Identification of Galpha 13 as One of the G-proteins That Couple to Human Platelet Thromboxane A2 Receptors*

Yasmine DjellasDagger , Jeanne M. ManganelloDagger , Kostas Antonakis§, and Guy C. Le BretonDagger

From the Dagger  Department of Pharmacology, University of Illinois, Chicago, Illinois 60612 and the § Institut de Recherches Scientifiques sur le Cancer, Chimie organique-biologique et spectroscopique (UMR 133), CNRS B.P. 8, Villejuif 94802, France

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Previous studies have shown that ligand or immunoaffinity chromatography can be used to purify the human platelet thromboxane A2 (TXA2) receptor-Galpha q complex. The same principle of co-elution was used to identify another G-protein associated with platelet TXA2 receptors. It was found that in addition to Galpha q, purification of TXA2 receptors by ligand (SQ31,491)-affinity chromatography resulted in the co-purification of a member of the G12 family. Using an antipeptide antibody specific for the human G13 alpha -subunit, this G-protein was identified as Galpha 13. In separate experiments, it was found that the TXA2 receptor agonist U46619 stimulated [35S]guanosine 5'-O-(3-thiotriphosphate) incorporation into G13 alpha -subunit. Further evidence for functional coupling of G13 to TXA2 receptors was provided in studies where solubilized platelet membranes were subjected to immunoaffinity chromatography using an antibody raised against native TXA2 receptor protein. It was found that U46619 induced a significant decrease in Galpha q and Galpha 13 association with the receptor protein. These results indicate that both Galpha q and Galpha 13 are functionally coupled to TXA2 receptors and dissociate upon agonist activation. Furthermore, this agonist effect was specifically blocked by pretreatment with the TXA2 receptor antagonist, BM13.505. Taken collectively, these data provide direct evidence that endogenous Galpha 13 is a TXA2 receptor-coupled G-protein, as: 1) its alpha -subunit can be co-purified with the receptor protein using both ligand and immunoaffinity chromatography, 2) TXA2 receptor activation stimulates GTPgamma S binding to Galpha 13, and 3) Galpha 13 affinity for the TXA2 receptor can be modulated by agonist-receptor activation.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES

Interaction of the prostaglandin endoperoxide analogue, TXA2 1 (1, 2) with platelet receptors (3-5) has been shown to modulate not only hemostasis but also the development of thromboembolic diseases (6-9). However, despite recent progress, the TXA2-mediated signal transduction pathway is not completely understood. In this regard, previous studies have shown that one mechanism by which TXA2 receptors act is through stimulation of phospholipase C (PLC) leading to inositol 1,4,5-triphosphate (IP3) production, and subsequent intracellular Ca2+ mobilization (10-14). Furthermore, separate studies have linked this stimulation of PLC activity to TXA2 receptor signal transduction through the pertussis toxin-insensitive guanine nucleotide-binding protein (G-protein) Gq (16, 17). On the other hand, experiments conducted in our laboratory provided evidence for the existence of intraplatelet Ca2+ mobilization, which is independent of IP3 production (15). This finding raised the possibility that TXA2 receptors may also couple to a G-protein family separate from Gq. Additional evidence in support of this notion was provided by experiments showing that a C-terminal antibody which recognizes the alpha -subunit of Gq and G11 was not able to completely inhibit U46619-stimulated GTPase activity (16). Moreover, ligand and immunoaffinity chromatography purification of the TXA2 receptor-G-protein complex allowed co-purification of G-proteins distinct from Gq (17). Taken together, these results led to the hypothesis that TXA2 receptors might couple to a G-protein(s) to stimulates platelet aggregation independently of the Gq-PLC-IP3 pathway.

