Proc. Nati. Acad. Sci. USA Vol. 89, pp. 2784-2788, April 1992 Biochemistry
Epinephrine suppresses raplB.GAP-activated GTPase activity in human platelets (ras oncogenes/ras GTPase-activating protein/ras superfamily of proteins/platelet activation)
KIM BENCKE MARTI AND EDUARDO G. LAPETINA* Division of Cell Biology, Burroughs Wellcome Co., Research Triangle Park, NC 27709
Communicated by George H. Hitchings, December 20, 1991
ABSTRACT Lysate from quiescent platelets promotes rapid hydrolysis of [Iv32P]GTP bound to rap1B. Various platelet agonists, including platelet-activating factor, phorbol 12,13-dibutyrate, a-thrombin, epinephrine, ADP, and ioprost, that affect platelet metabolism by different sigal transduction pathways were used to stimulate intact platelets and study their effects on raplB.GAP-activated GTPase activity (GAP, GTPase-activating protein). Only epinephrine was found to dramatically decrease not only the rate but also the amount of hydrolysis of raplB-bound GTP activated by raplB.GAP. This effect was dose dependent and occurred rapidly. The suppression of GTPase activity was specific for raplB.GAP in that ras.GAP- and rap2B.GAP-activated GTPase activity were not affected by epinephrine stimulation. This effect appears to be mediated by the a-adrenergic receptor, as evidenced by a similar suppression of GTPase activity by stimulating platelets with the synthetic a2-adrenergic receptor agonist UK14304 (bromoxidine). Furthermore, the selective a2-adrenergic receptor antagonist yohimbine blocked the suppression of GTPase activity expressed in epinephrine-stimulated cell lysates. No apparent changes in the patterns of protein expression or tyrosine phosphorylation were observed. Although the migration characteristics upon anion-exchange chromatography of raplB.GAP and ras.GAP activities were unaffected by epinephrine stimulation, the specific activity of raplB.GAP was noticeably decreased with 250 and 500 FtM epinephrine. These results suggest a possible role for rap1B and raplB.GAP in epinephrine-stimulated signal transduction.
inhibition of ras-induced germinal vesicle breakdown in oocytes (16) suggest that rapi may serve as an antagonist to ras action, such that binding of rapi to ras.GAP interferes with ras effector function (14). Binding of rapl.GAP activates rapl GTPase activity, resulting in rapl.GDP. The binding of rapi to ras.GAP has been shown to be GTP dependent (14). Therefore, the interaction of rapi with its own GAP serves to regulate the antagonism of rapl towards ras.GAP. rapl has also been implicated in the cAMP-mediated inhibition of platelet metabolism (17, 18). raplB becomes phosphorylated in response to hormones that elevate intracellular cAMP (18-21), and this phosphorylation is coupled to the translocation of raplB from a membrane to a cytosolic fraction (17, 18). In contrast, agonists such as a-thrombin that also activate platelets promote translocation of raplB from a cytosolic to a cytoskeletal fraction (22). Epinephrine is a unique platelet agonist in that it induces primary aggregation independent of the biochemical responses typically associated with activation, such as inositolphospholipid turnover, intracellular calcium mobilization, or protein kinase C activation (23-25). Little is known about epinephrine's mechanism of action; however, it has been shown to activate a2-adrenergic receptors (a2-adrenoceptors) (26, 27). This class of receptors is known to be coupled to the GTP-binding regulatory protein Ghz2 and to mediate the inhibition of adenylate cyclase (28, 29). Epinephrine also potentiates aggregation induced by other agonists such as thrombin, collagen, and ADP (24, 30). We found that in human platelets, epinephrine suppresses the raplB.GAP-activated GTPase activity of rapiB. Human platelets possess only the a2A-adrenoceptor (31, 32). Our data provide evidence for a role of raplB and raplB.GAP in a2-adrenoceptor-mediated signal transduction.
Recent reports have identified GTPase-activating proteins (GAPs) specific for several members of the ras superfamily of proteins. In addition to ras.GAP (1-4), there is a GAP specific for the rab3A gene product (5). rho.GAPs have been purified from spleen (6) and bovine adrenal gland (7). Two GAPs specific for rapi have been found in bovine brain (8), and a membrane-associated rapl.GAP has been purified from HL60 cells (9). Most recently, the molecular cloning and expression of a cDNA encoding a rapl.GAP was reported (10). Although the function for the rapl.GAPs is unknown, they may play a role in the down-regulation of rapi activity. However, the function of rapi itself remains unclear. Both of the rapi proteins (raplA and rap1B) contain effector site sequences identical to the effector site sequence of ras (11); this region is important for GAP interaction (12). The rapi proteins associate with both ras.GAP and rapl.GAP with high affinity in vitro (13); however, the binding of rapi to ras.GAP does not affect rapi GTPase activity (13, 14). It has been suggested that the function of ras.GAP and rapl.GAP in vivo is related to the interplay of rapi with these two proteins (10). Observations of the phenotypic reversion of Kirsten ras-transformed NIH 3T3 cells (15) and the
EXPERIMENTAL PROCEDURES Materials. Recombinant bacterially expressed H-ras was a gift from Paul Ray (Burroughs Wellcome). [_y32P]GTP and [a-32P]GTP were from ICN. Anti-phosphotyrosine antibody was obtained from Upstate Biotechnology (Lake Placid, NY). Mono Q HR5/5 FPLC columns were from Pharmacia. Nitrocellulose filter disks were from Schleicher & Schuell. Cellulose triacetate membrane was obtained from Sartorius. UK14304 was purchased from Pfizer. All other reagents were from Sigma. Isolation of Human Platelets. Platelets were prepared as previously described (18, 24). Aliquots of platelets were incubated with various agonists for the indicated times and samples were added to an equal volume of either Nonidet P-40 (NP-40) lysis buffer (150 mM NaCI/50 mM Tris HCl, pH 7.5/5 mM EDTA/50 Ag of leupeptin per ml/100 p.M phenylmethylsulfonyl fluoride/2 mM NaVO4/1% NP-40) or Triton
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Abbreviations: GAP, GTPase-activating protein; NP-40, Nonidet P.40; PAF, platelet-activating factor. *To whom reprint requests should be addressed.
