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

Roberta Vezza, Aida HabibDagger , and Garret A. FitzGerald§

From the Center for Experimental Therapeutics, University of Pennsylvania, Philadelphia, Pennsylvania 19104

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES

Thromboxane A2 acts via G protein-coupled receptors; two splice variants of the thromboxane A2 receptor (TPalpha and TPbeta ) 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 TPalpha and TPbeta in transfected COS-7 cells resulted in the co-immunoprecipitation of Gh, indicating that TPs may associate Gh in vivo. Finally, agonist activation of TPalpha , but not of TPbeta , 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.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Thromboxane A2 (TxA2)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. TxA2 exerts potent biological activity, causing platelet aggregation and secretion, vasoconstriction, and mitogenesis (1). These biological effects are the consequence of the interaction of TxA2 with membrane receptors. Although pharmacological studies suggested that different TxA2 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, TPalpha , was cloned from a placental library (5) and subsequently from megakaryocytic cell lines (6, 7). The second, TPbeta , 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 EP3 receptor for prostaglandin E2 (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 Gq (17-20) and G12/G13 families (7, 21), whereas conflicting data have been reported on their ability to couple with Gi proteins (7, 17, 21-23). TPs do not appear to signal via Gs. 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, Gh, has been identified and cloned (26-28). Gh may be activated via the alpha 1B and alpha 1D adrenoreceptor isoforms; this subtype specificity involves determinants in their third intracellular loops (27, 29). In turn, Gh activates a 69-kDa phosphatidylinositol PLC (28) that has been identified as the PLCdelta 1 subtype (30) through an 8-amino acid portion near the carboxyl terminus (28). Gh may also function as a tissue transglutaminase. The capacity of Gh to function as a transglutaminase varies inversely with GTP binding in vitro. However, the relevance of this "switch" function to the role of Gh in vivo is unknown. Indeed, the role of Gh 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 Gh. We report that Gh is present in cardiovascular cells that express both TP isoforms and that either may be immunoprecipitated with Gh. However, whereas both isoforms signal via Gq, only TPalpha signals via Gh to activate PLC-dependent inositol phosphate formation.

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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Materials-- Rat Gh cDNA was donated by Robert M. Graham of The Victor Chang Cardiac Research Institute, New South Wales, Australia. cDNAs for the human TPalpha and TPbeta isoforms were contributed by Colin D. Funk of the University of Pennsylvania, Philadelphia, and by J. Anthony Ware of the Beth Israel Hospital, Boston, respectively. Gqalpha cDNA, originally provided in pCMV5 vector, was donated by Gary L. Johnson, National Jewish Center, Denver, CO. The cDNA for rat Gsalpha was provided by David R. Manning of the University of Pennsylvania.

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-9alpha ,11alpha -methano-epoxy prostaglandin F2alpha (U46619) and SQ29,548 were obtained from Cayman Chemical Co., Ann Arbor, MI. Nonidet P-40 was obtained from BDH, Poole, UK. [3H]SQ29,548 was obtained from NEN Life Science Products.

Cell Culture and Transfection-- All cDNAs used for COS-7 cell transfections, except for Gqalpha , 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 µg/ml streptomycin under 5% CO2 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 × 104 cells/well for determination of inositol phosphate formation, as described (19), grown overnight, and transfected with a total amount of 0.4 µg of plasmid DNA, mixed with 2 µl of LipofectAMINE (Life Technologies, Inc.) in 250 µl 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 × 105 cells/chamber, and transfected with a total amount of 0.8 µg of plasmid DNA mixed with 5 µl of LipofectAMINE in 500 µl of Opti-MEM. The cells were plated in 60-mm dishes for immunoblotting, at 4 × 105 cells/dish, and transfected with a total of 4 µg of plasmid DNA mixed with 20 µl of LipofectAMINE in 2 ml of Opti-MEM. COS-7 cells were seeded in 100-mm culture dishes for co-immunoprecipitation experiments, at 1.2 × 106 cells/dish and transfected with a total amount of 12 µg of plasmid DNA mixed with 50 µl of LipofectAMINE in 8 ml of OptiMEM. The cells were seeded in 150-mm dishes at 3 × 106 cells/dish and transfected with 20 µg of total plasmid DNA mixed with 140 µl of LipofectAMINE in 14 ml of Opti-MEM for protein purification and binding experiments. The total amount of DNA was kept constant in control experiments by adding DNA from the vector pcDNA3.

