this consideration, experiments that employed this technique were conducted to determine whether 38-45-kDa phosphoproteins are TXA₂ receptor-associated. In these studies, platelet membranes were phosphorylated by [-³²P]ATP in the presence or absence of cAMP (1 µM). After a 30-min incubation, the membranes were solubilized in CHAPS and were incubated with the TXA₂ receptor affinity matrix. After washing the column to remove unbound proteins, TXA₂ receptor-G-protein complexes were specifically eluted using the TXA₂ receptor antagonist BM13.177. The column eluate was subjected to SDS-PAGE to resolve the purified proteins, and the gel was silver-stained. It can be seen from the autoradiogram (Fig. 3) that phosphoproteins at approximately 44 kDa co-eluted with the TXA₂ receptors upon ligand affinity chromatography purification. Analysis of the autoradiogram by densitometry revealed that cAMP induced a 97 ± 13% increase in phosphorylation of these TXA₂ receptor-associated proteins. To verify that the increase in phosphorylation was not due to an increase in the amount of receptor eluted from each column, the silver-stained receptor bands were analyzed by densitometry. Thus, it was confirmed that the same amount (±4%) of receptor protein was eluted from each column.

View larger version (34K): [in this window] [in a new window]   Fig. 3.   Purification of TXA2 receptor-G-protein complexes from cAMP-phosphorylated platelet membranes. Platelet membranes were phosphorylated by [-32P]ATP in the presence or absence of 1 µM cAMP and were solubilized in CHAPS. TXA2 receptor-G-protein complexes were then purified by ligand affinity chromatography as described under "Experimental Procedures," and the affinity column eluate was subjected to SDS-PAGE. The autoradiogram shown is representative of three separate experiments. Lane 1, vehicle; lane 2, 1 µM cAMP.

Similar phosphorylation results were obtained using a preparation of solubilized platelet membranes. In these experiments, CHAPS-solubilized platelet membranes were phosphorylated by [-³²P]ATP in the presence or absence of cAMP (1 µM). The phosphorylated, solubilized membranes were then purified by ligand affinity chromatography and were separated by SDS-PAGE. Autoradiograms from these experiments also revealed the presence of TXA₂ receptor-associated phosphoproteins in the 38-45-kDa range (Fig. 4). Densitometric analysis of the 38-45-kDa region demonstrated that cAMP induced a 64 ± 9% increase in phosphorylation of the TXA₂ receptor-associated proteins. Therefore, in both intact membranes and in solubilized membranes, it appears that cAMP causes phosphorylation of proteins that are in the molecular weight region of  subunits and that co-purify with TXA₂ receptors.

View larger version (29K): [in this window] [in a new window]   Fig. 4.   Purification of TXA2 receptor-G-protein complexes from cAMP-phosphorylated, solubilized platelet membranes. Platelet membranes were first solubilized in CHAPS and were subsequently incubated with [-32P]ATP in the presence of 1 µM cAMP (lane 2) or vehicle (lane 1). TXA2 receptor-G-protein complexes were purified by ligand affinity chromatography, and the column eluate was subjected to SDS-PAGE. The autoradiogram shown is representative of seven separate experiments.

Immunopurification of Phosphorylated G₁₃ from Solubilized Membranes-- Experiments were next performed to identify these TXA₂ receptor-associated phosphoproteins. Recent results from our laboratory have demonstrated that G₁₃ co-purifies with platelet TXA₂ receptors by ligand affinity chromatography (17). Based on this observation, we probed the ligand affinity column eluate for the presence of cAMP-phosphorylated G₁₃. Specifically, solubilized platelet membranes, which were phosphorylated by [-³²P]ATP in the presence or absence of the hydrolysis-resistant cAMP analog 8-Br-cAMP (1 mM), were purified by ligand affinity chromatography, as described previously. The column eluate was then probed with G₁₃-N IgG, which is an anti-peptide antibody that is specific for the amino terminus of human G₁₃ (Table I). Following incubation of the column eluate with this antibody, the ³²P-labeled immune complexes were bound to protein A-Sepharose beads and were precipitated by centrifugation. The washed, immunoprecipitated phosphoproteins were then quantified by scintillation spectrometry. Control experiments were performed in parallel using a preimmune IgG in order to determine nonspecific phosphorylation. Specific phosphorylation is represented as the difference between G₁₃ antibody counts and preimmune antibody counts from the immunoprecipitated proteins. It was found in these experiments that treatment with 8-Br-cAMP caused an 87 ± 27% increase in specific phosphorylation of G₁₃ (Fig. 5A). Immunoblot analysis of proteins precipitated by the G₁₃-N antibody verified that G₁₃ was precipitated from both control and phosphorylated solubilized membranes, indicating that this antibody recognizes both the non-phosphorylated and phosphorylated form of the native G-protein (Fig. 5B). These results, therefore, provide evidence that cAMP causes phosphorylation of G₁₃  subunits and, furthermore, indicate that these phosphorylated  subunits are in physical association with platelet TXA₂ receptors.