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Biochemistry: Marti and Lapetina X-100 lysis buffer [25 mM Tris HCI/0.223 mM Tris base/10%o (vol/vol) glycerol/2% (vol/vol) Triton X-100/5 mM EGTA/ 1.1 mM phenylmethylsulfonyl fluoride, pH 7.4]. GAP Assay. The raplB.[y-32P]GTP complex was formed by adding 13 Al of purified rapiB [3.0 mg/ml in 50 mM Tris-HCI, pH 8.0/10 mM MgCl2/1 mM dithiothreitol/100 mM KCI/10 mM EDTA/10% (vol/vol) glycerol/12.5 ,uM GTP] to 32 A.l of loading buffer [32 mM Tris HCI, pH 8.0/0.2 M (NH4)2SO4/ 0.5 mM dithioerythritol/0.5 mM NaN3] containing 0.5 p.M [y-32P]GTP (5.9 x 105 cpm/pmol) and incubating the mixture at room temperature for 5 min. The buffer was then changed by microdialysis on a 1.5 x 1.5 cm square cellulose triacetate membrane against 20 mM Tris HCI, pH 7.5, containing 0.1 M NaCl, 1 mM MgCI2, and 1 mM dithiothreitol for 1 hr at 4°C. Typical sample dilution was in the range of 5-6%. Two microliters of the raplB[y-32P]GTP complex was added to 60 p.1 of 25 mM Tris HCI, pH 7.5, containing 6.25 mM MgCl2, 1 mM dithiothreitol, 625 p.M GTP, 1.25 mg of bovine serum albumin per ml, 0.06% deoxycholate, and 0.06% NP-40. After the addition of unstimulated or variously stimulated lysate containing 10 ,ug of total protein, the samples were incubated at 30°C for 10 min. Reactions were stopped by diluting 5- to 10-,ul samples in 4 ml of ice-cold 25 mM Tris HCI, pH 7.5, containing 0.1 M NaCI and 5 mM MgCl2 at various times, followed by rapid vacuum filtration through nitrocellulose. Filters were washed three times with 4 ml of the same buffer and the radioactivity was measured by liquid scintillation counting. Typical aliquots contained 50,000-100,000 cpm. Greater than 90% of the radioactivity remained associated with the protein in the absence of added GAP-containing lysate. Activities are expressed as the percentage of [y-32P]GTP bound to rap that is hydrolyzed with respect to buffer control. The GAP assay used in this study is based on the measurement of [y-32P]GTP that remains bound to raplB; an increased dissociation of [.y-32P]GTP from the protein might also appear to be increased GTP hydrolysis. Therefore, a raplB-[a-32P]GTP complex was also tested. No loss of bound radioactivity was observed in response to lysate containing GAP activity. ras-[y-32P]GTP and rap2Bt[y-32P]GTP complexes were formed and assayed as described for raplB. -Mono Q Anion-Exchange Chromatography. Lysates containing 2 mg of protein from quiescent platelets and platelets that had been stimulated with 250 or 500 p.M epinephrine were injected onto a Mono Q HR5/5 FPLC column (5 x 50 mm) equilibrated with Mono Q buffer (20 mM Tris HCI, pH 8.5/0.1% NP-40/5 mM EDTA/0.5 mM dithiothreitol and pepstatin and leupeptin each at 1 ,ug/ml). The column was washed with 12 ml of buffer and then developed with a 40-ml gradient of 0-0.3 M NaCI in the same buffer, and 1-ml fractions were collected at a flow rate of 1 ml/min. Aliquots (25 p.l) of fractions were assayed for both raplB.GAP- and ras.GAP-stimulated GTPase activity. Total protein was determined by using the method of Bradford (33) with bovine serum albumin as standard.
RESULTS Epinephrine Inhibits raplB.GAP-Activated GTPase Activity in Platelets. Whole platelet lysate from quiescent cells promoted rapid hydrolysis of [Y-32P]GTP bound to raplB, with greater than 65% hydrolysis in 10 min when 10 ,ug of total protein was used. Various platelet agonists affecting platelet metabolism by different signaling pathways were used in intact platelets to determine if their effects are reflected in changes of raplB.GAP-activated GTPase activity. There was no apparent effect on raplB-bound GTPase activity in equivalent platelet lysates that had been maximally stimulated with platelet-activating factor (PAF), phorbol 12,13-dibutyrate, a-thrombin, or iloprost (Fig. 1A). How-
Proc. Natl. Acad. Sci. USA 89 (1992)
2785
ever, when platelets were stimulated with 100 ,uM epinephrine for 2 min, only 25% of the [-32P]GTP bound to raplB was hydrolyzed in 10 min. In fact, dramatic decreases in hydrolytic rate and percentage hydrolysis of raplB-bound GTP were observed. In addition, when 10 ,AM ADP was used to stimulate platelets, an increase in the rate of GTP hydrolysis was observed (Fig. 1B). GTPase activity is typically defined as the percent of 100
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FIG. 1. Effects of stimulation by various platelet agonists on GTPase activity rapiB GTPase activity. (A) Time courses of raplB after stimulation of platelets with agonists that do not affect GTPase activity. Washed platelets incubated at 370C were either unstimulated (in) or stimulated with 1 &M PAF for 2 mmi(i), 100 nM phorbol 12,13-dibutyrate for 5 mmn (A), thrombin at 5 units/mi for 1 mmn (o), or 10 MM were lysed and 10pg of iloprost for 30 mn ().uPlatelets protein from the cleared lysates was assayed for raplB.GAP activity. Activities are expressed as percent [y-32P]GTP bound to raplB that is hydrolyzed relative to buffer control. Similar results were obtained in at least three other experiments. (B) Time courses of raplB GTPase activity after stimulation with epinephrine and ADP. Platelet treatment was as in A with no stimulation (m) or stimulation by 100 A&M epinephrine (A) or 10 A.M ADP (o) for 2 mmn. Results represent +the mean ±& SDT oftilctedtriaiosotie infeseart
experiments. (C) Epinephrine suppresses and ADP enhances raplB GTPase activity. Bars illustrate the mean percentage of GTP hydrolyzed in 10 min per 10 pAg of total protein in unstimulated (basal) and epinephrine- (100 ,M) and ADP- (10 ,M) stimulated platelet lysates.
Biochemistry: Marti and Lapetina
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Proc. Natl. Acad Sci. USA 89 (1992)
[y-32P]GTP bound to protein that is hydrolyzed in 10 min relative to buffer control. Although the increase in percentage of GTP hydrolyzed upon ADP stimulation was not as dramatic as the decrease observed with epinephrine, the observed differences in GTPase activity were real and significant (Fig. 1C). The mean percent ofraplB-bound GTP hydrolyzed in 10 min per 10 Iug total protein in unstimulated lysate was 68.5 + 0.825; GTPase activity in epinephrine- and ADP-stimulated platelet lysates was 25.8 + 1.3 and 78 1.25, respectively. Data are the mean SEM of 15 experiments, each performed in triplicate. Epinephrine suppressed the GTPase activity of raplB by -6o, whereas ADP increased GTPase activity -12%. Epinephrine Has No Effect on ras.GAP or rap2B.GAP. The GAP-activated GTPase activities associated with ras and rap2B were also assayed in lysates from quiescent and agonist-stimulated platelets. As shown in Fig. 2, there was no significant suppression of either of these GTPase activities in response to epinephrine stimulation, nor was there an increase upon ADP stimulation. It is also of interest to note that the GTPase activity observed in quiescent platelet lysate at 10 min was -30% of bound GTP hydrolyzed for both ras and rap2B. In addition, the initial rates of hydrolysis were significantly less than the rate observed for raplB.GAPstimulated hydrolysis. Epinephrine Action Appears to Be Receptor Mediated. To investigate whether the observed suppression of raplBassociated GTPase activity by epinephrine is a receptormediated process, platelets were exposed to the synthetic a2-adrenoceptor agonist UK14304 (bromoxidine). GTPase activity was completely suppressed by 20 ,uM UK14304 for up to 10 min at 30°C; low-level hydrolysis was observed at later time points (Fig. 3). Furthermore, the selective a2adrenoceptor antagonist yohimbine blocked suppression of GTPase activity resulting from epinephrine stimulation. As shown in Fig. 3, yohimbine, at a concentration reported to inhibit epinephrine's action in human platelets (34), exhibited a rate and level of raplB-associated GTPase activity similar to that found in unstimulated platelet lysates. To further test the possibility of receptor mediation of epinephrine's effect, equal amounts of lysate of unstimulated platelets and lysates of platelets stimulated with 100 ,uM epinephrine were mixed and GTPase activity was monitored. In addition, exogenous epinephrine was added to an unstimulated lysate to eliminate the possibility of a non-receptor-mediated effect on raplBassociated GTPase activity. The mixture of stimulated and ±
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FIG. 3. Effect of epinephrine on raplB.GAP is mimicked by UK14304 and blocked by yohimbine. Platelets were incubated at 37°C in the absence (o) or presence of 100 ,uM epinephrine (o), 20 ,uM UK14304 (A), or 100 ,uM epinephrine plus 20 AM yohimbine (U) for 2 min. After lysis of the cells, 10 Ag of protein was assayed for GTPase activity. Results represent the mean SD of triplicate determinations in three separate experiments. ±
unstimulated lysates, as well as the addition of exogenous epinephrine, did not result in suppression of raplBassociated GTPase activity (data not shown). Epiephrine's Suppression of raplB.GAP-AcP-Ated GTPase Activity Is Dose Dependent. All epinephrine stimulation data presented thus far were obtained by using epinephrine in the absence of a reducing agent. Dose dependence experiments were performed in the absence or presence of sodium metabisulfite (Na2S205) to ensure that the observed suppression was not due to oxidation of epinephrine and that the concentrations used were precise. Fig. 4 shows that suppression of raplB.GAP GTPase activity by epinephrine is dose dependent. Moreover, there is a
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FIG. 2. Epinephrine and ADP do not affect the GAP-stimulated GTPase activities associated with ras and rap2B. (A) Time courses of ras-bound GTP hydrolysis. Platelet treatment was as in the legend to Fig. 1 with no stimulation (-) or stimulation by 100 jLM epinephrine (e) or 10 ,uM ADP (A). Results represent the mean SD of triplicate determinations in three separate experiments. (B) Time courses of rap2B-bound GTP hydrolysis by unstimulated (m), 100 jM epinephrine-stimulated (9), or 10 ,uM ADP-stimulated (A) platelet lysates. Results represent the mean SD of triplicate determinations in three separate experiments. ±
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Epinephrlne, ,uM FIG. 4. Epinephrine suppresses raplB.GAP-stimulated GTPase activity in a dose-dependent manner. Platelets were stimulated for 2 min with various concentrations of epinephrine in the absence (o) or the presence (e) of 20 ,uM Na2S205. Cells were then lysed and assayed for raplB.GAP-stimulated GTPase activity in 10 min with 10 ,Bg of total protein. Percent inhibition is defined as the percent decrease in GTPase activity compared with unstimulated platelet lysate. Similar results were obtained in two other experiments.
