Differential Signaling by the Thromboxane Receptor Isoforms via the Novel GTP-binding Protein, Gh *

Thromboxane A2 acts via G protein-coupled receptors; two splice variants of the thromboxane A2 receptor (TPα and TPβ) have been cloned. It is unknown whether they differ in their capacity to activate intracellular signaling pathways. Recently, a high molecular weight G protein, Gh, that can also function as a tissue transglutaminase, has been described. We investigated whether Gh functions as a signaling protein in association with thromboxane receptors. First, we sought Gh expression in cells known to express TPs. Reverse transcription-polymerase chain reaction and immunoblotting demonstrated Gh expression in platelets, megakaryocytic cell lines, and endothelial and vascular smooth muscle cells. Second, immunoprecipitation of both TPα and TPβ in transfected COS-7 cells resulted in the co-immunoprecipitation of Gh, indicating that TPs may associate Gh in vivo. Finally, agonist activation of TPα, but not of TPβ, resulted in stimulation of phospholipase C-mediated inositol phosphate production in cells cotransfected with Gh. By contrast, agonist activation of both TP isoforms resulted in Gq-mediated inositol phosphate signaling. Gh is expressed in platelets and vascular cells and may associate with both TP isoforms. However, stimulation of TP isoforms results in differential activation of downstream signaling pathways via this novel G protein.

Thromboxane A 2 acts via G protein-coupled receptors; two splice variants of the thromboxane A 2 receptor (TP␣ and TP␤) have been cloned. It is unknown whether they differ in their capacity to activate intracellular signaling pathways. Recently, a high molecular weight G protein, G h , that can also function as a tissue transglutaminase, has been described. We investigated whether G h functions as a signaling protein in association with thromboxane receptors. First, we sought G h expression in cells known to express TPs. Reverse transcriptionpolymerase chain reaction and immunoblotting demonstrated G h expression in platelets, megakaryocytic cell lines, and endothelial and vascular smooth muscle cells.

Second, immunoprecipitation of both TP␣ and TP␤ in transfected COS-7 cells resulted in the co-immunoprecipitation of G h , indicating that TPs may associate G h in vivo. Finally, agonist activation of TP␣, but not of TP␤, resulted in stimulation of phospholipase C-mediated inositol phosphate production in cells cotransfected with G h . By contrast, agonist activation of both TP isoforms resulted in G q -mediated inositol phosphate signaling. G h is expressed in platelets and vascular cells and may associate with both TP isoforms. However, stimulation of TP isoforms results in differential activation of downstream signaling pathways via this novel G protein.
Thromboxane A 2 (TxA 2 ) 1 is a product of arachidonic acid metabolism and is synthesized upon activation of a variety of cells, including platelets, vascular smooth muscle cells, and macrophages. TxA 2 exerts potent biological activity, causing platelet aggregation and secretion, vasoconstriction, and mitogenesis (1). These biological effects are the consequence of the interaction of TxA 2 with membrane receptors. Although pharmacological studies suggested that different TxA 2 receptors (TPs) are expressed in different cell types or even in a single cell (2), only one gene, encoding a heptahelical G protein-coupled receptor, has been cloned (3). Deletion of this gene renders mouse platelets unresponsive to thromboxane analogues and abolishes the pressor response to infusion of TP agonists (4). Two splice variants of the carboxyl-terminal tail of TP have been identified. The first isoform, TP␣, was cloned from a placental library (5) and subsequently from megakaryocytic cell lines (6,7). The second, TP␤, was cloned from an umbilical vein endothelial cell library (8). Distinct functions for the two isoforms remain to be established. Alternative splicing of the carboxyl-terminal tail may be relevant to coupling of receptors with distinct G proteins. Examples include the splice variants of the EP 3 receptor for prostaglandin E 2 (9) and the angiotensin-II receptors (10 -13). Other regions of G protein-coupled receptors, including the second and third intracellular loops, may also influence their interaction with G proteins (14 -16).