View this table: [in this window] [in a new window]   Table I Specificity of G13 antibodies

View larger version (18K): [in this window] [in a new window]   Fig. 5.   Specific phosphorylation of TXA2 receptor-associated G13. A, solubilized platelet membranes were phosphorylated by [-32P]ATP in the presence (filled column) or absence (open column) of 1 mM 8-Br-cAMP, and TXA2 receptor-G-protein complexes were then purified by ligand affinity chromatography as described. The column eluate was incubated with G13-N or preimmune IgG, and immunoprecipitation was performed as described under "Experimental Procedures." The precipitated, labeled protein was quantified by liquid scintillation counting, and the specifically eluted counts were determined as described in the text. Results are the average of six separate experiments ± the S.E. Statistical analysis was performed using the Student's paired t test. *, p < 0.05. B, immunoblot of phosphorylated and non-phosphorylated proteins precipitated by the G13-N antibody. Solubilized platelet membranes were phosphorylated in the absence (lane 1) or presence (lane 2) of 8-Br-cAMP and were incubated with G13-N or preimmune IgG (PI IgG). Immunoprecipitation was performed as described, and the precipitated proteins were detected by immunoblot analysis using a 1:100 dilution of the G13-N antibody, as described under "Experimental Procedures."

Further evidence demonstrating the ability of cAMP to phosphorylate G₁₃ was provided using a different antibody against this G-protein as well as a separate technique to specifically isolate phosphorylated G₁₃  subunits. Briefly, a rabbit polyclonal, anti-peptide antibody was raised against an internal amino acid sequence that is unique to G₁₃ (Table I) (17), and the G-13 IgG was coated onto microtiter wells. Solubilized platelet membranes were then phosphorylated by [-³²P]ATP in the presence or absence of 8-Br-cAMP (1 mM) and were subsequently incubated on the antibody-coated microtiter wells. After washing the wells to remove unbound proteins, specifically immobilized ³²P-labeled proteins were eluted and were quantified by liquid scintillation counting. In parallel experiments, a preimmune antibody control was used to determine nonspecific phosphorylation. Specific phosphorylation is represented as the difference between G₁₃ antibody counts and preimmune antibody counts. In these experiments it was found that 8-Br-cAMP induced a 53 ± 19% increase in specific phosphorylation of G₁₃ (Fig. 6).

View larger version (12K): [in this window] [in a new window]   Fig. 6.   Specific phosphorylation of G13 immunopurified from solubilized platelet membranes. Solubilized platelet membranes were incubated with [-32P]ATP in the presence (filled column) or absence (open column) of 1 mM 8-Br-cAMP and were added to G-13 or preimmune antibody-coated microtiter wells. Specifically immobilized, 32P-labeled protein was eluted and quantified by scintillation counting, and the specific counts were determined as described under "Experimental Procedures." Results are the average of five separate experiments ± the S.E. Statistical analysis was performed using the Student's paired t test. *, p < 0.05.

Immunopurification of G₁₃ from ³²P-Labeled Whole Platelets-- The ability of G₁₃ to undergo cAMP-mediated phosphorylation in intact cells was next examined by treating platelets with the physiological inhibitor PGI₂. In these experiments, platelets were labeled with [³²P]P_i and were subsequently incubated for 5 min with either PGI₂ (1 µM) in the presence of the phosphodiesterase inhibitor IBMX (1 mM) or with vehicle. The cells were then cooled in an ice slurry and were solubilized in CHAPS buffer. G₁₃ was immunoprecipitated from the solubilized cells using the G₁₃-N IgG, and the precipitated ³²P counts were quantified by scintillation counting, and the counts were normalized for the amount of precipitated G₁₃ protein. In these experiments, it was found that treatment of platelets with PGI₂ caused a 90 ± 14% increase in phosphorylation of G₁₃ as compared with control platelets (Fig. 7). Finally, when adenylate cyclase activity was blocked using the cell-permeable specific cyclase inhibitor MDL-12, PGI₂-induced phosphorylation of G₁₃ was completely abolished, i.e. 17 ± 16% (n = 2). These findings, therefore, establish that activation of platelet PGI₂ receptors leads to cAMP-dependent phosphorylation of G₁₃.