Proc. Natl. Acad. Sci. USA 89 (1992)
Biochemistry: Marti and Lapetina shift in the dose-response curve toward more effective suppression of GTPase activity when epinephrine is maintained in a reduced state. Suppression can be observed at epinephrine concentrations as low as 25 ,uM in the absence of reducing agent and 10 uM in the presence of reducing agent. Complete suppression of GTPase activity can be obtained by using 500 A&M epinephrine and 20 AM Na2S2O5. Epinephrine Causes a Decrease in the Specific Activity of raplB.GAP-Stimulated GTPase Activity. To determine whether epinephrine specifically modifies raplB.GAP activity, lysates from quiescent and epinephrine-stimulated platelets were subjected to SDS/PAGE and either stained with Coomassie blue or immunoblotted with anti-phosphotyrosine antibody. No differences were observed in the patterns of proteins of whole cell lysates or tyrosine phosphorylation. In a recent study, ras.GAP and rapl.GAP activities migrated differently on anion-exchange chromatography (9). Lacking a specific antibody for raplB.GAP, we looked for a possible change in migration of raplB.GAP activity after epinephrine stimulation. Platelets were stimulated with 250 or 500 ,uM epinephrine for 2 min and the cells were lysed. Equal amounts of lysate (2 mg) from unstimulated or epinephrinestimulated platelets were subjected to anion-exchange chromatography on a Mono Q FPLC column. Fractions were assayed for both raplB.GAP and ras.GAP GTPase activities. As shown in Fig. 5 A and B, raplB.GAP and ras.GAP GTPase activities were well resolved in both unstimulated and stimulated lysates. In both unstimulated and epinephrinestimulated lysates, raplB.GAP activity eluted at 200-215 mM NaCl and ras.GAP activity eluted at about 150 mM NaCl. However, the peak GTPase activity associated with rapiB dropped dramatically from 65% in unstimulated lysate to 25% in epinephrine-stimulated lysate. A further reduction to 12% GTP hydrolysis was observed when a lysate from platelets stimulated with 500 AM epinephrine was subjected to Mono Q chromatography (data not shown). In contrast, ras.GAP GTPase activity remained essentially unchanged at around 30%. More important, the amount of protein in both raplB.GAP and ras.GAP peak fractions in the epinephrinestimulated lysate was not significantly different from that in the unstimulated lysate. Fig. 5C shows that the specific activity of peak raplB.GAP activity decreased as the concentration of epinephrine used to stimulate the platelets increased. Recoveries of raplB.GAP and ras.GAP activities from either unstimulated or epinephrine-stimulated platelet lysates were in the range of 70-90%o of the activity loaded onto the Mono Q column.
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DISCUSSION Platelets possess GAPs specific for raplB, rap2B, and ras. The specificity of these GAPs is evidenced not only by a difference in the rates of hydrolysis of GTP bound to these proteins but also by a difference in the amount of proteinbound hydrolysis observed in a typical 10-min period. The most convincing evidence for this specificity, however, is our finding that epinephrine suppressed the otherwise rapid hydrolysis of raplB-bound GTP observed in lysates of quiescent platelets. Moreover, ADP stimulation of intact platelets slightly increased raplB.GAP-activated GTPase activity but not rap2B.GAP or ras.GAP activity. Other agonists that activate platelets by different signal transduction pathways had no effect on the GAP-activated GTPase activity of raplB, rap2B, or ras. Epinephrine action appears to be a2A-adrenoceptor mediated. Several lines of evidence support this hypothesis. First, the suppression of GTPase activity can be mimicked by stimulating platelets with the potent synthetic a2-adrenoceptor agonist UK14304. This analog of clonidine has been shown to produce an aggregatory effect on platelets compa-
Basal
Epi 250
Epi 500
FIG. 5. Epinephrine decreases the relative specific activity of raplB.GAP. (A and B) Mono Q anion-exchange column profiles of quiescent platelet lysate (A) or lysate from platelets stimulated with 250 AtM epinephrine (B). *, raplB.GAP activity; o, ras.GAP activity; solid line, protein concentration; broken line, NaCl concentration. (C) Decreased specific activity of epinephrine-stimulated platelet lysate. Specific activity is defined as the ratio of the GTPase activity (percent hydrolyzed) to protein concentration (j.g/ml) of pooled peak fractions of raplB.GAP (cross-hatched bars) and ras.GAP (hatched bars) activity after anion-exchange chromatography of lysate of unstimulated platelets (Basal) or lysates of platelets stimulated with 250 jAM epinephrine (Epi 250) or 500 ,.M epinephrine (Epi 500). rable to that seen with epinephrine (35). Furthermore, the selective a2-adrenoceptor antagonist yohimbine efficiently blocked epinephrine-stimulated suppression of GTPase activity. This compound also blocks platelet aggregation in-
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Biochemistry: Marti and Lapetina
duced by epinephrine (36). In addition, when lysates from unstimulated and epinephrine-stimulated platelets were mixed or exogenous epinephrine was added to unstimulated lysate, no suppression of GTPase activity was observed, eliminating the possibility of the presence of a raplB.GAPspecific phosphatase. We have shown that the effect of epinephrine suppression of raplB.GAP activity is dose dependent and that suppression is more effective in the presence of a mild reducing agent such as Na2S205, thereby eliminating the possibility that epinephrine's action results from oxidation of epinephrine itself. Our preliminary experiments to assess the mechanism of epinephrine's effect on raplB.GAP show that no obvious modifications occur. Appearance or disappearance of a particular protein or change in tyrosine phosphorylated proteins was not observed after epinephrine stimulation. Epinephrine did not change the migration behavior of raplB.GAP or ras.GAP during anion-exchange chromatography, indicating that the ionic state of these proteins was not significantly affected. However, the level of peak GTPase activity associated with raplB was notably reduced, in contrast to the essentially unchanged peak level of ras.GAP activity. In addition, the amounts of protein in raplB.GAP and ras.GAP peaks in unstimulated and epinephrine-stimulated lysate did not significantly differ. Thus, the specific activity of peak raplB.GAP decreased as the concentration of epinephrine used to stimulate the platelets increased. These results indicate that epinephrine's effect is truly suppression of GTPase activity and not reduction in the amount of raplB.GAP available for activation of raplB GTPase by modification, degradation, or otherwise selective elimination of raplB.GAP. The reason for the observed increase in specific activity of peak ras.GAP is unclear, given that no enhancement of ras.GAP activity was observed in epinephrinestimulated lysate prior to separation on the Mono Q column. In addition, there was no significant difference in recovery of either raplB.GAP or ras.GAP activity after anion-exchange chromatography. Considering the highly reproducible and consistent effect of epinephrine on raplB.GAP activity, we believe this apparent increase in ras.GAP activity is an artifactual consequence of the chromatography and is not related to the suppression of raplB.GAP activity observed in epinephrine-stimulated platelet lysates. It is known that a2-adrenergic receptors couple to an inhibition of adenylate cyclase. This inhibition is mediated by Gia-2. However, many a2-adrenoceptor-mediated responses do not result in lower cAMP. In fact, epinephrine's aggregatory action does not appear to occur as a result of adenylate cyclase inhibition (21). Some evidence suggests that a2adrenoceptors may also couple to other second messenger systems (31, 37). The a2-adrenoceptor has been implicated in the modulation of cellular signaling pathways such as Na+/H' exchange (38), K+ channels (39), and voltagesensitive Ca2+ channels (40). It is unclear whether these effects were due to decreased cAMP caused by additional receptor or to effector activation. The data presented here may be indicative of a pathway in platelets initiated by epinephrine binding to its receptor but not involving inhibition of adenylate cyclase. None of the members of the ras superfamily of proteins has yet been implicated as playing a role in platelet activation. Our observations suggest that raplB.GAP and rap1B appear to actually play a role in platelet response to agonist stimulation of the a2-adrenoceptor in vivo. Suppression of raplB.GAP-activated GTPase activity by epinephrine results in raplB remaining in the GTP-bound or activated form.