TPs have been shown to couple to members of the G q (17)(18)(19)(20) and G 12 /G 13 families (7,21), whereas conflicting data have been reported on their ability to couple with G i proteins (7,17,(21)(22)(23). TPs do not appear to signal via G s . Although the domains of this receptor that regulate interactions with G proteins remain to be defined, a naturally occurring mutation in the first intracellular loop results in a mild bleeding disorder and defective activation of phospholipase C (PLC) (24).
Partial purification of the human platelet TP by ourselves and others (18,25) suggests its association with very high molecular weight G protein(s). However, the identity of these protein(s) remains unknown. Recently, a high molecular weight G protein, G h , has been identified and cloned (26 -28). G h may be activated via the ␣1B and ␣1D adrenoreceptor isoforms; this subtype specificity involves determinants in their third intracellular loops (27,29). In turn, G h activates a 69-kDa phosphatidylinositol PLC (28) that has been identified as the PLC␦1 subtype (30) through an 8-amino acid portion near the carboxyl terminus (28). G h may also function as a tissue transglutaminase. The capacity of G h to function as a transglutaminase varies inversely with GTP binding in vitro. However, the relevance of this "switch" function to the role of G h in vivo is unknown. Indeed, the role of G h as a G protein has remained controversial. This results in part from the restriction of studies of its G protein function to a single receptor subfamily. Given our prior copurification of the platelet TP with a high molecular weight G protein, we have sought evidence for the association of this receptor with G h . We report that G h is present in cardiovascular cells that express both TP isoforms and that either may be immunoprecipitated with G h . However, whereas both isoforms signal via G q , only TP␣ signals via G h to activate PLC-dependent inositol phosphate formation. * This study was supported by a Grant HL4500 from the National Institutes of Health and by a grant from the Southeastern Pennsylvania Affiliate of the American Heart Association (to R. V.). Preliminary reports of these data have been presented to the XVIth Congress of the International Society on Thrombosis and Haemostasis (June 6 -12, 1997, Florence, Italy) and to the 70th Scientific Session of the American Heart Association (November 9 -12, 1997, Orlando, FL). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18  Oligonucleotides were synthesized by Genosys Biotechnologies Inc., The Woodlands, TX. All the cell culture media were purchased from Life Technologies Inc. Anion exchange resin AG 1-X8 and 30% acrylamide/ bisacrylamide solution were purchased from Bio-Rad. Aprotinin, leupeptin, pefabloc, dithiothreitol, and restriction enzymes were obtained from Roche Molecular Biochemicals, Mannheim, Germany. Phenylmethylsulfonyl fluoride (PMSF), CHAPS, and benzamidine were obtained from Sigma. 9,11-Dideoxy-9␣,11␣-methano-epoxy prostaglandin F 2␣ (U46619) and SQ29,548 were obtained from Cayman Chemical Co., Ann Arbor, MI. Nonidet P-40 was obtained from BDH, Poole, UK. Cell Culture and Transfection-All cDNAs used for COS-7 cell transfections, except for G q ␣, were subcloned into the mammalian expression vector pcDNA3 (Invitrogen, San Diego, CA). COS-7 cells (ATCC, Rockville, MD) were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 2 mM glutamine, 50 units /ml penicillin, and 100 g/ml streptomycin under 5% CO 2 at 37°C. The cells were seeded at different densities, for transient transfection, depending on the experiment to be performed. Cells were seeded in 24-well plates at a density of 4 ϫ 10 4 cells/well for determination of inositol phosphate formation, as described (19), grown overnight, and transfected with a total amount of 0.4 g of plasmid DNA, mixed with 2 l of Lipo-fectAMINE (Life Technologies, Inc.) in 250 l of serum-free medium (Opti-MEM). COS-7 cells were seeded in a two-well Lab-Tek slide culture chamber (Nunc, Naperville, IL) for immunofluorescence staining, at 1 ϫ 10 5 cells/chamber, and transfected with a total amount of 0. HEL (ATCC) and CHRF-288 cells (donated by Lawrence F. Brass of the University of Pennsylvania) were cultured in RPMI 1640 medium containing glutamine, penicillin/streptomycin, and 20% fetal bovine serum. Primary cultures of human aortic smooth muscle cells (HASMC) were purchased from Clonetics (San Diego, CA) and used at passages 7-9. Human umbilical vein endothelial cells (HUVEC) were grown as described (31); human umbilical vein smooth muscle cells (HUVSMC) were donated by Elliot S. Barnathan and cultured as described (32). The hepatoma cells HepG2 were donated by Rebecca A. Taub of the University of Pennsylvania and cultured in Dulbecco's modified Eagle's medium containing glutamine, penicillin/streptomycin, and 10% fetal bovine serum.