Although this putative G-protein has not been identified, recent reports have provided indirect evidence that it may belong to the G12 family (18, 19). In one study, it was shown that activation of platelet TXA2 receptors led to increased incorporation of the photo reactive GTP analogue [alpha -32P]GTP azidoanilide into both G12 and G13 alpha -subunits, which may suggest coupling of TXA2 receptors to these alpha -subunits (20). On the other hand, as all the agonists tested (U46619, thrombin, ADP, and vasopressin) produced [alpha -32P]GTP azidoanilide incorporation, this labeling could also have been due to activation of a downstream signaling event or to cross-talk between these separate signal transduction pathways. In separate studies, it was shown that the affinity state of TXA2 receptors transfected in COS-7 cells could be influenced by co-expression of Galpha 13 (21). Although this finding is consistent with the notion that TXA2 receptors have the capacity to couple with G13, it is not clear whether such coupling occurs at physiological concentrations of receptor and/or G-protein. Consequently, two independent reports have provided indirect evidence that TXA2 receptors may couple to a Galpha subunit in the G12/13 family. Based on these considerations, in the present study we performed experiments to determine whether this phenomenon occurs in a native platelet preparation using endogenous concentrations of TXA2 receptor and Galpha subunits. To this end, affinity purification of the receptor-G-protein complex was employed to measure direct physical association of TXA2 receptors and Galpha 12/13. This approach has been previously applied in our laboratory to the identification of Galpha q as one of the G-proteins associated with the platelet TXA2 receptors (17). It was found that, in addition to Galpha q, purification of the TXA2 receptors resulted in co-elution of Galpha 13. Furthermore, agonist activation of TXA2 receptors caused an increase in GTPgamma S binding to G13 alpha -subunit as well as dissociation of the receptor-Galpha 13 complex, providing evidence that Galpha 13 is indeed functionally coupled to platelet TXA2 receptors.

    EXPERIMENTAL PROCEDURES
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INTRODUCTION
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Materials-- Outdated platelet concentrates were obtained from Heartland Blood Services (Aurora, IL). SQ intermediate (ethyl-[1S [1alpha , 2alpha -(Z),3alpha ,4alpha ]]-7-[[3-aminomethyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoate) for the synthesis of SQ31,491 was provided by Bristol-Myers Squibb Institute for Medical Research. BM13.177 and BM13.505 were generously supplied by Dr. K. Stegmeier, Roche Molecular Biochemicals (Mannheim, Germany). [35S]GTPgamma S (1000-1300 Ci/mmol) was purchased from Amersham Pharmacia Biotech. U44619 was purchased from Cayman Chemicals; asolectin was from the American Lecithin Co. (Atlanta, GA); CHAPS, protein A-Sepharose CL-4B, GTPgamma S, o-phenylenediamine, and rabbit preimmune IgG were from Sigma; Affi-Gel 102 and 4-chloro-1-naphthol (horseradish peroxidase color development reagent) were from Bio-Rad; and horseradish peroxidase-conjugated goat anti-rabbit IgG (H+L), biotinylated goat anti-rabbit IgG (H+L), and the Vectastain ABC kit were purchased from Vector Laboratories (Burlingame, CA).

Antibodies-- A 9-amino acid peptide corresponding to residues 40-48 of the human Galpha 13 (P21, Table I) (22), with a cysteine added at the N terminus to facilitate coupling to carrier protein was synthesized by Chiron Mimotropes (Raleigh, NC). The peptide was coupled to keyhole limpet hemocyanin using m-maleimidobenzoic acid N-hydroxysuccinimide ester and injected into White New Zealand Pasteurella multocida-free rabbits, according to previously described procedures (23). Rabbit polyclonal antibodies against the C-terminal region of Galpha q (G-QL, Table I) were produced as described previously (16). Antibodies were purified from rabbit serum by chromatography on protein A-Sepharose CL-4B, and the IgG fractions were labeled with carrier-free Na125I (Amersham Pharmacia Biotech) using the IODO-BEADS iodination reagent (Pierce). Rabbit polyclonal IgG raised against residues 2-21 of Galpha 12 (G-12) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit polyclonal IgG directed against the C-terminal region of Galpha 12 was a generous gift from J. Sylvio Gutkind (National Institutes of Health, Bethesda, MD) (24). Although this antibody has been suggested to be specific for Galpha 12, C-terminal segments of G12 and G13 differ only by 4 amino acids (18, 19). Consequently, this antibody may have limited cross-reactivity with G13 and is named G-12/13. Galpha common, a rabbit polyclonal IgG recognizing G-protein alpha -subunits of the Gs and Gi families, was purchased from Calbiochem.