Proc. Natl. Acad. Sci. USA 89 (1992)
Further characterization of this effect will provide a better understanding of the implications of these observations. 1. Halenbeck, R., Crosier, W. J., Clark, R., McCormick, F. & Koths, K. (1990) J. Biol. Chem. 265, 21922-21928. 2. Trahey, M., Wong, G., Halenbeck, R., Rubinfeld, B., Martin, G. A., Ladner, M., Long, C. M., Crosier, W. J., Watt, K., Koths, K. & McCormick, F. (1988) Science 242, 1697-1700. 3. Vogel, U. S., Dixon, R. A. F., Schaber, M. D., Diehl, R. E., Marshall, M. S., Scolnick, E. M., Sigal, I. S. & Gibbs, J. B. (1988) Nature (London) 335, 90-93. 4. Gibbs, J. B., Schaber, M. D., Allard, W. J., Sigal, I. S. & Scolnick, E. M. (1988) Proc. Nail. Acad. Sci. USA 85, 5026-5030. 5. Burstein, E. S., Linko-Stentz, K., Lu, Z. & Macara, I. G. (1991) J. Biol. Cheml. 266, 2689-2692. 6. Garrett, M. D., Major, G. N., Totty, N. & Hall, A. (1991) Biochem. 1. 276, 833-836. 7. Morii, N., Kawano, K., Sekine, A., Yamada, T. & Narumiya, S. (1991) J. Biol. Chem. 266, 7646-7650. 8. Kikuchi, A., Sasaki, T., Araki, T., Hata, Y. & Takai, Y. (1989) J. Biol. Chem. 264, 9133-9136. 9. Polakis, P. G., Rubinfeld, B., Evans, T. & McCormick, F. (1991) Proc. Nadl. Acad. Sci. USA 88, 239-243. 10. Rubinfeld, B., Munemitsu, S., Clark, R., Conroy, L., Watt, K., Crosier, W. J., McCormick, F. & Polakis, P. (1991) Cell 65, 1033-1042. 11. Pizon, V., Lerosey, I., Chardin, P. & Tavitian, A. (1988) Nucleic Acids Res. 16, 7719. 12. Adari, H., Lowy, D. R., Willumsen, B. M., Der, C. J. & McCormick, F. (1988) Science 240, 518-521. 13. Hata, Y., Kikuchi, A., Sasaki, T., Schaber, M. D., Gibbs, J. B. & Takai, Y. (1990) 1. Biol. Chem. 265, 7104-7107. 14. Frech, M., John, J., Pizon, V., Chardin, P., Tavitian, A., Clark, R., McCormick, F. & Wittinghofer, A. (1990) Science 249, 169-171. 15. Kitayama, H., Sugimoto, Y., Matsuzaki, T., Ikawa, Y. & Noda, M. (1989) Cell 56, 77-84. 16. Campa, M. J., Chang, K.-J., Molina y Vedia, L., Reep, B. R. & Lapetina, E. G. (1991) Biochem. Biophys. Res. Commun. 174, i-5. 17. Kawata, M., Kikuchi, A., Hoshijima, M., Yamamoto, K., Hashimoto, E., Yamamura, H. & Takai, Y. (1989) J. Biol. Chem. 264, 15688-15695. 18. Lapetina, E. G., Lacal, J. C., Reep, B. R. & Molina y Vedia, L. (1989) Proc. Nail. Acad. Sci. USA 86, 3131-3134. 19. Lazarowski, E. R., Lacal, J.-C. & Lapetina, E. G. (1989) Biochem. Biophys. Res. Commun. 161, 972-978. 20. Siess, W., Winegar, D. A. & Lapetina, E. G. (1990) Biochem. Biophys. Res. Commun. 170, 944-950. 21. Siess, W. & Lapetina, E. G. (1990) Biochem. J. 271, 815-819. 22. Fischer, T. H., Gatling, M. N., Lacal, J.-C. & White, G. C., III (1990) J. Biol. Chem. 265, 19405-19408. 23. Seiss, W., Weber, P. C. & Lapetina, E. G. (1984) J. Biol. Chem. 259, 8286-8292. 24. Crouch, M. F. & Lapetina, E. G. (1988) J. Biol. Chem. 263, 3363-3371. 25. Lapetina, E. G. (1986) in Phosphoinositides and Receptor Mechanisms, ed. Putney, J. W., Jr. (Liss, New York), Vol. 7, pp. 271-286. 26. Jakobs, K. H., Saur, W. & Schultz, G. (1976) J. Cyclic Nucleotide Res. 2, 381-392. 27. Macfarlane, D. E. & Stump, D. C. (1982) Mol. Pharmacol. 22, 574-579. 28. Cerione, R. A., Regan, J. W., Nakata, H., Codina, J., Benovie, J. L., Gierschik, P., Somers, R. L., Spiegel, A. M., Birnbaumer, L., Lefikowitz, R. J. & Caron, M. G. (1986) J. Biol. Chem. 261, 3901-3909. 29. Aktories, K. & Jakobs, K. H. (1985) in The Platelet: Physiology and Pharmacology, ed. Langenecker, E. (Academic, New York), pp. 243260. 30. Steen, V. M., Tysnes, O.-B. & Holmsen, H. (1988) Biochem. J. 253, 581-586. 31. Cotecchia, S., Kobilka, B. K., Daniel, K. W., Nolan, R. D., Lapetina, E. G., Caron, M. G., Lefkowitz, R. J. & Regan, J. W. (1990) J. Biol. Chem. 265, 63-69. 32. Bylund, D. B., Ray-Prenger, C. & Murphy, T. J. (1988) J. Pharmacol. Exp. Ther. 245, 600-607. 33. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254. 34. Crouch, M. F., Winegar, D. A. & Lapetina, E. G. (1989) Proc. Natl. Acad. Sci. USA 86, 1776-1780. 35. Grant, J. A. & Scrutton, M. C. (1980) Br. J. Pharmacol. 71, 121-134. 36. Hsu, C. Y., Knapp, D. R. & Halushka, P. V. (1979) J. Pharmacol. Exp. Ther. 206, 366-370. 37. Michel, M. C., Brass, L. F., Williams, A., Bokoch, G. M., La Morte, V. J. & Motulsky, H. J. (1989) J. Biol. Chem. 264, 4986-4991. 38. Sweatt, J. D., Blair, I. A., Cragoe, E. J. & Limbird, L. E. (1986) J. Biol. Chem. 261, 8660-8666. 39. North, R. A., Williams, J. T., Surprenant, A. & Christie, M. J. (1987) Proc. Natl. Acad. Sci. USA 84, 5487-5491. 40. Hescheler, J., Rosenthal, W., Trautwein, W. & Schultz, G. (1987) Nature (London) 325, 445-447.