Extraction and Amplification of RNA-Total RNA was extracted from HepG2, HEL, CHRF-288, HUVEC, HUVSMC, and HASMC by the acid guanidinium/phenol/chloroform method using the Trizol reagent (Life Technologies Inc.). Fifty ml of human blood was collected from healthy volunteers in 9 ml of a sterile solution containing 1.5% citric acid and 2.5% sodium citrate, pH 6.5. Platelet-rich plasma was obtained by centrifugation and filtered through a PXL TM 8 leukocyte removal filter (Pall Biomedical Corp., Fajardo, Puerto Rico), as described (33). Total RNA was extracted as described above and resuspended in 100 l of diethylpropylcarbonate-treated water.
One g of total RNA or, in the case of platelets, 8 l of the RNA solution, was reverse-transcribed (RT) in a 20-l volume using the 1st strand cDNA synthesis kit from Roche Molecular Biochemicals. Five l of the RT mixture were amplified by polymerase chain reaction (PCR) in a volume of 50 l using the Expand TM High Fidelity PCR System (Roche Molecular Biochemicals) and 0.5 g of each primer.
The products of the RT were also amplified, using primers based on the sequence of the human TP, upstream of the splicing site and, thus, we were able to amplify both TP␣ and TP␤. The following primers were used: 5Ј-CCTTCCTGCTGAACACGGTCA-3Ј (nt 572-592) and 5Ј-GATATACACCCAGGGGTCCAG-3Ј (nt 847-867).
The absence of leukocyte contamination in the platelet preparations was confirmed by using the platelet cDNA in PCR reactions with primers based on the sequence of the leukocyte marker HLA-DQb as described previously (33). The HLA-DQb primers used were 5Ј-GTCT-CAATTATGTCTTGGAA-3Ј and 5Ј-TGCCACTCAGCATCTTGCT-3Ј corresponding to nt 37-56 and 730 -748, respectively.
Control experiments were carried out using human genomic DNA (0.5 g/reaction) or platelet RNA (not reverse-transcribed, 2 l/reaction) as templates in PCR reactions with human G h primers.
cDNA was denatured at 94°C for 3 min, and then 30 amplification cycles were performed. The denaturation step was at 94°C for 1 min and the elongation step at 72°C for 1 min. The annealing conditions were 60°C for 1 min when using human G h or TP primers and 55°C for 1 min when using HLA-DQb or rat G h primers. PCR products were electrophoresed, and the gel was blotted on nylon transfer membranes (Hybond TM -Nϩ, Amersham Pharmacia Biotech).
Immunoblotting-Immunoblotting was performed under reducing conditions as described previously (35) using an anti-G h Ab (NeoMarkers, Fremont, CA) (1 g/ml) followed by a peroxidase-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch) diluted 1:5000. Anti-G q ␣, anti-G q/11 ␣, and anti-G s ␣ Abs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and were used at 0.5 g/ml; a peroxidase-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch) was used as secondary Ab diluted 1:5000.