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 PXLTM8 leukocyte removal filter (Pall Biomedical Corp., Fajardo, Puerto Rico), as described (33). Total RNA was extracted as described above and resuspended in 100 µl of diethylpropylcarbonate-treated water.

One µg of total RNA or, in the case of platelets, 8 µl of the RNA solution, was reverse-transcribed (RT) in a 20-µl volume using the 1st strand cDNA synthesis kit from Roche Molecular Biochemicals. Five µl of the RT mixture were amplified by polymerase chain reaction (PCR) in a volume of 50 µl using the ExpandTM High Fidelity PCR System (Roche Molecular Biochemicals) and 0.5 µg of each primer.

Primers used for Gh amplification were designed on the basis of human tissue transglutaminase (5'-ATCTACCAGGGCTCGGCCAA-3' and 5-ACTCCACCCAGCAGTGGAAG-3') (34) corresponding to nucleotides (nt) 634-653 and nt 1134-1153 (28, 34); when using the cDNA of rat Gh, primers were designed on the basis of the corresponding sequences of rat Gh corresponding to nt 504-523 and 1004-1023, respectively (5'-ATCTACCAGGGCTCTGTCAA-3' and 5'-ACTCCACCCAGCAGTGGAAA-3') (26).

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 TPalpha and TPbeta . The following primers were used: 5'-CCTTCCTGCTGAACACGGTCA-3' (nt 572-592) and 5'-GATATACACCCAGGGGTCCAG-3' (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 5'-GTCTCAATTATGTCTTGGAA-3' and 5'-TGCCACTCAGCATCTTGCT-3' corresponding to nt 37-56 and 730-748, respectively.

Control experiments were carried out using human genomic DNA (0.5 µg/reaction) or platelet RNA (not reverse-transcribed, 2 µl/reaction) as templates in PCR reactions with human Gh 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 Gh or TP primers and 55 °C for 1 min when using HLA-DQb or rat Gh primers. PCR products were electrophoresed, and the gel was blotted on nylon transfer membranes (HybondTM-N+, Amersham Pharmacia Biotech).

Hybridization was carried out at 45 °C from 3 h to overnight using 32P-labeled oligonucleotide probes. The Gh oligonucleotide probe was 5'-GTGGCATGGTCAACTGCAACGAT-3' corresponding to nt 809-831. The HLA-DQb oligonucleotide probe was 5'-TCGGTGGACACCGTATGCAGACAC-3' corresponding to nt 361-384. After hybridization, the membranes were washed and autoradiographed.

Immunoblotting-- Immunoblotting was performed under reducing conditions as described previously (35) using an anti-Gh Ab (NeoMarkers, Fremont, CA) (1 µg/ml) followed by a peroxidase-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch) diluted 1:5000. Anti-Gqalpha , anti-Gq/11alpha , and anti-Gsalpha Abs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and were used at 0.5 µg/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 Gh in platelets by immunoblotting. Outdated human platelets obtained from the Philadelphia American Red Cross were filtered through a PXLTM8 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 µg/ml prostaglandin E1 and sonicated 3 times for 30 s on ice. After a centrifugation at 1,000 × g to remove unbroken cells, the platelet membranes were centrifuged at 100,000 × 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 µg/ml leupeptin, and 10 µg/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 Gh. Cells were washed twice with phosphate-buffered saline and scraped in 25 mM HEPES, pH 6.5, 150 mM NaCl, 10 mM MgCl2, 5 mM KCl containing 1 mM benzamidine and 128 µg/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 (~3 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 µg/ml leupeptin, 10 µg/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 Gh was performed as described previously.

Binding Assays-- Binding assays were performed in COS-7 cells transfected with either TPalpha or TPbeta , in the presence or in the absence of Gh. 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 MgCl2, 5 mM KCl) and scraped in buffer B (as buffer A, plus 1 mM benzamidine, 128 µg/ml pefabloc, and 10 µM indomethacin). Cells were homogenized with a glass-glass homogenizer and unbroken cells removed by centrifugation. Membranes were centrifuged at 100,000 × g for 45 min, resuspended in buffer B at a concentration of 300 µg/ml, and stored at -80 °C until use.