Epinephrine suppresses raplB.GAP-activated GTPase activity in human platelets (ras oncogenes/ras GTPase-activating protein/ras superfamily of proteins/platelet activation)
KIM BENCKE MARTI AND EDUARDO G. LAPETINA* Division of Cell Biology, Burroughs Wellcome Co., Research Triangle Park, NC 27709
Communicated by George H. Hitchings, December 20, 1991
ABSTRACT Lysate from quiescent platelets promotes rapid hydrolysis of [Iv32P]GTP bound to rap1B. Various platelet agonists, including platelet-activating factor, phorbol 12,13-dibutyrate, a-thrombin, epinephrine, ADP, and ioprost, that affect platelet metabolism by different sigal transduction pathways were used to stimulate intact platelets and study their effects on raplB.GAP-activated GTPase activity (GAP, GTPase-activating protein). Only epinephrine was found to dramatically decrease not only the rate but also the amount of hydrolysis of raplB-bound GTP activated by raplB.GAP. This effect was dose dependent and occurred rapidly. The suppression of GTPase activity was specific for raplB.GAP in that ras.GAP- and rap2B.GAP-activated GTPase activity were not affected by epinephrine stimulation. This effect appears to be mediated by the a-adrenergic receptor, as evidenced by a similar suppression of GTPase activity by stimulating platelets with the synthetic a2-adrenergic receptor agonist UK14304 (bromoxidine). Furthermore, the selective a2-adrenergic receptor antagonist yohimbine blocked the suppression of GTPase activity expressed in epinephrine-stimulated cell lysates. No apparent changes in the patterns of protein expression or tyrosine phosphorylation were observed. Although the migration characteristics upon anion-exchange chromatography of raplB.GAP and ras.GAP activities were unaffected by epinephrine stimulation, the specific activity of raplB.GAP was noticeably decreased with 250 and 500 FtM epinephrine. These results suggest a possible role for rap1B and raplB.GAP in epinephrine-stimulated signal transduction.
inhibition of ras-induced germinal vesicle breakdown in oocytes (16) suggest that rapi may serve as an antagonist to ras action, such that binding of rapi to ras.GAP interferes with ras effector function (14). Binding of rapl.GAP activates rapl GTPase activity, resulting in rapl.GDP. The binding of rapi to ras.GAP has been shown to be GTP dependent (14). Therefore, the interaction of rapi with its own GAP serves to regulate the antagonism of rapl towards ras.GAP. rapl has also been implicated in the cAMP-mediated inhibition of platelet metabolism (17, 18). raplB becomes phosphorylated in response to hormones that elevate intracellular cAMP (18-21), and this phosphorylation is coupled to the translocation of raplB from a membrane to a cytosolic fraction (17, 18). In contrast, agonists such as a-thrombin that also activate platelets promote translocation of raplB from a cytosolic to a cytoskeletal fraction (22). Epinephrine is a unique platelet agonist in that it induces primary aggregation independent of the biochemical responses typically associated with activation, such as inositolphospholipid turnover, intracellular calcium mobilization, or protein kinase C activation (23-25). Little is known about epinephrine's mechanism of action; however, it has been shown to activate a2-adrenergic receptors (a2-adrenoceptors) (26, 27). This class of receptors is known to be coupled to the GTP-binding regulatory protein Ghz2 and to mediate the inhibition of adenylate cyclase (28, 29). Epinephrine also potentiates aggregation induced by other agonists such as thrombin, collagen, and ADP (24, 30). We found that in human platelets, epinephrine suppresses the raplB.GAP-activated GTPase activity of rapiB. Human platelets possess only the a2A-adrenoceptor (31, 32). Our data provide evidence for a role of raplB and raplB.GAP in a2-adrenoceptor-mediated signal transduction.
Recent reports have identified GTPase-activating proteins (GAPs) specific for several members of the ras superfamily of proteins. In addition to ras.GAP (1-4), there is a GAP specific for the rab3A gene product (5). rho.GAPs have been purified from spleen (6) and bovine adrenal gland (7). Two GAPs specific for rapi have been found in bovine brain (8), and a membrane-associated rapl.GAP has been purified from HL60 cells (9). Most recently, the molecular cloning and expression of a cDNA encoding a rapl.GAP was reported (10). Although the function for the rapl.GAPs is unknown, they may play a role in the down-regulation of rapi activity. However, the function of rapi itself remains unclear. Both of the rapi proteins (raplA and rap1B) contain effector site sequences identical to the effector site sequence of ras (11); this region is important for GAP interaction (12). The rapi proteins associate with both ras.GAP and rapl.GAP with high affinity in vitro (13); however, the binding of rapi to ras.GAP does not affect rapi GTPase activity (13, 14). It has been suggested that the function of ras.GAP and rapl.GAP in vivo is related to the interplay of rapi with these two proteins (10). Observations of the phenotypic reversion of Kirsten ras-transformed NIH 3T3 cells (15) and the
EXPERIMENTAL PROCEDURES Materials. Recombinant bacterially expressed H-ras was a gift from Paul Ray (Burroughs Wellcome). [_y32P]GTP and [a-32P]GTP were from ICN. Anti-phosphotyrosine antibody was obtained from Upstate Biotechnology (Lake Placid, NY). Mono Q HR5/5 FPLC columns were from Pharmacia. Nitrocellulose filter disks were from Schleicher & Schuell. Cellulose triacetate membrane was obtained from Sartorius. UK14304 was purchased from Pfizer. All other reagents were from Sigma. Isolation of Human Platelets. Platelets were prepared as previously described (18, 24). Aliquots of platelets were incubated with various agonists for the indicated times and samples were added to an equal volume of either Nonidet P-40 (NP-40) lysis buffer (150 mM NaCI/50 mM Tris HCl, pH 7.5/5 mM EDTA/50 Ag of leupeptin per ml/100 p.M phenylmethylsulfonyl fluoride/2 mM NaVO4/1% NP-40) or Triton
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Abbreviations: GAP, GTPase-activating protein; NP-40, Nonidet P.40; PAF, platelet-activating factor. *To whom reprint requests should be addressed.
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Biochemistry: Marti and Lapetina X-100 lysis buffer [25 mM Tris HCI/0.223 mM Tris base/10%o (vol/vol) glycerol/2% (vol/vol) Triton X-100/5 mM EGTA/ 1.1 mM phenylmethylsulfonyl fluoride, pH 7.4]. GAP Assay. The raplB.[y-32P]GTP complex was formed by adding 13 Al of purified rapiB [3.0 mg/ml in 50 mM Tris-HCI, pH 8.0/10 mM MgCl2/1 mM dithiothreitol/100 mM KCI/10 mM EDTA/10% (vol/vol) glycerol/12.5 ,uM GTP] to 32 A.l of loading buffer [32 mM Tris HCI, pH 8.0/0.2 M (NH4)2SO4/ 0.5 mM dithioerythritol/0.5 mM NaN3] containing 0.5 p.M [y-32P]GTP (5.9 x 105 cpm/pmol) and incubating the mixture at room temperature for 5 min. The buffer was then changed by microdialysis on a 1.5 x 1.5 cm square cellulose triacetate membrane against 20 mM Tris HCI, pH 7.5, containing 0.1 M NaCl, 1 mM MgCI2, and 1 mM dithiothreitol for 1 hr at 4°C. Typical sample dilution was in the range of 5-6%. Two microliters of the raplB[y-32P]GTP complex was added to 60 p.1 of 25 mM Tris HCI, pH 7.5, containing 6.25 mM MgCl2, 1 mM dithiothreitol, 625 p.M GTP, 1.25 mg of bovine serum albumin per ml, 0.06% deoxycholate, and 0.06% NP-40. After the addition of unstimulated or variously stimulated lysate containing 10 ,ug of total protein, the samples were incubated at 30°C for 10 min. Reactions were stopped by diluting 5- to 10-,ul samples in 4 ml of ice-cold 25 mM Tris HCI, pH 7.5, containing 0.1 M NaCI and 5 mM MgCl2 at various times, followed by rapid vacuum filtration through nitrocellulose. Filters were washed three times with 4 ml of the same buffer and the radioactivity was measured by liquid scintillation counting. Typical aliquots contained 50,000-100,000 cpm. Greater than 90% of the radioactivity remained associated with the protein in the absence of added GAP-containing lysate. Activities are expressed as the percentage of [y-32P]GTP bound to rap that is hydrolyzed with respect to buffer control. The GAP assay used in this study is based on the measurement of [y-32P]GTP that remains bound to raplB; an increased dissociation of [.y-32P]GTP from the protein might also appear to be increased GTP hydrolysis. Therefore, a raplB-[a-32P]GTP complex was also tested. No loss of bound radioactivity was observed in response to lysate containing GAP activity. ras-[y-32P]GTP and rap2Bt[y-32P]GTP complexes were formed and assayed as described for raplB. -Mono Q Anion-Exchange Chromatography. Lysates containing 2 mg of protein from quiescent platelets and platelets that had been stimulated with 250 or 500 p.M epinephrine were injected onto a Mono Q HR5/5 FPLC column (5 x 50 mm) equilibrated with Mono Q buffer (20 mM Tris HCI, pH 8.5/0.1% NP-40/5 mM EDTA/0.5 mM dithiothreitol and pepstatin and leupeptin each at 1 ,ug/ml). The column was washed with 12 ml of buffer and then developed with a 40-ml gradient of 0-0.3 M NaCI in the same buffer, and 1-ml fractions were collected at a flow rate of 1 ml/min. Aliquots (25 p.l) of fractions were assayed for both raplB.GAP- and ras.GAP-stimulated GTPase activity. Total protein was determined by using the method of Bradford (33) with bovine serum albumin as standard.