Partial Protein Purification-We carried out a partial protein purification of the plasma membrane proteins to detect G h in platelets by immunoblotting. Outdated human platelets obtained from the Philadelphia American Red Cross were filtered through a PXL™8 filter to remove contaminating leukocytes. Low speed centrifugation was carried out to remove red blood cells. Platelets were resuspended in 5 mM Tris-HCl, pH 7.4, containing 0.1 g/ml prostaglandin E 1 and sonicated 3 times for 30 s on ice. After a centrifugation at 1,000 ϫ g to remove unbroken cells, the platelet membranes were centrifuged at 100,000 ϫ g for 40 min. The pellet was resuspended by sonication in a buffer containing 20% glycerol, 5 mM EDTA, pH 7.4, 0.5 mM dithiothreitol, 0.5 mM 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (pefabloc), 1 g/ml leupeptin, and 10 g/ml aprotinin. CHAPS was then added at a final concentration of 10 mM.
Membranes were prepared for partial protein purification from COS-7 cells transfected with rat G h . Cells were washed twice with phosphate-buffered saline and scraped in 25 mM HEPES, pH 6.5, 150 mM NaCl, 10 mM MgCl 2 , 5 mM KCl containing 1 mM benzamidine and 128 g/ml pefabloc. Cells were sonicated, and membranes were prepared as described previously for platelets.
Partial protein purification was performed using pre-swollen DE52 (diethylaminoethyl cellulose) ion exchanger (Whatman) washed with 200 mM Tris-HCl, pH 8, and 1 mM EDTA. The columns were equilibrated with 2 volumes (ϳ3 ml) of column buffer (20 mM Tris-HCl, pH 8, 0.1 mM EDTA, 5 mM CHAPS) and finally washed with the same buffer that contained 0.5 mM dithiothreitol, 1 g/ml leupeptin, 10 g/ml aprotinin, and 0.5 mM pefabloc. Two mg of platelet membrane or 0.5 mg of membrane from COS-7 cells were loaded on the columns. The columns were washed 3 times with 0.5 ml of column buffer, and the proteins were eluted three times with 0.5 ml of the same buffer containing increasing concentrations of NaCl (0.1-0.5 M). The proteins eluted at each step were trichloroacetic acid-precipitated, washed with ice-cold acetone, and dissolved in Laemmli sample buffer. Immunoblotting for G h was performed as described previously.
Binding Assays-Binding assays were performed in COS-7 cells transfected with either TP␣ or TP␤, in the presence or in the absence of G h . Membranes were prepared as described previously (7). Briefly, the cells were washed twice in buffer A (25 mM HEPES, pH 6.5, 125 mM NaCl, 10 mM MgCl 2 , 5 mM KCl) and scraped in buffer B (as buffer A, plus 1 mM benzamidine, 128 g/ml pefabloc, and 10 M indomethacin). Cells were homogenized with a glass-glass homogenizer and unbroken cells removed by centrifugation. Membranes were centrifuged at 100,000 ϫ g for 45 min, resuspended in buffer B at a concentration of 300 g/ml, and stored at Ϫ80°C until use.
We tested for the significance of differences using Student's twotailed t test.
The DNA was shorn by passing the cell lysate through a 21-gauge needle. The lysate was then centrifuged at 4°C at 3,000 rpm for 10 min and then at 13,000 rpm for 30 min at 4°C. Sodium deoxycholate and sodium dodecyl sulfate (SDS) were added to the supernatant at a final concentration of 1% and 0.1%, respectively. After a further centrifugation at 13,000 rpm at 4°C for 10 min, the clear supernatant was used for immunoprecipitation experiments which were performed in Eppendorf tubes precoated for 10 min with lysis buffer (10 mM Tris-HCl, pH 8, 140 mM NaCl, 0.5% Triton X-100).