Membrane Preparation and Solubilization-- Human platelet membranes were prepared from platelet concentrates and solubilized using 10 mM CHAPS as described previously (5). Typically, this method resulted in a 60-70% solubilization of platelet membrane protein, yielding a final protein concentration of 2-3 mg/ml.

Ligand Affinity Chromatography Purification of the Thromboxane A2 Receptor-G-protein Complex-- TXA2 receptors were purified as described previously (5). Briefly, the TXA2 receptor antagonist SQ31,491 was immobilized to Affi-Gel 102, and CHAPS-solubilized membranes (4 mg of protein) in buffer A (20% glycerol, 500 mM KCl, 0.2 mM EGTA, and 0.5 mg/ml asolectin) were incubated with the matrix overnight. Unbound proteins were eluted as flow-through, and the column was washed with buffer D (20 mM Tris-HCl, 10 mM CHAPS, 20% glycerol, 500 mM KCl, 0.2 mM EGTA, 0.5 mg/ml asolectin, pH 7.4). TXA2 receptors and receptor-associated proteins were then eluted with buffer D containing 50 mM TXA2 receptor antagonist, BM13.177 (25, 26). After elution of the first 1-ml fraction, the flow was stopped for 30 min and restarted to elute the subsequent 1-ml fractions. TXA2 receptor binding activity as well as Galpha q immunoreactivity were found to be concentrated in the first fraction following the 30-min incubation (17). A modification of this method was used in order to allow further identification of the TXA2 receptor-associated G-proteins (27). Specifically, after unbound proteins were washed with buffer D, 3 µg/ml 125I-G-QL IgG, 125I-G-12 IgG, or 125I-G-13 IgG (or the same protein concentration of 125I-labeled preimmune IgG (PI IgG)) was added, and the reaction mixture was allowed to incubate for 1 h at 20 °C. Unbound antibodies were washed with buffer D, and elution of TXA2 receptors and receptor-associated proteins was performed as described above. The elution fractions were counted for 125I activity and specific binding attributable to Galpha q, Galpha 12, or Galpha 13 was defined as the difference between the counts eluted from the 125I-antibody columns minus the counts eluted from the 125I-PI IgG column.

Immunoaffinity Chromatography Purification of the Thromboxane A2 Receptor-G-protein Complex-- Solubilized platelet membranes were prepared as described (5), and the CHAPS concentration was adjusted to 2 mM. The preparation (4 mg of protein) was then incubated with an immunoaffinity matrix coupled to an anti-TXA2 receptor antibody (TxAb) for 1 h at 20 °C (28). 125I-Labeled G-QL IgG, G-13 IgG, or PI IgG was added (final concentration 150 µg/ml), and the reaction mixture was allowed to incubate for 5 min. The preparation was then incubated with vehicle or the TXA2 agonist U46619 (100 nM) (29) for an additional 5 min. The matrix was loaded on a column and washed with buffer D to elute unbound proteins. The column was eluted with 100 mM glycine (pH 2.5), and the 3-ml elution fraction was counted for 125I activity. Specific binding attributable to Galpha q or Galpha 13 was defined as the difference between the counts eluted from the 125I-G-QL or 125I-G-13 columns, respectively, minus the counts eluted from the 125I-PI IgG column. Eluted counts were normalized to the amount of purified TXA2 receptor protein, as measured by densitometric analysis of the immunoaffinity column elution fractions immunoblotted with TxAb. In experiments where the TXA2 receptor antagonist BM13.505 (30) was used to block U46619 effects, BM13.505 (10 µM) was incubated for 30 min before addition of U46619.