Radioligand binding was performed, incubating the thromboxane receptor antagonist [3H]SQ29,548 (1-40 nM) with 50 µg of membrane protein in a total volume of 200 µl for 2 h at 4 °C (36).

We tested for the significance of differences using Student's two-tailed t test.

Immunofluorescence Staining-- Immunofluorescence staining was performed as we have described previously (36), using anti-TPalpha Ab diluted 1:1000 or anti-TPbeta Ab diluted 1:500 plus or minus anti-Gh Ab diluted at 2 µg/ml. Fluorescein isothiocyanate-conjugated donkey anti-rabbit IgG and tetramethylrhodamine-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch) diluted 1:1000 were used as secondary Abs.

Co-immunoprecipitation-- Transfected COS-7 cells, HEL cells, or HASMC were washed twice in PBS and lysed with 10 mM Tris-HCl, pH 8, 140 mM NaCl, 0.5% Triton X-100, 1 mM PMSF, 10 µg/ml aprotinin, and 1 µg/ml leupeptin. When indicated, cells were stimulated with the TP agonist, 300 nM U46619, for 5 min at 37 °C before lysis.

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).

Five hundred µg of HASMC lysate, 400-600 µg of HEL cell lysate, or 300-400 µg of transfected COS-7 cell lysate were used for immunoprecipitation experiments, and the volume was adjusted to 1 ml with lysis buffer. Samples were precleared for 30 min at 4 °C with normal rabbit serum covalently coupled to Sepharose CL-4B and immunoprecipitated with immunoaffinity Sepharose for either anti-TPalpha or anti-TPbeta Abs, as described (36). The beads were washed twice with 1 ml of ice-cold radioimmune precipitation buffer (50 mM Tris-HCl, pH 8, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS), once with 1 ml of ice-cold Tris/saline/azide (10 mM Tris-HCl, pH 7.5, 140 mM NaCl), and once with 1 ml of ice-cold 0.05 M Tris-HCl, pH 6.8. The beads were resuspended in 80 µl of Laemmli sample buffer, boiled, and loaded onto 10% SDS-polyacrylamide gels. The proteins were transferred onto nitrocellulose membranes (Schleicher & Schuell, Göttingen, Germany) in 25 mM Tris-HCl, 192 mM glycine buffer, pH 8.3, containing 0.01% SDS and 20% methanol. Immunoblotting with anti-Gh antibody was performed as described above.

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 µl of a 20% slurry of protein G-Sepharose 4B (Amersham Pharmacia Biotech, Uppsala, Sweden) in 50 mM Tris-HCl, pH 7.5, 20 mM MgCl2, 150 mM NaCl, 0.5% Nonidet P-40, 0.01 units/ml aprotinin were incubated with 2.5 µg of the anti-rhodopsin Ab or 10 µl 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 µg of HEL cell lysate, or 300 µg of the lysate of COS-7 cells transfected with Gh 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 (~500 µg 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 µg/ml aprotinin, and 1 µg/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 µg/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 µl of ice-cold 10 mM formic acid (36). Agonist-stimulated inositol phosphate formation is expressed as a percentage of the non-stimulated sample. We tested for significant differences using analysis of variance followed by Bonferroni's multiple comparison test between all pairs.

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Identification of Gh Message-- Total RNA was extracted from human platelets, HUVEC, HUVSMC, and HASMC, that are known to express TPs. We also sought Gh 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 Gh expression, since it is known that Gh is highly expressed in liver (26). Using primers specific for human Gh, 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 Gh, cloned into pcDNA3. The specificity of the PCR products was confirmed by hybridization with a 32P-labeled internal oligonucleotide (Fig. 1B). Sequence analysis of the PCR product obtained with HepG2 cDNA confirmed that the 520-bp fragment corresponds to human Gh (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 Gh mRNA (Fig. 1B).