Control co-immunoprecipitation experiments were carried out using an anti-rhodopsin antibody obtained from Biodesign International, Kennebunkport, ME. Coupling of the antibodies to protein G-Sepharose beads was then performed. Briefly, 500 l of a 20% slurry of protein G-Sepharose 4B (Amersham Pharmacia Biotech, Uppsala, Sweden) in 50 mM Tris-HCl, pH 7.5, 20 mM MgCl 2 , 150 mM NaCl, 0.5% Nonidet P-40, 0.01 units/ml aprotinin were incubated with 2.5 g of the antirhodopsin Ab or 10 l of normal rabbit serum for 5 h at 4°C under constant agitation. The protein G-Sepharose was washed three times with radioimmune precipitation buffer and used for the immunoprecipitation. Three to five hundred micrograms of HASMC lysate, 500 -700 g of HEL cell lysate, or 300 g of the lysate of COS-7 cells transfected with G h were precleared for 30 min and immunoprecipitated overnight. After washing as described above, samples were analyzed by electrophoresis and immunoblotted. The same immunoprecipitation procedure was performed using a bovine retina preparation, which was kindly provided by Dr. John H. Parks, University of Pennsylvania. Proteins (ϳ500 g at a concentration of 3 mg/ml) were solubilized by adding 1/10 volume of 10% sodium deoxycholate and radioimmune precipitation buffer (containing 1 mM PMSF, 10 g/ml aprotinin, and 1 g/ml leupeptin) up to 1 ml and by shaking at 4°C for 5 h. Proteins were precleared for 30 min and immunoprecipitated overnight with an anti-rhodopsin antibody coupled to protein G-Sepharose, as described above. Immunoblotting was performed using the anti-rhodopsin Ab diluted at 2.5 g/ml, followed by a peroxidase-conjugated donkey anti-mouse IgG diluted 1:5000.
Inositol Phosphate Formation-Inositol phosphate formation was measured in COS-7 cells stimulated with U46619 for 30 min at 37°C.
Reactions were terminated by aspiration of the medium and by the addition of 750 l of ice-cold 10 mM formic acid (36). Agonist-stimulated inositol phosphate formation is expressed as a percentage of the nonstimulated sample. We tested for significant differences using analysis of variance followed by Bonferroni's multiple comparison test between all pairs.

Identification of G h Message-Total
RNA was extracted from human platelets, HUVEC, HUVSMC, and HASMC, that are known to express TPs. We also sought G h expression in megakaryocytic cell lines (HEL and CHRF-288) that have several platelet characteristics and have been used as models to study platelet structure and function (37,38). We used the HepG2 hepatoma cell line as a positive control for G h expression, since it is known that G h is highly expressed in liver (26). Using primers specific for human G h , a fragment of 520 bp was amplified as demonstrated in Fig. 1A. A fragment of 520 bp was also amplified from the cDNA of rat G h , cloned into pcDNA3. The specificity of the PCR products was confirmed by hybridization with a 32 P-labeled internal oligonucleotide (Fig. 1B). Sequence analysis of the PCR product obtained with HepG2 cDNA confirmed that the 520-bp fragment corresponds to human G h (data not shown). When platelet cDNA was used, no bands were evident by staining of the gel, although a positive band was detected after hybridization, demonstrating that platelets also express G h mRNA (Fig. 1B).
No fragments were obtained when using human genomic DNA as template, indicating that the PCR products were derived from the respective cDNAs. Amplification of the G h fragment from platelets depended strictly on cDNA synthesis, since no products were formed when using platelet RNA in the PCR reaction.
The purity of our platelet preparation was tested by performing RT-PCR, followed by Southern blotting and hybridization, with primers specific for the leukocyte marker HLA-DQb. However, a product was not formed, indicating that contaminating leukocytes were absent from the platelet preparation.
The expression of TPs in the cell types used in these experiments was confirmed by RT-PCR using oligonucleotides common to TP␣ and TP␤: a positive band of 296 bp was obtained with all the cDNA preparations tested (data not shown).