Assay of [35S]GTPgamma S Binding-- Solubilized platelet membranes (4 mg of protein) were prepared as described (5) and incubated with 10 µM GTPgamma S (5 × 106 cpm [35S]GTPgamma S) for 5 min at room temperature in the presence of 1 µM GDP. The incubation was then allowed to proceed for an additional 15 min at room temperature in the presence or absence of 10 nM U46619. The preparation was added to 20 µl of G-13 IgG, which had been preincubated with 55 µl of a 10% (w/v) suspension of protein A-Sepharose. The immunoprecipitates were collected, washed to remove nonspecifically bound proteins and incubated with 1 mM G-13 peptide for 1 h at room temperature to specifically elute G13 alpha -subunits. The fractions were then added to 5 ml of EconosafeTM (Research Product International, IL) and analyzed by scintillation spectrometry. Eluted counts were normalized to the amount of immunoprecipitated G13 alpha -subunit, as measured by densitometric analysis of the elution fractions immunoblotted with G-13 IgG.

ELISA-- Immulon 2 microtiter plates were coated with either 12.5 µg of synthetic peptide or 125 µg of solubilized platelet membranes. Following incubation for 1 h at room temperature, the plates were washed three times with modified Tyrode's buffer containing 0.1% bovine serum albumin, 5 mM dextrose, 1 mM CaCl2, 5 mM HEPES, pH 7.4, and then blocked by incubation for 1 h with 5% bovine serum albumin in the same buffer. Serial dilutions of antisera were applied to the wells and incubated for an additional 1 h at room temperature. The wells were washed three times with the modified Tyrode's buffer, and bound antibodies were detected by incubation for 1 h with goat anti-rabbit IgG (H+L) conjugated to horseradish peroxidase. After extensive washing, the color reaction was developed by addition of 50 µl of 0.4 mg/ml o-phenylenediamine, 0.012% H2O2 in 80 mM citrate phosphate, pH 5. An equal volume of 2 N H2SO4 was then added, and the presence of specific antibodies was measured by absorbance at 490 nm.

Polyacrylamide Gel Electrophoresis and Immunoblot Assay-- The affinity column eluates were first concentrated using Millipore Ultrafree-MC filters. 20-40 µl of sample (30-40 µg of protein) was then subjected to SDS-PAGE according to the method of Laemmli (31) using 10% minigels, under nonreducing conditions, and the proteins were electrophoretically transferred onto nitrocellulose membranes according to the method of Towbin et al. (32). After transfer, the nitrocellulose membranes were blocked with 3% gelatin in Tris-buffered saline and incubated overnight at room temperature with the indicated dilution of G-QL, G-12/13, or Galpha common IgG. The blots were washed and treated with biotinylated goat anti-rabbit IgG (H+L) as the secondary antibody. The immunoreactive proteins were detected with avidin and horseradish peroxidase, followed by 0.5 mg/ml 4-chloro-1-naphthol.

Statistical Analysis-- Data were analyzed according to Student's paired t test (*, p < 0.05; **, p < 0.005).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In order to purify TXA2 receptor-associated G-proteins, solubilized platelet membranes were subjected to ligand affinity chromatography, and the column was eluted with the TXA2 receptor antagonist BM13.177 (5, 25, 26). Elution fractions were then immunoblotted with G-protein alpha -subunit specific antibodies. As illustrated in Fig. 1, G-QL IgG, which recognizes Gq and G11 alpha -subunits, blotted a major protein band in the molecular mass region of 42 kDa and two minor bands at approximately 38 kDa in solubilized platelet membranes and the ligand column elution fraction. This pattern of primary labeling at 42 kDa and secondary labeling at lower molecular masses has been previously described in human platelets and other tissues and has been attributed to proteolytic fragments of Galpha q (16). Furthermore, blotting with G-12/13 IgG also revealed immunoreactivity for the alpha -subunit of G12/13. Thus, a single band in the molecular mass range of 43-44 kDa was observed both in solubilized platelet membranes and the ligand affinity column eluate (Fig. 1). On the other hand, an antibody against Galpha common revealed that members of the Galpha s and Galpha i families were only present in solubilized platelet membranes but not in the affinity column eluate (data not shown). These results indicate that the ligand chromatography-purified TXA2 receptor-G-protein complex is selectively enriched in both Galpha q and a member(s) of the Galpha 12 family.