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Fig. 1.   Identification of Gh message by RT-PCR. RNA from HepG2, HEL, CHRF-288, HUVEC, HUVSMC, HASMC, and platelets was reverse-transcribed and amplified by PCR. Rat Gh cDNA was used as positive control. A, agarose gel stained with ethidium bromide; B, Southern blot of the gel followed by hybridization with a specific internal oligonucleotide.

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 Gh 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 TPalpha and TPbeta : a positive band of 296 bp was obtained with all the cDNA preparations tested (data not shown).

Identification of Platelet Gh by Immunoblotting-- The results obtained by RT-PCR demonstrated the presence of Gh 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 Gh by Western blotting, using a whole cell lysate or a platelet membrane preparation. To increase the concentration of Gh 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 purification steps were performed using membranes from COS-7 cells transfected with rat Gh. Immunoblotting revealed a band corresponding to Gh 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 Gh (Fig. 2). The differential retention of rat versus human Gh is attributable to species divergence (39).


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Fig. 2.   Identification of Gh protein by immunoblotting. Membrane proteins from human platelets and from COS-7 cells transfected with rat Gh cDNA were partially purified by chromatography through a DE52 anion exchanger and eluted with different concentrations of NaCl (0-0.5 M). Crude cell lysate of Gh-transfected COS-7 cells (C) was also loaded on the gel, as a control. Molecular mass markers (kDa) are indicated in the left margin of the figure.

Overexpression of TPs and G Proteins in COS-7 Cells-- To address the interaction of TPs with Gh, we used a cotransfection approach in COS cells that has been described previously (14, 40-42). Binding of [3H]SQ29,548 to both TPalpha and TPbeta was saturable, and receptor density in both TPalpha - and TPbeta -transfected cells was similar (Fig. 3). In addition, neither density nor affinity of either TP isoform, as detected by binding of the antagonist [3H]SQ29,548, was altered by cotransfection with Gh (Fig. 3). COS-7 cells that were not transfected failed to bind [3H]SQ29,548 detectably, although TP message could be amplified from these cells (data not shown).


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Fig. 3.   Binding of [3H]SQ29,548 to membranes of transfected COS-7 cells. Membranes from COS-7 cells transfected with TPalpha  + vector (+vec), TPalpha  + Gh, TPbeta  + vector, or TPbeta  + Gh (congruent 50 µg) were incubated with increasing concentrations of [3H]SQ29,548 for 2 h at 4 °C. Nonspecific binding was measured in the presence of 25 µM unlabeled ligand. Results represent the mean ± S.E. of 6-7 experiments.

The expression of G proteins in non-transfected COS-7 cells and in cells transfected with TPalpha or TPbeta plus Gh, Gqalpha , or Gsalpha was confirmed by immunoblotting. A strong signal corresponding to Gh was observed when Gh was overexpressed together with either TPalpha or TPbeta . A band of slightly higher molecular weight, usually also observed in non-transfected cells, may represent monkey Gh, probably expressed constitutively in COS-7 cells. Both Gqalpha and Gsalpha were detected when they were overexpressed with either TPalpha or TPbeta , whereas no signal was detected in non-transfected cells. When non-transfected cells were probed with an anti-Gq/11alpha Ab, a band of the expected molecular weight was observed. Detection of a positive band with the anti-Gq/11alpha Ab, but not with the anti Gqalpha Ab, may reflect higher expression of G11alpha than Gqalpha in COS-7 cells (Fig. 4). Immunofluorescence staining was performed to assess the coexpression of the TP isoforms with Gh in these transiently transfected cells. The majority of the transfected cells stained positive both for the respective TPs and Gh (data not shown).


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Fig. 4.   Identification of G proteins in transfected COS-7 cells. Lysates from mock-transfected (M) COS-7 cells or from cells transfected with TPalpha (alpha ) or TPbeta (beta ) together with Gh (lanes 2 and 3), Gq (lanes 5 and 6), or Gs (lanes 8 and 9) were analyzed by SDS-PAGE and immunoblotting. Anti-Gh (lanes 1-3), anti-Gqalpha (lanes 4-6), anti-Gs (lanes 7-9), or anti-Gq/11 (lane 10) antibodies were used for immunoblotting. Molecular mass markers (kDa) are indicated in the left margin of the figure.