Identification of Platelet G h by Immunoblotting-The results obtained by RT-PCR demonstrated the presence of G h message in platelets, but the level of expression appears to be low, since a positive band was observed only after Southern blotting and hybridization. This is consistent with our failure to detect clear bands corresponding to G h by Western blotting, using a whole cell lysate or a platelet membrane preparation. To increase the concentration of G h to a level detectable by immunoblotting, we carried out a partial purification of the membrane proteins by ion-exchange chromatography. As a control, the same purifica- tion steps were performed using membranes from COS-7 cells transfected with rat G h . Immunoblotting revealed a band corresponding to G h in the 0.1 M NaCl fraction from human platelets and in the 0.3 M NaCl fraction from COS-7 cells transfected with rat G h (Fig. 2). The differential retention of rat versus human G h is attributable to species divergence (39).
Overexpression of TPs and G Proteins in COS-7 Cells-To address the interaction of TPs with G h , we used a cotransfection approach in COS cells that has been described previously (14, 40 -42). Binding of [ 3 H]SQ29,548 to both TP␣ and TP␤ was saturable, and receptor density in both TP␣-and TP␤-transfected cells was similar (Fig. 3). In addition, neither density nor affinity of either TP isoform, as detected by binding of the antagonist [ 3 H]SQ29,548, was altered by cotransfection with G h (Fig. 3). COS-7 cells that were not transfected failed to bind [ 3 H]SQ29,548 detectably, although TP message could be amplified from these cells (data not shown).
The expression of G proteins in non-transfected COS-7 cells and in cells transfected with TP␣ or TP␤ plus G h , G q ␣, or G s ␣ was confirmed by immunoblotting. A strong signal corresponding to G h was observed when G h was overexpressed together with either TP␣ or TP␤. A band of slightly higher molecular weight, usually also observed in non-transfected cells, may represent monkey G h , probably expressed constitutively in COS-7 cells. Both G q ␣ and G s ␣ were detected when they were overexpressed with either TP␣ or TP␤, whereas no signal was detected in non-transfected cells. When non-transfected cells were probed with an anti-G q/11 ␣ Ab, a band of the expected molecular weight was observed. Detection of a positive band with the anti-G q/11 ␣ Ab, but not with the anti G q ␣ Ab, may reflect higher expression of G 11 ␣ than G q ␣ in COS-7 cells (Fig.  4). Immunofluorescence staining was performed to assess the coexpression of the TP isoforms with G h in these transiently transfected cells. The majority of the transfected cells stained positive both for the respective TPs and G h (data not shown).
Physical Coupling of TPs with G h -TPs were immunoprecipitated using specific Abs which we previously raised against TP␣ and TP␤ (36). These experiments were carried out in COS-7 cells transfected with TP␣ ϩ G h , TP␤ ϩ G h , or only with G h , as well as in control cells, such as HEL and HASMC.
TP␣ ϩ G h and TP␤ ϩ G h -transfected COS-7 cells were stimulated or not with the TP agonist, U46619. G h was detected in TP␣ and TP␤ immunoprecipitates, irrespective of whether the cells had been previously stimulated with the agonist (Fig. 5A). COS-7 cells transfected with G h only express very low levels of their endogenous TPs, detectable by RT-PCR but not by [ 3 H]SQ29,548 binding.
Even under these circumstances, G h was readily detectable in immunoprecipitates of either isoform (Fig. 5B). Thus, G h appears to possess high affinity for TPs, as it can be immunoprecipitated even when TP expression is low. It is unlikely that this reflects nonspecific immunoprecipitation of G h , as G h was not detected in the normal rabbit serum sample that we used for the preclearing step (Fig. 5B) and was not immunoprecipitated by a control anti-rhodopsin Ab (Fig. 5A).