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Fig. 1.   Immunoblot of solubilized platelet membranes and the ligand affinity column eluate against G-QL and G-12/13 antisera. Proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes as described under "Experimental Procedures." The membranes were incubated overnight with 1:300 dilution of G-QL IgG or 1:200 dilution of G-12/13 IgG. Lanes 1 and 3, solubilized platelet membranes; lanes 2 and 4, ligand affinity column eluate.

In order to determine whether TXA2 receptors purified in complex with either Galpha 12 or Galpha 13, the ligand affinity eluate was first probed using a new antibody specifically directed against Galpha 13. This polyclonal antibody was generated by immunizing rabbits with a peptide sequence unique to an internal segment of the human G13 alpha -subunit (G-13, Table I) (22). Evaluation of the sera and IgG revealed that specific antibodies against Galpha 13 were successfully raised. Specifically, the anti-G-13 serum was shown by ELISA to react in a concentration-dependent manner with its cognate peptide (Fig. 2A). Moreover, evaluation of solubilized platelet membranes by ELISA revealed positive immunoreactivity against an endogenous platelet protein (Fig. 2B). In addition, immunoblotting of solubilized platelet membranes with G-13 serum and IgG demonstrated a predominant 43-44 kDa protein (Fig. 3, lanes 1 and 2, respectively), consistent with the molecular mass previously described for G13 alpha -subunit (18, 19, 22). The blotting of this band could be prevented by preincubation of G-13 IgG with its cognate peptide (Fig. 3, lane 3). To utilize this antibody for quantitative evaluation of Galpha 13, G-13 IgG was iodinated by standard procedures.

                              
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Table I
Definition of anti-G-protein alpha -subunit antibodies


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Fig. 2.   Reactivity of G-13 antiserum against its cognate peptide and solubilized platelet membranes. Microtiter wells were coated with 12.5 µg of G-13 peptide (panel A) or 125 µg of solubilized platelet membrane proteins (panel B). Various dilutions of G-13 antiserum () or preimmune antiserum (open circle ) were added to the wells. Immunoreactivities were detected by ELISA, as described under "Experimental Procedures." Each point is the mean of triplicate values obtained from two separate experiments. Standard error of the mean was typically less than 10%.


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Fig. 3.   Immunoblot of solubilized platelet membranes against G-13 antiserum and G-13 IgG. Proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes as described under "Experimental Procedures." The membranes were incubated overnight with 1:50 dilution of G-13 antiserum (lane 1), 1:50 dilution of G-13 IgG (lane 2), or 1:50 dilution of G-13 IgG preincubated with 14.4 µg/ml (100 µM) G-13 peptide (lane 3).

In the next experiments, solubilized platelet membranes were incubated with the ligand affinity matrix (5). The column was washed with buffer and then equilibrated with 125I-G-13 IgG. The TXA2 receptor-G-protein complex was next eluted by BM13.177 (25, 26), and the elution fractions were quantitated for 125I. As Galpha q is known to couple to TXA2 receptors (16, 17), a positive control experiment was conducted using 125I-G-QL IgG. Control experiments were also performed using 125I-labeled preimmune IgG to determine nonspecific binding of both G-QL and G-13 IgG. Reported specific binding represents the difference between the counts eluted from the 125I-anti-alpha -subunit IgG column and the counts eluted from the 125I-labeled preimmune IgG column. Using this procedure, it was found that TXA2 receptors co-purified not only with Galpha q but also with Galpha 13. Thus, Fig. 4 illustrates that the affinity column eluate contained 53 ± 5% and 24 ± 5% specific binding for Galpha q (solid bar) and Galpha 13 (open bar), respectively. These findings, therefore, provide evidence that both Galpha q and Galpha 13 are in direct physical association with endogenous platelet TXA2 receptors.