Physical Coupling of TPs with Gh-- TPs were immunoprecipitated using specific Abs which we previously raised against TPalpha and TPbeta (36). These experiments were carried out in COS-7 cells transfected with TPalpha  + Gh, TPbeta  + Gh, or only with Gh, as well as in control cells, such as HEL and HASMC.

TPalpha  + Gh and TPbeta  + Gh-transfected COS-7 cells were stimulated or not with the TP agonist, U46619. Gh was detected in TPalpha and TPbeta immunoprecipitates, irrespective of whether the cells had been previously stimulated with the agonist (Fig. 5A). COS-7 cells transfected with Gh only express very low levels of their endogenous TPs, detectable by RT-PCR but not by [3H]SQ29,548 binding.


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Fig. 5.   Immunoprecipitation of Gh with TPalpha and TPbeta in the lysate of COS-7 cells transfected with TPalpha  + Gh (A, left panel), TPbeta  + Gh (A, right panel), or Gh alone (B). Cells transfected with TPalpha  + Gh or TPbeta  + Gh were either stimulated with U46619 before lysis (stim.) or not stimulated (unstim.). Immunoprecipitations were carried out using anti-TPalpha (TPalpha Ab), anti-TPbeta (TPbeta 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.

Even under these circumstances, Gh was readily detectable in immunoprecipitates of either isoform (Fig. 5B). Thus, Gh 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 Gh, as Gh 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, Gh was again detected in samples immunoprecipitated with either TPalpha or TPbeta Abs from both HEL (Fig. 6A) and HASM cells (Fig. 6B). Again, Gh was not detected either in the normal rabbit serum samples used for preclearing or when the anti-rhodopsin Ab was used for immunoprecipitation (Fig. 6, A and B). Thus, Gh appears to favor immunoprecipitation with either TPalpha or TPbeta .


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Fig. 6.   Immunoprecipitation of Gh with TPalpha and TPbeta in the lysate of HEL (A) and of HASMC (B). Cell lysates were immunoprecipitated with anti-TPalpha (TPalpha Ab), anti-TPbeta (TPbeta Ab), or anti-rhodopsin (rhod Ab) antibodies. Normal rabbit serum (nrs) samples used for 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.

Functional Coupling of TPs with Gh-- COS-7 cells were transfected with either TPalpha or TPbeta with or without Gh, Gqalpha , or Gsalpha . Gqalpha and Gsalpha were used as positive and negative controls for TP-mediated signaling events, respectively (2). U46619 dose-dependently increased inositol phosphate production in cells transfected with TPalpha alone, with an EC50 of 3.48 ± 0.34 nM (n = 6). When cells had been transfected with both TPalpha and Gh, a further increase of inositol phosphate was observed (Fig. 7A); however, the EC50 (4.27 ± 0.81 nM) was not significantly altered. The agonist-stimulated increase of inositol phosphate formation was higher in TPalpha  + Gqalpha -transfected cells than in TPalpha  + vector-transfected cells. Consequently, the EC50 (0.83 ± 0.08 nM) was significantly (p < 0.05) lower than in cells transfected with TPalpha  + vector. As expected, TPalpha did not activate PLC via Gs. Agonist-stimulated TPalpha  + Gsalpha -transfected cells did not produce more inositol phosphate than cells transfected with TPalpha  + vector. The EC50 (6.87 ± 2.48 nM) for agonist was also similar (p = not significant) to that observed in TPalpha  + vector-transfected cells.


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Fig. 7.   U46619-stimulated production of IP. IP production in COS-7 cells transfected with TPalpha (A) or TPbeta (B) along with the vector pcDNA3 (+vec), as a control, Gh (+Gh), Gqalpha (+Gq), or Gsalpha (+Gs) was measured after 30 min incubation with different concentrations of U46619. Results are expressed as percentage of the inosital phosohate (IPs) production measured in the non-stimulated sample (control) and represent the mean ± S.E. of six experiments performed in duplicate. * p < 0.05; ** p < 0.01, compared with control.

Similar experiments were carried out in cells transfected with TPbeta . Agonist-stimulated inositol phosphate production only exceeded that in cells transfected TPbeta  + vector in those cotransfected with TPbeta  + Gqalpha (Fig. 7B). Although Gqalpha caused a shift of the U46619 dose-response curve to the left, the EC50 calculated in TPbeta  + Gqalpha -transfected cells was not significantly different from that found in TPbeta  + vector-transfected cells (TPbeta  + vector: EC50 = 5.36 ± 1.89 nM; TPbeta  + Gqalpha : EC50 = 1.97 ± 0.29 nM, p = not significant). No significant increase in agonist-induced inositol phosphate production was observed in either TPbeta  + Gh or TPbeta  + Gsalpha -transfected cells versus TPbeta  + vector-transfected cells. Similarly, no differences were found in the EC50 (TPbeta  + Gh: EC50 = 5.75 ± 0.75 nM; TPbeta  + Gsalpha EC50 = 5.10 ± 2.08 nM, p = not significant).

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Gh is a newly characterized high molecular weight G protein that can be activated via the alpha 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 (~70 kDa) molecular mass (25). Given these observations, we decided to address the possibility that TPs might actually transmit intracellular signals via Gh. Furthermore, given the uncertain role of the TP isoforms, we sought to investigate whether they might couple with differential affinity to either Gh or Gqalpha . Gh is expressed in cells of the cardiovascular system which express TPs. Thus, platelets express message for both isoforms, although they appear to translate only TPalpha (43). Vascular smooth muscle cells express both isoforms, whereas endothelial cells appear to express only TPbeta (8). All of these cell types, as well as megakaryocytic cell lines, express Gh.

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 Gh, together with TPalpha or TPbeta , demonstrated that Gh 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 TPalpha ). 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 Gh seems to be high. For example, immunoprecipitation of Gh with TPs occurs even when using a lysate of COS-7 cells overexpressing Gh 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 [3H]SQ29,548. To address the specificity of the co-immunoprecipitation of Gh 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. Gh 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 Gh with TPalpha and TPbeta appears to reflect a specific interaction.

To address the possibility that the TP-Gh 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 Gqalpha amplified the response to agonist. By contrast, cotransfection with Gsalpha had no effect, as expected. Similar to our observation with Gqalpha , cotransfection of Gh with TPalpha enhanced the agonist-dependent increase in inositol phosphate formation over that found in cells transfected with TPalpha alone. By contrast, a similar effect was not observed in cells transfected with TPbeta .

The role of Gh 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 alpha  adrenoreceptors. We now provide evidence for association of this protein with a distinct receptor, the TP, a member of the eicosanoid receptor subfamily. Gh 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 Gh in cardiovascular cells and associate with this protein. However, their differential ability to signal via Gh provides the first suggestion of distinct roles for these receptor isoforms in vivo.

    ACKNOWLEDGEMENTS

We thank Drs. David R. Manning, Anthony J. Ware, Colin D. Funk, Robert M. Graham, and Gary L. Johnson for providing some of the cDNAs used in this study. We thank Dr. Padraig V. Nestor and Dr. Paolo Gresele for helpful suggestions and Yu-Min Shen and Ginger J. Griffis for technical assistance.

    FOOTNOTES

* 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. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Current address: INSERM U348, Paris, France.

§ Robinette Foundation Professor of Cardiovascular Medicine. To whom correspondence should be addressed: Center for Experimental Therapeutics, 153 Johnson Pavilion, 3600 Hamilton Walk, Philadelphia, PA 19104. Tel.: 215-898-1184; Fax: 215-573-9135; E-mail: garret{at}spirit.gcrc.upenn.edu.

    ABBREVIATIONS

The abbreviations used are: TxA2, thromboxane A2; Ab, antibody; G protein, GTP-binding protein; HASMC, human aortic smooth muscle cells; HEL, human erythroleukemia; HUVEC, human umbilical vein endothelial cells; HUVSMC, human umbilical vein smooth muscle cells; PMSF, phenylmethylsulfonyl fluoride; PCR, polymerase chain reaction; PLC, phospholipase C; nt, nucleotide; RT, reverse transcription; TP, thromboxane receptor; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; bp, base pair; SDS, sodium dodecyl sulfate.

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