Rhodopsin is specifically expressed in retina and is absent in COS-7 cells. In contrast to our findings in COS-7 cells, we could immunoprecipitate rhodopsin from a retinal preparation with the anti-rhodopsin Ab (data not shown). Consistent with our observations in COS-7 cells, G h was again detected in samples immunoprecipitated with either TP␣ or TP␤ Abs from both HEL (Fig. 6A) and HASM cells (Fig. 6B). Again, G h was not detected either in the normal rabbit serum samples used for  (lanes 2 and 3), G q (lanes 5 and 6), or G s (lanes 8 and 9) were analyzed by SDS-PAGE and immunoblotting. Anti-G h (lanes 1-3), anti-G q ␣ (lanes 4 -6), anti-G s (lanes 7-9), or anti-G q/11 (lane 10) antibodies were used for immunoblotting. Molecular mass markers (kDa) are indicated in the left margin of the figure. ؉ G h (A, left panel), TP␤ ؉ G h (A, right panel), or G h alone (B). Cells transfected with TP␣ ϩ G h or TP␤ ϩ G h were either stimulated with U46619 before lysis (stim.) or not stimulated (unstim.). Immunoprecipitations were carried out using anti-TP␣ (TP␣ Ab), anti-TP␤ (TP␤ Ab), or anti-rhodopsin (rhod Ab) antibodies, and the immunoprecipitated samples (ip) were analyzed by SDS-polyacrylamide gel electrophoresis. Normal rabbit serum (nrs) samples used for the preclearing, an aliquot of the supernatant (s) of the immunoprecipitations, and a crude cell lysate sample (C) were also run in the gel. Molecular mass markers (kDa) are indicated in the left margin of the figure. preclearing or when the anti-rhodopsin Ab was used for immunoprecipitation (Fig. 6, A and B). Thus, G h appears to favor immunoprecipitation with either TP␣ or TP␤.

FIG. 5. Immunoprecipitation of G h with TP␣ and TP␤ in the lysate of COS-7 cells transfected with TP␣
Functional Coupling of TPs with G h -COS-7 cells were transfected with either TP␣ or TP␤ with or without G h , G q ␣, or G s ␣. G q ␣ and G s ␣ were used as positive and negative controls for TP-mediated signaling events, respectively (2). U46619 dosedependently increased inositol phosphate production in cells transfected with TP␣ alone, with an EC 50 of 3.48 Ϯ 0.34 nM (n ϭ 6). When cells had been transfected with both TP␣ and G h , a further increase of inositol phosphate was observed (Fig. 7A); however, the EC 50 (4.27 Ϯ 0.81 nM) was not significantly altered. The agonist-stimulated increase of inositol phosphate formation was higher in TP␣ ϩ G q ␣-transfected cells than in TP␣ ϩ vector-transfected cells. Consequently, the EC 50 (0.83 Ϯ 0.08 nM) was significantly (p Ͻ 0.05) lower than in cells transfected with TP␣ ϩ vector. As expected, TP␣ did not activate PLC via G s . Agonist-stimulated TP␣ ϩ G s ␣-transfected cells did not produce more inositol phosphate than cells transfected with TP␣ ϩ vector. The EC 50 (6.87 Ϯ 2.48 nM) for agonist was also similar (p ϭ not significant) to that observed in TP␣ ϩ vector-transfected cells.