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Fig. 4.   Ligand affinity chromatography purification of TXA2 receptor-G-protein complex. Solubilized platelet membranes were subjected to ligand affinity chromatography as described under "Experimental Procedures," and the purified TXA2 receptor-G-protein complex was probed using 125I-labeled G-QL, G-13 and G-12. Results are expressed in counts per minute (cpm) of specifically eluted 125I-labeled IgG and represent the mean ± S.E. of four separate experiments. Statistical significance was evaluated using a two-sample Student's t test (*, p < 0.05; **, p < 0.005).

The next series of experiments was performed to determine whether Galpha 12 also copurified as part of the TXA2 receptor-G-protein complex. These studies employed a specific antibody directed at a unique N-terminal segment of Galpha 12 (G-12, Table I), which was also iodinated for quantitative purposes. Again, the TXA2 receptor-G-protein complex was purified by ligand affinity chromatography and the amount of Galpha 12 present in the eluate was determined using 125I-G-12 IgG. As above, specific binding was determined by parallel experiments using 125I-labeled preimmune IgG. It was found that, in contrast to Galpha 13, Galpha 12 did not appear to co-purify with TXA2 receptors (Fig. 4, hatched bar). In these experiments, it can be seen that the counts attributable to Galpha 12 are less than the counts representing nonspecific binding by preimmune IgG. Although this decrease is not significant, it can be explained on the basis that G-12 IgG is enriched in immunoglobulins against Galpha 12 and consequently contains a lesser percentage of nonspecific proteins than preimmune IgG. As nonspecific protein is presumably responsible for nonspecific binding observed with preimmune IgG, the difference between 125I-G-12 IgG counts and 125I-labeled preimmune IgG counts yields a negative number. The same phenomenon would also suggest that the specific binding observed for both G-QL and G-13 IgG (Fig. 4) is probably underestimated, as each of these IgG fractions contain less nonspecific proteins than their preimmune IgG controls. Furthermore, this consideration would indicate that the relative percentage of specific binding with G-QL and G-13 IgG may not necessarily represent the actual distribution of Galpha q and Galpha 13 within the TXA2 receptor-G-protein complex. Taken together, the above results provide evidence that in addition to Galpha q, platelet TXA2 receptors are coupled to endogenous Galpha 13.

In the next series of experiments, the agonist U46619 was used to determine whether Galpha 13 is functionally coupled to TXA2 receptors. In these studies, solubilized platelet membranes were incubated with [35S]GTPgamma S in the presence and absence of U46619 (10 nM) and subjected to immunoprecipitation with G-13 IgG. Immunoprecipitated Galpha 13 was eluted using 1 mM G-13 peptide, the elution fractions were counted for 35S and the counts were normalized for the amount of purified G13 alpha -subunit. It can be seen in Fig. 5A that TXA2 receptor activation led to a 44 ± 18% (p = 0.05) increase in [35S]GTPgamma S binding to Galpha 13.


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Fig. 5.   A, effect of U46619 treatment on [35S]GTPgamma S binding to Galpha 13. Solubilized platelet membranes were incubated with [35S]GTPgamma S in the presence and absence of U46619 (10 nM) and subjected to immunoprecipitation using G-13 IgG as described under "Experimental Procedures." Results are expressed as percentage of increase in [35S]GTPgamma S binding to immunoprecipitated Galpha 13 compared with vehicle and represent the mean ± S.E. of six separate experiments. Statistical analysis measuring the effect of U46619 on [35S]GTPgamma S binding to Galpha 13 was performed using a two-sample Student's t test. B, effect of U46619 treatment on Galpha q and Galpha 13 association with TXA2 receptors. Solubilized platelet membranes were subjected to immunoaffinity chromatography purification as described under "Experimental Procedures," and the purified TXA2 receptor-G-protein complex was probed with 125I-labeled G-QL and G-13 IgG in the presence and absence of U46619 (100 nM). Results are expressed in counts per minute (cpm) normalized for the amount of purified TXA2 receptor protein and represent the mean ± S.E. of five separate experiments. Statistical analysis measuring the effect of U46619 on Galpha q and Galpha 13 association with TXA2 receptors was performed using a two-sample Student's t test (*, p < 0.05; **, p < 0.005).

In separate experiments, an affinity column matrix coupled to an antibody raised against native TXA2 receptor protein (TxAb) was used to purify the receptor-G-protein complex (16, 28). Briefly, solubilized platelet membranes were incubated with the affinity matrix and the coupling of G-protein alpha -subunits to TXA2 receptors was evaluated in the presence and absence of U46619 (29). Evidence has been previously provided that agonist activation of a seven-transmembrane receptor results in the dissociation of its coupled G-protein alpha -subunits (33). Based on these considerations, excess 125I-labeled anti-G-protein antibody was added to the column matrix prior to U46619 treatment in order to trap alpha -subunits that may be released upon receptor activation. The excess 125I-labeled anti-alpha -subunit antibody was then washed from the column matrix and the TXA2 receptor-G-protein complex was eluted with glycine 100 mM, pH 2.5. Elution fractions were then counted for 125I activity and normalized to the amounts of purified TXA2 receptor protein. Again, specific binding was determined in parallel experiments using 125I-labeled preimmune IgG. Consistent with the results obtained with ligand affinity chromatography, both Galpha q and Galpha 13 co-purified with platelet TXA2 receptors. Specific binding attributable to G-QL IgG and G-13 IgG was 42 ± 11% and 54 ± 10%, respectively (Fig. 5B, open bars). Furthermore, treatment with 100 nM U46619 (Fig. 5B, solid bars) caused a significant decrease in the amount of specifically eluted 125I-labeled G-QL and G-13 IgG. The magnitude of the agonist-induced decrease in G-protein-receptor association was 44 ± 15% and 32 ± 1% for Galpha q and Galpha 13, respectively. Finally, pretreatment with a TXA2 receptor antagonist, BM13.505 (10 µM) (30), completely inhibited U46619-induced receptor-G-protein complex dissociation (Fig. 5B, hatched bars). Taken together, these results demonstrate that in addition to Galpha q, human platelet TXA2 receptors are functionally coupled to Galpha 13.

    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The present study employed ligand affinity (5) and immunoaffinity chromatography techniques (28) to purify and identify G-proteins associated with human platelet TXA2 receptors. These techniques have previously been used for the purification of TXA2 receptor-G-protein complexes from solubilized platelet membranes (17). Using both ligand and immunoaffinity chromatography, it was found that in addition to Galpha q, a member of the Galpha 12 family of G-proteins co-purifies with platelet TXA2 receptors. This G-protein was identified as Galpha 13 using an antibody raised against a unique internal sequence of human G13 alpha -subunit (22). Additional studies demonstrated that this G13 alpha -subunit was functionally coupled to TXA2 receptors. Specifically, the TXA2 receptor agonist U46619 stimulated [35S]GTPgamma S incorporation into G13 alpha -subunit as well as caused dissociation of this subunit from TXA2 receptors.

The Galpha 12 family of G-proteins defines the fourth and the most recently discovered class of alpha -subunits (18, 19). The members of this family share high sequence homology and are ubiquitous and immunodetectable in most membranes of various mammalian cells and tissues (22, 34, 35). However, despite intensive research in the past years, no definitive effector(s) has been assigned to either Galpha 12 or Galpha 13 (36, 37). Both Galpha 12 and Galpha 13 are oncogenic, and expression of their mutationally activated forms stimulates cell proliferation and induces neoplastic transformation in NIH3T3 and Rat1 cells (24, 38-40). Furthermore, GTPase-deficient mutants of Galpha 12 and Galpha 13 have been shown to stimulate Jun kinase/stress-activated protein kinase (JNK/SAPK) in NIH3T3, HEK293, and COS-1 cells (41, 42). In addition, both activated alpha -subunits have been shown to stimulate stress fiber formation/focal adhesion assembly in Swiss 3T3 cells (43) and induce apoptosis when transfected in Chines hamster ovary or COS-7 cells (44, 45). Finally, signal transduction through Galpha 12 and Galpha 13 appears to involve small molecular weight GTP-binding proteins such as RhoA, cdc42, and Ras (46-48). However, even though there is similarity between Galpha 12 and Galpha 13-associated pathways, evidence has been provided that both subunits seem to fulfill distinct cellular and biological functions. Specifically, Galpha 12 but not Galpha 13 has been shown to be involved in the transcriptional activation of the serum response element (47). On the other hand, Galpha 13 but not Galpha 12 is involved in the induction of inducible nitric-oxide synthase in MCT cells (49) and in lysophosphatidic acid-induced activation of Rho (50). Other studies showed that, whereas the guanine nucleotide exchange factor (GEF) for Rho, p155RhoGEF, was able to act as a GTPase-activating protein toward both Galpha 12 and Galpha 13, only Galpha 13 bound to p155RhoGEF and stimulated its capacity to catalyze nucleotide exchange on Rho (51, 52). In addition, disruption of the gene encoding G13 alpha -subunit in mice impaired the ability of endothelial cells to develop into organized vascular system, resulting in intrauterine death and demonstrating a role for Galpha 13 in the regulation of cell movement and developmental angiogenesis (53).

Two potential effectors for Galpha 13 have been proposed that would be of interest in the signal transduction pathways associated with TXA2 receptors in platelets. In this connection, Galpha 13 has been shown to stimulate the ubiquitously distributed Na/H exchanger isoform, NHE1 (46, 53-55). Moreover, substitution of C-terminal residues from alpha z conferred on alpha 13 the ability to respond to stimulation by the D2-dopamine receptor and to activate NHE1 in an agonist-dependent manner (54).

In platelets, regulation of Na/H exchange has been shown to modulate receptor-mediated phospholipase A2 and phospholipase C activation as well as intracellular Ca2+ mobilization (57, 58). In regard to platelet TXA2 receptors, it was found that U46619 caused an increase in intracellular pH, which was required for full U46619-induced Ca2+ mobilization (59). Thus, coupling of TXA2 receptors to NHE1 activity stimulation could be a possible mechanism by which Galpha 13 is involved in TXA2-mediated signal transduction in platelets.

In addition to its indirect effects on intracellular Ca2+ via Na/H exchanger activity stimulation, Galpha 13 has also been implicated in the activation of L-type Ca2+ channels (60, 61). Specifically, in rat portal vein myocytes, the heterotrimer alpha 13beta 1gamma 3 couples to the angiotensin AT1A receptors to increase cytoplasmic Ca2+ concentration (60). Furthermore, it was found that the beta gamma dimer released from alpha 13 upon angiotensin AT1A receptor activation was responsible for the activation of L-type Ca2+ channels (61). Although extracellular Ca2+ influx through L-type and non-L-type Ca2+ channels has been associated with TXA2 receptor-mediated contraction in rat aorta (62), no such channels have been identified on the platelet surface thus far.

In summary, the present data demonstrate that platelet TXA2 receptors are functionally coupled to Galpha 13. The physiological significance of the signal transduction pathway associated with such coupling requires further investigation.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grant HL-24530, NATO Grant CRG-940595, and Training Grant "Cellular Signaling in the Cardiovascular System" T32 HL07692, and was conducted under the auspices of the Association for U.S.-French Biomedical Cooperation. This work was presented in part at the annual meeting of the American Society of Hematology, Miami Beach, FL, December, 1998.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 Ave. (M/C 868), Chicago, IL 60612. Tel.: 312-996-4929; Fax: 312-996-1225; E-mail: gcl{at}tigger.uic.edu.

    ABBREVIATIONS

The abbreviations used are: TXA2, thromboxane A2; PLC, phospholipase C; IP3, inositol 1,4,5-triphosphate; GTPgamma S, guanosine 5'-O-(3-thiotriphosphate); CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; TxAb, TXA2 antibody; ELISA, enzyme-linked immunosorbent assay; PI, preimmune; GEF, guanine nucleotide exchange factor.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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