RESULTS Epinephrine Inhibits raplB.GAP-Activated GTPase Activity in Platelets. Whole platelet lysate from quiescent cells promoted rapid hydrolysis of [Y-32P]GTP bound to raplB, with greater than 65% hydrolysis in 10 min when 10 ,ug of total protein was used. Various platelet agonists affecting platelet metabolism by different signaling pathways were used in intact platelets to determine if their effects are reflected in changes of raplB.GAP-activated GTPase activity. There was no apparent effect on raplB-bound GTPase activity in equivalent platelet lysates that had been maximally stimulated with platelet-activating factor (PAF), phorbol 12,13-dibutyrate, a-thrombin, or iloprost (Fig. 1A). How-
Proc. Natl. Acad. Sci. USA 89 (1992)
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ever, when platelets were stimulated with 100 ,uM epinephrine for 2 min, only 25% of the [-32P]GTP bound to raplB was hydrolyzed in 10 min. In fact, dramatic decreases in hydrolytic rate and percentage hydrolysis of raplB-bound GTP were observed. In addition, when 10 ,AM ADP was used to stimulate platelets, an increase in the rate of GTP hydrolysis was observed (Fig. 1B). GTPase activity is typically defined as the percent of 100
100
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FIG. 1. Effects of stimulation by various platelet agonists on GTPase activity rapiB GTPase activity. (A) Time courses of raplB after stimulation of platelets with agonists that do not affect GTPase activity. Washed platelets incubated at 370C were either unstimulated (in) or stimulated with 1 &M PAF for 2 mmi(i), 100 nM phorbol 12,13-dibutyrate for 5 mmn (A), thrombin at 5 units/mi for 1 mmn (o), or 10 MM were lysed and 10pg of iloprost for 30 mn ().uPlatelets protein from the cleared lysates was assayed for raplB.GAP activity. Activities are expressed as percent [y-32P]GTP bound to raplB that is hydrolyzed relative to buffer control. Similar results were obtained in at least three other experiments. (B) Time courses of raplB GTPase activity after stimulation with epinephrine and ADP. Platelet treatment was as in A with no stimulation (m) or stimulation by 100 A&M epinephrine (A) or 10 A.M ADP (o) for 2 mmn. Results represent +the mean ±& SDT oftilctedtriaiosotie infeseart
experiments. (C) Epinephrine suppresses and ADP enhances raplB GTPase activity. Bars illustrate the mean percentage of GTP hydrolyzed in 10 min per 10 pAg of total protein in unstimulated (basal) and epinephrine- (100 ,M) and ADP- (10 ,M) stimulated platelet lysates.
Biochemistry: Marti and Lapetina
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Proc. Natl. Acad Sci. USA 89 (1992)
[y-32P]GTP bound to protein that is hydrolyzed in 10 min relative to buffer control. Although the increase in percentage of GTP hydrolyzed upon ADP stimulation was not as dramatic as the decrease observed with epinephrine, the observed differences in GTPase activity were real and significant (Fig. 1C). The mean percent ofraplB-bound GTP hydrolyzed in 10 min per 10 Iug total protein in unstimulated lysate was 68.5 + 0.825; GTPase activity in epinephrine- and ADP-stimulated platelet lysates was 25.8 + 1.3 and 78 1.25, respectively. Data are the mean SEM of 15 experiments, each performed in triplicate. Epinephrine suppressed the GTPase activity of raplB by -6o, whereas ADP increased GTPase activity -12%. Epinephrine Has No Effect on ras.GAP or rap2B.GAP. The GAP-activated GTPase activities associated with ras and rap2B were also assayed in lysates from quiescent and agonist-stimulated platelets. As shown in Fig. 2, there was no significant suppression of either of these GTPase activities in response to epinephrine stimulation, nor was there an increase upon ADP stimulation. It is also of interest to note that the GTPase activity observed in quiescent platelet lysate at 10 min was -30% of bound GTP hydrolyzed for both ras and rap2B. In addition, the initial rates of hydrolysis were significantly less than the rate observed for raplB.GAPstimulated hydrolysis. Epinephrine Action Appears to Be Receptor Mediated. To investigate whether the observed suppression of raplBassociated GTPase activity by epinephrine is a receptormediated process, platelets were exposed to the synthetic a2-adrenoceptor agonist UK14304 (bromoxidine). GTPase activity was completely suppressed by 20 ,uM UK14304 for up to 10 min at 30°C; low-level hydrolysis was observed at later time points (Fig. 3). Furthermore, the selective a2adrenoceptor antagonist yohimbine blocked suppression of GTPase activity resulting from epinephrine stimulation. As shown in Fig. 3, yohimbine, at a concentration reported to inhibit epinephrine's action in human platelets (34), exhibited a rate and level of raplB-associated GTPase activity similar to that found in unstimulated platelet lysates. To further test the possibility of receptor mediation of epinephrine's effect, equal amounts of lysate of unstimulated platelets and lysates of platelets stimulated with 100 ,uM epinephrine were mixed and GTPase activity was monitored. In addition, exogenous epinephrine was added to an unstimulated lysate to eliminate the possibility of a non-receptor-mediated effect on raplBassociated GTPase activity. The mixture of stimulated and ±
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FIG. 3. Effect of epinephrine on raplB.GAP is mimicked by UK14304 and blocked by yohimbine. Platelets were incubated at 37°C in the absence (o) or presence of 100 ,uM epinephrine (o), 20 ,uM UK14304 (A), or 100 ,uM epinephrine plus 20 AM yohimbine (U) for 2 min. After lysis of the cells, 10 Ag of protein was assayed for GTPase activity. Results represent the mean SD of triplicate determinations in three separate experiments. ±
unstimulated lysates, as well as the addition of exogenous epinephrine, did not result in suppression of raplBassociated GTPase activity (data not shown). Epiephrine's Suppression of raplB.GAP-AcP-Ated GTPase Activity Is Dose Dependent. All epinephrine stimulation data presented thus far were obtained by using epinephrine in the absence of a reducing agent. Dose dependence experiments were performed in the absence or presence of sodium metabisulfite (Na2S205) to ensure that the observed suppression was not due to oxidation of epinephrine and that the concentrations used were precise. Fig. 4 shows that suppression of raplB.GAP GTPase activity by epinephrine is dose dependent. Moreover, there is a
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FIG. 2. Epinephrine and ADP do not affect the GAP-stimulated GTPase activities associated with ras and rap2B. (A) Time courses of ras-bound GTP hydrolysis. Platelet treatment was as in the legend to Fig. 1 with no stimulation (-) or stimulation by 100 jLM epinephrine (e) or 10 ,uM ADP (A). Results represent the mean SD of triplicate determinations in three separate experiments. (B) Time courses of rap2B-bound GTP hydrolysis by unstimulated (m), 100 jM epinephrine-stimulated (9), or 10 ,uM ADP-stimulated (A) platelet lysates. Results represent the mean SD of triplicate determinations in three separate experiments. ±
±
100
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Epinephrlne, ,uM FIG. 4. Epinephrine suppresses raplB.GAP-stimulated GTPase activity in a dose-dependent manner. Platelets were stimulated for 2 min with various concentrations of epinephrine in the absence (o) or the presence (e) of 20 ,uM Na2S205. Cells were then lysed and assayed for raplB.GAP-stimulated GTPase activity in 10 min with 10 ,Bg of total protein. Percent inhibition is defined as the percent decrease in GTPase activity compared with unstimulated platelet lysate. Similar results were obtained in two other experiments.
Proc. Natl. Acad. Sci. USA 89 (1992)
Biochemistry: Marti and Lapetina shift in the dose-response curve toward more effective suppression of GTPase activity when epinephrine is maintained in a reduced state. Suppression can be observed at epinephrine concentrations as low as 25 ,uM in the absence of reducing agent and 10 uM in the presence of reducing agent. Complete suppression of GTPase activity can be obtained by using 500 A&M epinephrine and 20 AM Na2S2O5. Epinephrine Causes a Decrease in the Specific Activity of raplB.GAP-Stimulated GTPase Activity. To determine whether epinephrine specifically modifies raplB.GAP activity, lysates from quiescent and epinephrine-stimulated platelets were subjected to SDS/PAGE and either stained with Coomassie blue or immunoblotted with anti-phosphotyrosine antibody. No differences were observed in the patterns of proteins of whole cell lysates or tyrosine phosphorylation. In a recent study, ras.GAP and rapl.GAP activities migrated differently on anion-exchange chromatography (9). Lacking a specific antibody for raplB.GAP, we looked for a possible change in migration of raplB.GAP activity after epinephrine stimulation. Platelets were stimulated with 250 or 500 ,uM epinephrine for 2 min and the cells were lysed. Equal amounts of lysate (2 mg) from unstimulated or epinephrinestimulated platelets were subjected to anion-exchange chromatography on a Mono Q FPLC column. Fractions were assayed for both raplB.GAP and ras.GAP GTPase activities. As shown in Fig. 5 A and B, raplB.GAP and ras.GAP GTPase activities were well resolved in both unstimulated and stimulated lysates. In both unstimulated and epinephrinestimulated lysates, raplB.GAP activity eluted at 200-215 mM NaCl and ras.GAP activity eluted at about 150 mM NaCl. However, the peak GTPase activity associated with rapiB dropped dramatically from 65% in unstimulated lysate to 25% in epinephrine-stimulated lysate. A further reduction to 12% GTP hydrolysis was observed when a lysate from platelets stimulated with 500 AM epinephrine was subjected to Mono Q chromatography (data not shown). In contrast, ras.GAP GTPase activity remained essentially unchanged at around 30%. More important, the amount of protein in both raplB.GAP and ras.GAP peak fractions in the epinephrinestimulated lysate was not significantly different from that in the unstimulated lysate. Fig. 5C shows that the specific activity of peak raplB.GAP activity decreased as the concentration of epinephrine used to stimulate the platelets increased. Recoveries of raplB.GAP and ras.GAP activities from either unstimulated or epinephrine-stimulated platelet lysates were in the range of 70-90%o of the activity loaded onto the Mono Q column.
0A 1
2787
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DISCUSSION Platelets possess GAPs specific for raplB, rap2B, and ras. The specificity of these GAPs is evidenced not only by a difference in the rates of hydrolysis of GTP bound to these proteins but also by a difference in the amount of proteinbound hydrolysis observed in a typical 10-min period. The most convincing evidence for this specificity, however, is our finding that epinephrine suppressed the otherwise rapid hydrolysis of raplB-bound GTP observed in lysates of quiescent platelets. Moreover, ADP stimulation of intact platelets slightly increased raplB.GAP-activated GTPase activity but not rap2B.GAP or ras.GAP activity. Other agonists that activate platelets by different signal transduction pathways had no effect on the GAP-activated GTPase activity of raplB, rap2B, or ras. Epinephrine action appears to be a2A-adrenoceptor mediated. Several lines of evidence support this hypothesis. First, the suppression of GTPase activity can be mimicked by stimulating platelets with the potent synthetic a2-adrenoceptor agonist UK14304. This analog of clonidine has been shown to produce an aggregatory effect on platelets compa-
Basal
Epi 250
Epi 500
FIG. 5. Epinephrine decreases the relative specific activity of raplB.GAP. (A and B) Mono Q anion-exchange column profiles of quiescent platelet lysate (A) or lysate from platelets stimulated with 250 AtM epinephrine (B). *, raplB.GAP activity; o, ras.GAP activity; solid line, protein concentration; broken line, NaCl concentration. (C) Decreased specific activity of epinephrine-stimulated platelet lysate. Specific activity is defined as the ratio of the GTPase activity (percent hydrolyzed) to protein concentration (j.g/ml) of pooled peak fractions of raplB.GAP (cross-hatched bars) and ras.GAP (hatched bars) activity after anion-exchange chromatography of lysate of unstimulated platelets (Basal) or lysates of platelets stimulated with 250 jAM epinephrine (Epi 250) or 500 ,.M epinephrine (Epi 500). rable to that seen with epinephrine (35). Furthermore, the selective a2-adrenoceptor antagonist yohimbine efficiently blocked epinephrine-stimulated suppression of GTPase activity. This compound also blocks platelet aggregation in-
2788
Biochemistry: Marti and Lapetina
duced by epinephrine (36). In addition, when lysates from unstimulated and epinephrine-stimulated platelets were mixed or exogenous epinephrine was added to unstimulated lysate, no suppression of GTPase activity was observed, eliminating the possibility of the presence of a raplB.GAPspecific phosphatase. We have shown that the effect of epinephrine suppression of raplB.GAP activity is dose dependent and that suppression is more effective in the presence of a mild reducing agent such as Na2S205, thereby eliminating the possibility that epinephrine's action results from oxidation of epinephrine itself. Our preliminary experiments to assess the mechanism of epinephrine's effect on raplB.GAP show that no obvious modifications occur. Appearance or disappearance of a particular protein or change in tyrosine phosphorylated proteins was not observed after epinephrine stimulation. Epinephrine did not change the migration behavior of raplB.GAP or ras.GAP during anion-exchange chromatography, indicating that the ionic state of these proteins was not significantly affected. However, the level of peak GTPase activity associated with raplB was notably reduced, in contrast to the essentially unchanged peak level of ras.GAP activity. In addition, the amounts of protein in raplB.GAP and ras.GAP peaks in unstimulated and epinephrine-stimulated lysate did not significantly differ. Thus, the specific activity of peak raplB.GAP decreased as the concentration of epinephrine used to stimulate the platelets increased. These results indicate that epinephrine's effect is truly suppression of GTPase activity and not reduction in the amount of raplB.GAP available for activation of raplB GTPase by modification, degradation, or otherwise selective elimination of raplB.GAP. The reason for the observed increase in specific activity of peak ras.GAP is unclear, given that no enhancement of ras.GAP activity was observed in epinephrinestimulated lysate prior to separation on the Mono Q column. In addition, there was no significant difference in recovery of either raplB.GAP or ras.GAP activity after anion-exchange chromatography. Considering the highly reproducible and consistent effect of epinephrine on raplB.GAP activity, we believe this apparent increase in ras.GAP activity is an artifactual consequence of the chromatography and is not related to the suppression of raplB.GAP activity observed in epinephrine-stimulated platelet lysates. It is known that a2-adrenergic receptors couple to an inhibition of adenylate cyclase. This inhibition is mediated by Gia-2. However, many a2-adrenoceptor-mediated responses do not result in lower cAMP. In fact, epinephrine's aggregatory action does not appear to occur as a result of adenylate cyclase inhibition (21). Some evidence suggests that a2adrenoceptors may also couple to other second messenger systems (31, 37). The a2-adrenoceptor has been implicated in the modulation of cellular signaling pathways such as Na+/H' exchange (38), K+ channels (39), and voltagesensitive Ca2+ channels (40). It is unclear whether these effects were due to decreased cAMP caused by additional receptor or to effector activation. The data presented here may be indicative of a pathway in platelets initiated by epinephrine binding to its receptor but not involving inhibition of adenylate cyclase. None of the members of the ras superfamily of proteins has yet been implicated as playing a role in platelet activation. Our observations suggest that raplB.GAP and rap1B appear to actually play a role in platelet response to agonist stimulation of the a2-adrenoceptor in vivo. Suppression of raplB.GAP-activated GTPase activity by epinephrine results in raplB remaining in the GTP-bound or activated form.
Proc. Natl. Acad. Sci. USA 89 (1992)
Further characterization of this effect will provide a better understanding of the implications of these observations. 1. Halenbeck, R., Crosier, W. J., Clark, R., McCormick, F. & Koths, K. (1990) J. Biol. Chem. 265, 21922-21928. 2. Trahey, M., Wong, G., Halenbeck, R., Rubinfeld, B., Martin, G. A., Ladner, M., Long, C. M., Crosier, W. J., Watt, K., Koths, K. & McCormick, F. (1988) Science 242, 1697-1700. 3. Vogel, U. S., Dixon, R. A. F., Schaber, M. D., Diehl, R. E., Marshall, M. S., Scolnick, E. M., Sigal, I. S. & Gibbs, J. B. (1988) Nature (London) 335, 90-93. 4. Gibbs, J. B., Schaber, M. D., Allard, W. J., Sigal, I. S. & Scolnick, E. M. (1988) Proc. Nail. Acad. Sci. USA 85, 5026-5030. 5. Burstein, E. S., Linko-Stentz, K., Lu, Z. & Macara, I. G. (1991) J. Biol. Cheml. 266, 2689-2692. 6. Garrett, M. D., Major, G. N., Totty, N. & Hall, A. (1991) Biochem. 1. 276, 833-836. 7. Morii, N., Kawano, K., Sekine, A., Yamada, T. & Narumiya, S. (1991) J. Biol. Chem. 266, 7646-7650. 8. Kikuchi, A., Sasaki, T., Araki, T., Hata, Y. & Takai, Y. (1989) J. Biol. Chem. 264, 9133-9136. 9. Polakis, P. G., Rubinfeld, B., Evans, T. & McCormick, F. (1991) Proc. Nadl. Acad. Sci. USA 88, 239-243. 10. Rubinfeld, B., Munemitsu, S., Clark, R., Conroy, L., Watt, K., Crosier, W. J., McCormick, F. & Polakis, P. (1991) Cell 65, 1033-1042. 11. Pizon, V., Lerosey, I., Chardin, P. & Tavitian, A. (1988) Nucleic Acids Res. 16, 7719. 12. Adari, H., Lowy, D. R., Willumsen, B. M., Der, C. J. & McCormick, F. (1988) Science 240, 518-521. 13. Hata, Y., Kikuchi, A., Sasaki, T., Schaber, M. D., Gibbs, J. B. & Takai, Y. (1990) 1. Biol. Chem. 265, 7104-7107. 14. Frech, M., John, J., Pizon, V., Chardin, P., Tavitian, A., Clark, R., McCormick, F. & Wittinghofer, A. (1990) Science 249, 169-171. 15. Kitayama, H., Sugimoto, Y., Matsuzaki, T., Ikawa, Y. & Noda, M. (1989) Cell 56, 77-84. 16. Campa, M. J., Chang, K.-J., Molina y Vedia, L., Reep, B. R. & Lapetina, E. G. (1991) Biochem. Biophys. Res. Commun. 174, i-5. 17. Kawata, M., Kikuchi, A., Hoshijima, M., Yamamoto, K., Hashimoto, E., Yamamura, H. & Takai, Y. (1989) J. Biol. Chem. 264, 15688-15695. 18. Lapetina, E. G., Lacal, J. C., Reep, B. R. & Molina y Vedia, L. (1989) Proc. Nail. Acad. Sci. USA 86, 3131-3134. 19. Lazarowski, E. R., Lacal, J.-C. & Lapetina, E. G. (1989) Biochem. Biophys. Res. Commun. 161, 972-978. 20. Siess, W., Winegar, D. A. & Lapetina, E. G. (1990) Biochem. Biophys. Res. Commun. 170, 944-950. 21. Siess, W. & Lapetina, E. G. (1990) Biochem. J. 271, 815-819. 22. Fischer, T. H., Gatling, M. N., Lacal, J.-C. & White, G. C., III (1990) J. Biol. Chem. 265, 19405-19408. 23. Seiss, W., Weber, P. C. & Lapetina, E. G. (1984) J. Biol. Chem. 259, 8286-8292. 24. Crouch, M. F. & Lapetina, E. G. (1988) J. Biol. Chem. 263, 3363-3371. 25. Lapetina, E. G. (1986) in Phosphoinositides and Receptor Mechanisms, ed. Putney, J. W., Jr. (Liss, New York), Vol. 7, pp. 271-286. 26. Jakobs, K. H., Saur, W. & Schultz, G. (1976) J. Cyclic Nucleotide Res. 2, 381-392. 27. Macfarlane, D. E. & Stump, D. C. (1982) Mol. Pharmacol. 22, 574-579. 28. Cerione, R. A., Regan, J. W., Nakata, H., Codina, J., Benovie, J. L., Gierschik, P., Somers, R. L., Spiegel, A. M., Birnbaumer, L., Lefikowitz, R. J. & Caron, M. G. (1986) J. Biol. Chem. 261, 3901-3909. 29. Aktories, K. & Jakobs, K. H. (1985) in The Platelet: Physiology and Pharmacology, ed. Langenecker, E. (Academic, New York), pp. 243260. 30. Steen, V. M., Tysnes, O.-B. & Holmsen, H. (1988) Biochem. J. 253, 581-586. 31. Cotecchia, S., Kobilka, B. K., Daniel, K. W., Nolan, R. D., Lapetina, E. G., Caron, M. G., Lefkowitz, R. J. & Regan, J. W. (1990) J. Biol. Chem. 265, 63-69. 32. Bylund, D. B., Ray-Prenger, C. & Murphy, T. J. (1988) J. Pharmacol. Exp. Ther. 245, 600-607. 33. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254. 34. Crouch, M. F., Winegar, D. A. & Lapetina, E. G. (1989) Proc. Natl. Acad. Sci. USA 86, 1776-1780. 35. Grant, J. A. & Scrutton, M. C. (1980) Br. J. Pharmacol. 71, 121-134. 36. Hsu, C. Y., Knapp, D. R. & Halushka, P. V. (1979) J. Pharmacol. Exp. Ther. 206, 366-370. 37. Michel, M. C., Brass, L. F., Williams, A., Bokoch, G. M., La Morte, V. J. & Motulsky, H. J. (1989) J. Biol. Chem. 264, 4986-4991. 38. Sweatt, J. D., Blair, I. A., Cragoe, E. J. & Limbird, L. E. (1986) J. Biol. Chem. 261, 8660-8666. 39. North, R. A., Williams, J. T., Surprenant, A. & Christie, M. J. (1987) Proc. Natl. Acad. Sci. USA 84, 5487-5491. 40. Hescheler, J., Rosenthal, W., Trautwein, W. & Schultz, G. (1987) Nature (London) 325, 445-447.