Similar experiments were carried out in cells transfected with TP␤. Agonist-stimulated inositol phosphate production only exceeded that in cells transfected TP␤ ϩ vector in those cotransfected with TP␤ ϩ G q ␣ (Fig. 7B). Although G q ␣ caused a shift of the U46619 dose-response curve to the left, the EC 50 calculated in TP␤ ϩ G q ␣-transfected cells was not significantly different from that found in TP␤ ϩ vector-transfected cells (TP␤ ϩ vector: EC 50 ϭ 5.36 Ϯ 1.89 nM; TP␤ ϩ G q ␣: EC 50 ϭ 1.97 Ϯ 0.29 nM, p ϭ not significant). No significant increase in agonist-induced inositol phosphate production was observed in either TP␤ ϩ G h or TP␤ ϩ G s ␣-transfected cells versus TP␤ ϩ vector-transfected cells. Similarly, no differences were found in the EC 50 (TP␤ ϩ G h : EC 50 ϭ 5.75 Ϯ 0.75 nM; TP␤ ϩ G s ␣ EC 50 ϭ 5.10 Ϯ 2.08 nM, p ϭ not significant). DISCUSSION G h is a newly characterized high molecular weight G protein that can be activated via the ␣1 adrenoreceptor (27,29). We have previously found that human platelet TPs, purified roughly 2,000-fold, were associated with an uncharacterized G protein of a similar, high (ϳ70 kDa) molecular mass (25). Given these observations, we decided to address the possibility that TPs might actually transmit intracellular signals via G h . Furthermore, given the uncertain role of the TP isoforms, we sought to investigate whether they might couple with differen-tial affinity to either G h or G q ␣. G h is expressed in cells of the cardiovascular system which express TPs. Thus, platelets express message for both isoforms, although they appear to translate only TP␣ (43). Vascular smooth muscle cells express both isoforms, whereas endothelial cells appear to express only TP␤ (8). All of these cell types, as well as megakaryocytic cell lines, express G h .
Both TP isoforms physically associate with Gh, as reflected by studies involving co-immunoprecipitation. Thus, immunoblotting of samples from lysates of COS-7 cells transfected with rat G h , together with TP␣ or TP␤, demonstrated that G h immunoprecipitates with either isoform. Similar results were obtained in HEL and HASMC, cells in which the proteins were not overexpressed. These results are consistent with our earlier finding that a high molecular weight G protein copurifies with platelet TPs (presumably TP␣). Stimulation of the cells with TP agonists, which promote receptor-G protein coupling, was not required for this association. However, this is unsurprising. Precedent for co-immunoprecipitation of receptors and G proteins in non-stimulated cells has already been established (44,45). Indeed, the affinity of TPs for G h seems to be high. For example, immunoprecipitation of G h with TPs occurs even when using a lysate of COS-7 cells overexpressing G h but not the receptors. COS-7 cells express low levels of endogenous TPs, which can be detected by RT-PCR, but not by binding of the TP antagonist [ 3 H]SQ29,548. To address the specificity of the co-immunoprecipitation of G h with TPs, we sought to replicate these findings using an antibody directed against rhodopsin. Rhodopsin is expressed specifically in retina and is absent from COS-7, HEL, or HASM cells. G h did not immunoprecipitate with either the anti-rhodopsin Ab or with the IgG fraction of the normal rabbit serum used for the preclearing step. Thus, immunoprecipitation of G h with TP␣ and TP␤ appears to reflect a specific interaction.
To address the possibility that the TP-G h association is of functional relevance, we utilized transfected COS-7 cells (14, 40 -42) measuring inositol phosphate production as an index of G protein-dependent PLC activation. Dose-dependent stimulation of inositol phosphate production was observed in response to agonist in cells transfected with either isoform alone, whereas cotransfection of the cells with G q ␣ amplified the response to agonist. By contrast, cotransfection with G s ␣ had no effect, as expected. Similar to our observation with G q ␣, cotransfection of G h with TP␣ enhanced the agonist-dependent increase in inositol phosphate formation over that found in cells transfected with TP␣ alone. By contrast, a similar effect was not observed in cells transfected with TP␤.
The role of G h as a conventional G protein has remained controversial, in part reflecting the restriction of evidence to a single subfamily of G protein-coupled receptors, the ␣ adrenoreceptors. We now provide evidence for association of this protein with a distinct receptor, the TP, a member of the eicosanoid receptor subfamily. G h also functions as a tissue transglutaminase, and it is now recognized that it binds and cross-links only specific substrates that may be relevant to cell death and survival (46). Interestingly, its transglutaminase function varies inversely with its binding of GTP (26), raising the possibility that the switch between these functions might be relevant to cellular survival. We have shown that TP isoforms are expressed with G h in cardiovascular cells and associate with this protein. However, their differential ability to signal via G h provides the first suggestion of distinct roles for these receptor isoforms in vivo.