View larger version (16K): [in this window] [in a new window]   Fig. 7.   In vivo phosphorylation of G13 by cAMP. Washed human platelets were labeled with [32P]H3PO4 and were incubated for 5 min in the absence (open column) or presence (filled column) of PGI2 (1 µM) and IBMX (1 mM). After incubation, platelets were place in an ice slurry, solubilized in CHAPS, and immunoprecipitated with the G13-N antibody as described under "Experimental Procedures." Precipitated proteins were quantified by scintillation counting. Results are expressed as the counts per min (cpm) normalized for the amount of precipitated G13 protein and represent the mean of three separate experiments ± S.E. Statistical analysis was performed using the Student's paired t test. *, p < 0.01.

Inhibition of Platelet Aggregation by PGI₂-- The above results demonstrate cAMP-mediated phosphorylation of TXA₂ receptor-associated G₁₃. Although the consequence of such phosphorylation on TXA₂-induced signal transduction is unknown, the following experiments demonstrate that platelet aggregation induced by TXA₂ is preferentially sensitive to inhibition by PGI₂. In these studies, platelets were treated with indomethacin to inhibit endogenous TXA₂ production. Subsequently, platelets were pretreated with PGI₂ for 1 min to elevate cAMP levels, and aggregation was measured in response to sub-maximal concentrations of the TXA₂ mimetic U46619, ADP, TRAP, and A23187 (Fig. 8A). The submaximal dose represents an agonist concentration that yields the same biological response for each agonist in the absence of PGI₂, i.e. 85% of the maximal response. As illustrated in Fig. 8B, a concentration of PGI₂ that caused significant inhibition of U46619-induced aggregation (62%) had only a minimal effect on aggregation induced by ADP (5%), TRAP (10%), or the calcium ionophore A23187 (15%). Furthermore, when various concentrations of PGI₂ were tested, U46619-induced aggregation was found to be far more sensitive to inhibition by PGI₂ than any of the other agonists tested (Fig. 8C).

View larger version (15K): [in this window] [in a new window]   Fig. 8.   Inhibition of U46619-, ADP-, TRAP- and A23187-induced platelet aggregation by PGI2. A, platelet-rich plasma was incubated for 1 min with 10 µM indomethacin. Platelets were then incubated for 1 min with prostacyclin (0.25-1 nM) and were subsequently activated with a sub-maximal dose of each agonist (at arrow) as described under "Experimental Procedures." Aggregation tracings shown are expressed as percent aggregation and are representative of three separate experiments. The control tracing represents aggregation induced by the sub-maximal dose of each agonist in the absence of PGI2. B, inhibition of aggregation by PGI2 is expressed as the average percent of inhibition in three experiments ± S.E. Statistical evaluation was performed using one-way analysis of variance. *, p < 0.05. C, dose-dependent inhibition of aggregation by PGI2. Indomethacin-treated platelets were incubated for 1 min with various concentrations of PGI2, as indicated, and were subsequently activated with a sub-maximal dose of U46619 (), ADP (), TRAP (), or A23187 (). The results shown are the average of three such experiments ± S.E.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

This study demonstrates that cAMP induces PKA-dependent phosphorylation of solubilized platelet membrane proteins in the molecular weight region of G-protein  subunits, i.e. 38-45 kDa, which is consistent with other reports (61, 62). Furthermore, it shows that platelet proteins that are phosphorylated by cAMP co-purify with TXA₂ receptors and are, therefore, closely associated with these receptors. Immunoaffinity purification experiments have identified, in both solubilized platelet membranes and intact platelets, that one of these phosphoproteins is TXA₂ receptor-associated G₁₃.

Previous reports have established that the G₁₃  subunit is a 43-44-kDa protein which is expressed ubiquitously among cells (14, 15, 60, 63). Although the role of G₁₃ in platelets is not understood, it has been linked to a number of cellular functions in other cell systems, including stimulation of the Na/H exchanger (18, 20), activation of L-type Ca²⁺ channels (23, 24), activation of phospholipase D (25), and organization of the cytoskeleton through Rho-dependent stress fiber formation and focal adhesion assembly (21). Based on these considerations, modulation of G₁₃ functional activity could potentially impact a number of different effector systems.

Classically, cellular activity is modulated by phosphorylation of signal transduction components. Thus, signaling through a receptor-G-protein complex can be modified by phosphorylation of either the receptor or the G-protein. In this regard, it is well established that phosphorylation of receptors by different protein kinases can mediate receptor desensitization (35-40). Previous reports have indicated that platelet TXA₂ receptors can indeed serve as substrates for PKC, PKA, and PKG (26-29). Specifically, Kinsella et al. (26) originally demonstrated that PKC and PKA caused phosphorylation of a fusion protein containing the latter third of the TXA₂ receptor. This finding suggested a possible mechanism by which phorbol 12-myristate 13-acetate (PMA) or cAMP may inhibit TXA₂-induced platelet activation is through phosphorylation of the receptor protein. However, subsequent results by the same group, using a transfected HEK 293 cell line, led them to conclude that this receptor phosphorylation was of "trivial" physiological significance and was not a major mechanism by which PMA or cAMP modifies TXA₂ receptor signal transduction (27). Recently, this group has reported that upon stimulation of platelets with various agonists, PKC can indeed induce phosphorylation of TXA₂ receptors (28). Thus, differing results appear to be obtained depending on the cell system and experimental conditions employed.

Regarding cGMP, however, a separate group has recently provided evidence that PKG causes TXA₂ receptor phosphorylation (29). Thus, 8-Br-cGMP-induced activation of PKG resulted in increased phosphorylation of immunoaffinity purified TXA₂ receptor protein. In addition, these studies also demonstrated that 8-Br-cGMP caused a decrease in U46619-stimulated GTPase activity in platelet membranes. Based on these results, the authors (29) proposed that cGMP-induced phosphorylation of TXA₂ receptors modulates receptor-G-protein coupling to regulate signaling through these receptors.

In addition to receptor phosphorylation, it has been recently recognized that G-protein phosphorylation may also be an important regulatory mechanism. In this regard, it was found that PKC-induced phosphorylation of purified G_z and G₁₂  subunits resulted in inhibition of their interaction with subunits (46, 64). In addition, PKC-induced phosphorylation of G_i was found to correlate with attenuation of adenylate cyclase inhibition in S49 cells (44) and inhibition of agonist-induced calcium mobilization in platelets (45). Finally, separate studies have found that PGD₂, which elevates cAMP, inhibits thrombin-induced dissociation of G_i from platelet thrombin receptors (65). Therefore, G-protein phosphorylation may functionally uncouple signal transduction by altering the stability of the heterotrimer or by tightening its association with the receptor.

In light of these findings, the present results demonstrating cAMP-mediated phosphorylation of G₁₃ suggest a potential mechanism for modulation of TXA₂ signal transduction in platelets. While effectors for G₁₃ in platelets are currently unknown, previous results have established that calcium mobilization in the TXA₂ receptor signaling pathway is modulated by cAMP (34, 66). Specifically these studies have shown that there is a direct relationship between platelet cAMP levels and inhibition of U46619-induced calcium release (34). Moreover, inhibition of TXA₂-induced calcium release from the platelet-dense tubular system was found to be PKA-dependent (66). The above findings, coupled with the notion that G₁₃ has been linked to calcium channels in other cells (23, 24), raises the possibility that G₁₃ phosphorylation may serve as one mechanism by which cAMP inhibits TXA₂-mediated calcium mobilization in platelets.

In summary, the present results provide the first evidence that PGI₂ can cause PKA-induced phosphorylation of an endogenous platelet G-protein  subunit and that phosphorylated G₁₃ is associated with platelet TXA₂ receptors. Furthermore, our results also demonstrate a preferential sensitivity of TXA₂ receptor signaling to PGI₂-stimulated increases in cAMP. Taken together, these results, therefore, suggest an alternative mechanism for cAMP inhibition of TXA₂ receptor signaling and may explain the preferential sensitivity of this signaling pathway to elevated cAMP.

	ACKNOWLEDGEMENTS

We thank Dr. Stephen Lam, Dr. R. Dale Brown, and Dr. Dorie Schwertz for their helpful advice and continuous support during these studies.

	FOOTNOTES

^* This work was supported in part by National Institutes of Health Grant HL-24530, the North Atlantic Treaty Organization Grant CRG-940595, and "Cellular Signaling in the Cardiovascular System" Training Grant T32 HL07692 and was conducted under the auspices of the Association for U. S.-French Biomedical Cooperation. A portion of this work was presented at the annual meeting of the American Society of Hematology, December 4-8, 1998, Miami Beach, FL.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: Dept. of Pharmacology, College of Medicine, University of Illinois, 835 S. Wolcott St. (M/C 868), Chicago, IL 60612. Tel.: 312-996-4929; Fax: 312-996-1225; E-mail: gcl@tigger.uic.edu.

	ABBREVIATIONS

The abbreviations used are: TXA₂, thromboxane A₂; PKA, protein kinase A; PKG, protein kinase G; PKC, protein kinase C; 8-Br-cAMP, 8-bromoadenosine 3':5'-cyclic monophosphate; TRAP, thrombin receptor-activating peptide; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]1-propanesulfonic acid; MDL-12, cis-N-(2-phenylcyclopentyl)azacyclotridec-1-en-2-amine, HCl; PAGE, polyacrylamide gel electrophoresis; PMA, phorbol 12-myristate 13-acetate; IP₂₀, inhibitor peptide 20; PRP, platelet-rich plasma; PGI₂, prostaglandin I₂ (prostacyclin); IBMX, isobutyl-methylxanthine.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES