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The G₁₂ Family of G Proteins as a Reporter of Thromboxane A₂ Receptor Activity

Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania

Received for publication October 7, 2005.

Accepted for publication January 13, 2006.

	Abstract

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Despite advances in the understanding of pathways regulated by the G₁₂ family of heterotrimeric G proteins, much regarding the engagement of this family by receptors remains unclear. We explore here, using the thromboxane A₂ receptor TP, the ability of G₁₂ and G₁₃ to report differences in the potency and efficacy of receptor ligands. We were interested especially in the potential of the isoprostane 8-iso-prostaglandin F (8-iso-PGF₂), among other ligands examined, to activate G₁₂ and G₁₃ through TP explicitly. We were also interested in the functionality of TP-G fusion proteins germane to G₁₂ and G₁₃. Using fusion proteins in Spodoptera frugiperda (Sf9) cells and independently expressed proteins in human embryonic kidney 293 cells, and using guanosine 5'-O-(3-[³⁵S]thio)triphosphate binding to evaluate G activation directly, we found for G that no ligand tested, including 8-iso-prostaglandin F (8-iso-PGF₂ and a purported antagonist (pinane thromboxane A₂), was silent. The activity of agonists was especially pronounced when evaluated for TP-G₁₃ and in the context of receptor reserve. Agonist activity for 8-iso-PGF₂ was diminished and that for pinane thromboxane A nonexistent when G₁₂ was the reporter. These data establish that G₁₂ and G₁₃ can report differentially potency and efficacy and underscore the relevance of receptor and G protein context.

Thromboxane A₂ is a member of the prostaglandin family of lipid mediators generated after cyclooxygenase-catalyzed conversion of arachidonic acid (Smith et al., 2000). As the principal product of the cyclooxygenase-1 isoform in platelets, thromboxane A₂ causes contraction and proliferation of vascular smooth muscle cells and activation of platelets, in the latter case evoking aggregation and amplifying the action of other platelet stimuli. These actions are concordant with studies implicating thromboxane A₂ in the pathology of a variety of cardiovascular diseases, including atherosclerosis (Kobayashi et al., 2004; Egan et al., 2005), stenosis after vascular injury (Cheng et al., 2002; Rudic et al., 2005), and hypertension (Francois et al., 2004). The biological actions of thromboxane A₂ are achieved through activation of one or both of its receptors, TP and TP, which are produced through differential splicing of a single gene product (Raychowdhury et al., 1994). Within the context of G protein signaling, TP, the predominant isoform in most if not all tissues studied (Miggin and Kinsella, 1998), is the more extensively characterized. TP exhibits the capacity in platelets to activate G_q, G₁₂, and G₁₃ (Offermanns et al., 1994).

Issues of agonism at TP and/or TP in general rest on effector activities or cell responses where G_q plays a prominent role. Of interest in this regard are the actions of isoprostanes, prostaglandin-like compounds produced primarily by oxygen free radical-induced peroxidation of arachidonic acid (Montuschi et al., 2004). Isoprostanes are in vivo markers of oxidant stress and are elevated coincident with perturbed cardiovascular function. The effects of two such isoprostanes, 8-iso-prostaglandin E₂ (8-iso-PGE₂) and 8-isoprostaglandin F₂ (8-iso-PGF₂), are blocked by TP receptor antagonists, suggesting a role for these compounds as TP receptor agonists (for review, see Janssen, 2001; Montuschi et al., 2004). Indeed, 8-iso-PGF mobilizes Ca²⁺2 in HEK293 cells when TP is introduced (Kinsella et al., 1997). Controversy remains, however, as to the identity of the receptor(s) activated by these mediators. Neither 8-iso-PGE₂ nor 8-iso-PGF₂ consistently displaces the binding of the TP antagonist [³H]SQ29548 efficiently (Pratico et al., 1996; Wilson et al., 2004), nor does 8-iso-PGF₂ mimic the actions of U46619 [GenBank] , a thromboxane A₂ mimetic, in several cells (Morrow et al., 1992), including platelets (Pratico et al., 1996). Furthermore, in contrast to HEK293 cells (Kinsella et al., 1997), expression of TP in Chinese hamster ovary cells does not reconstitute mobilization of Ca²⁺ by 8-iso-PGF₂ (Weber and Markillie, 2003). No study has examined the potential of isoprostanes, through TP, to activate G₁₂ and G₁₃ or has directly evaluated TP apart from the modifying influences of endogenously expressed receptors, for example, those in mammalian cells that might change functionality through receptor heterodimerization or competition for G proteins (Wilson et al., 2004).

We examine here, using TP, the extent to which potency and efficacy for ligands that operate through G₁₂ and G₁₃ can be evaluated. We address specifically the question of 8-iso-PGF₂ signaling through TP and the capacity of other ligands as well to signal as a function of the G protein examined and context of receptor expression.

	Materials and Methods

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Materials. U46619 [GenBank] , SQ29548, pinane thromboxane A₂ (PTA₂), U44069 [GenBank] , 8-iso-PGE₂, and 8-iso-PGF₂ were purchased from Cayman Chemical (Ann Arbor, MI). Protein A-Sepharose, aprotinin, normal rabbit serum, GTP, and GDP were purchased from Sigma-Aldrich (St. Louis, MO). Pansorbin cells and Nonidet P-40 were purchased from Fisher Scientific (Pittsburgh, PA). [³⁵S]GTPS (1250 Ci/mmol) and [³H]SQ29548 (58 Ci/mmol) were purchased from PerkinElmer Life and Analytical Sciences (Boston, MA). Sf900-II and Grace's insect medium (supplemented) were purchased from Invitrogen (Carlsbad, CA). cDNA for the hemagglutinin (HA)-tagged human TP receptor DNA was described previously (Wilson et al., 2004); the tag is at the N terminus. cDNAs for human G₁₂ and G₁₃ were purchased from Guthrie (Sayre, PA). Rabbit antisera for G₁₂ and G₁₃ were produced using peptides corresponding to the C-terminal 10 residues of the two proteins (Butkerait et al., 1995; Windh et al., 1999). The HA-directed monoclonal antibody HA.11 was purchased from Covance (Berkeley, CA).

Construction of Receptor-G Fusion Proteins. TP-G₁₃ was constructed by first introducing a BamH1 restriction site immediately 5' to the start codon of the (HA-tagged) TP cDNA and substituting the stop codon with an EcoR1 site by polymerase chain reactions. BamH1/Kpn and Kpn/EcoR1 fragments were subcloned into pcDNA(zeo) to reconstitute full-length TP cDNA, absent the stop codon, bounded by the two restriction sites. An EcoR1 site was introduced immediately 5' to codon 2 of the G₁₃ cDNA, eliminating the start codon. An EcoR1/XbaI restriction fragment, containing full-length G₁₃ absent the start codon, was subcloned into the pcDNA(zeo) containing HA-TP to form TP-G₁₃ cDNA. The TP-G₁₃ cDNA was subcloned into pcDNA3.1 using BamH1/BamH1 and then XhoI/XbaI digests. It was also subcloned into pFastbac using BamH1/BamH1 and then NotI/XbaI digests from TP-G₁₃ in pcDNA3.1. TP-G₁₂ was formed by appending an EcoR1 site immediately 5' to codon 2 of G₁₂ cDNA and then subcloning the EcoR1/XbaI fragment into pFastbac containing TP-G₁₃, with elimination of the G₁₃ cDNA. The TP-G₁₂ cDNA was formed in pcDNA3.1 by subcloning a NotI/XbaI fragment from pFastbac containing TP-G₁₂ into pcDNA3.1 containing HA-TP. Production of the recombinant baculoviruses was performed using the Bac-To-Bac baculovirus expression system (Invitrogen) according to the manufacturer's instructions.

Cell Culture and Membrane Preparation. Spodoptera frugiperda (Sf9) cells (American Type Culture Collection, Manassas, VA) were maintained in suspension culture in Grace's insect medium containing 10% heat-inactivated fetal bovine serum and 0.1% Pluronic F-68 at 27°C. For infection with recombinant baculoviruses, the cells were subcultured in monolayer and infected at a multiplicity usually of 1. The medium was replaced 16 h after infection with Sf900-II optimized serum-free medium (Invitrogen). The cells were harvested at 48 h, washed three times with 0.9% NaCl, and resuspended in 1 ml of ice-cold 20 mM HEPES, pH 8.0, 1 mM EDTA, 0.11% aprotinin, 0.02% leupeptin, and 0.1% phenylmethylsufonyl fluoride. After 5 min on ice, cells were homogenized by repeated passage through a 26-gauge needle. Homogenates were centrifuged at 110g for 5 min, and the resultant supernatants were centrifuged at 20,800g for 30 min at 4°C. The final pellets (membrane) were resuspended at 3 mg/ml protein.

Human embryonic kidney (HEK) 293 cells (American Type Culture Collection) were maintained in Dulbecco's modified Eagle's medium with 10% fetal bovine serum at 37°C with 5% CO₂. HEK293 cells stably expressing TP (4.8 pmol of receptor/mg of membrane protein; Wilson et al., 2004) were maintained in the above-mentioned medium but containing 1 mg/ml Geneticin (G418). Membranes were made from HEK293 cells in essentially the same manner as for Sf9 cells.

[³H]SQ29548 Binding. Membranes (20 µg of protein/assay point) were incubated with specified concentrations of [³H]SQ29548 in 20 mM HEPES, pH 7.4, 2 mM EDTA, and 5 mM NaCl for 30 min at 30°C. The incubation volume was 0.1 ml. Reactions were terminated by dilution with 10 mM HEPES, pH 7.4, and 0.01% bovine serum albumin at 0°C and rapid filtration over Whatman GF/C filters presoaked in the same buffer. The filters were washed three times with the same buffer at 0°C and dried. Filter-bound radioactivity was determined by scintillation spectrometry. Nonspecific binding, generally less that 10% at K_d, was defined as the binding of radioligand in the presence of 250 µM SQ29548.

Assay of [³⁵S]GTPS Binding. The assay for agonist-promoted binding of [³⁵S]GTPS to G₁₂ and G₁₃ was performed essentially as described previously (Windh et al., 1999). Membranes (20 µg of protein/assay point) were resuspended in 50 mM Tris-HCl, pH 7.5, 2 mM EDTA, 100 mM NaCl, 20 mM MgCl₂, 0.1 µM GDP, and 5 nM [³⁵S]GTPS in 1.5-ml Eppendorf Microfuge tubes on ice. Ligands, if any, were added, and the tubes were transferred immediately to a 30°C water bath for times ranging from 1 to 40 min as noted. The incubation was terminated by adding 600 µl of ice-cold 50 mM Tris-HCl, pH 7.5, 20 mM MgCl₂, 150 mM NaCl, 0.5% Nonidet P-40, 0.33% aprotinin, 0.1 mM GDP, and 0.1 mM GTP. The extract was transferred to a microcentrifuge tube containing 2 µl of nonimmune serum preincubated with 100 µl of a 12% suspension of Pansorbin cells. Nonspecifically bound proteins were removed after 20 min by centrifugation. The extract was then incubated for 1h at 4°C with 10 µl of a G-directed antiserum or HA-directed antibody, or nonimmune serum, all of which had been preincubated with 100 µl of a 5% suspension of protein A-Sepharose. Immunoprecipitates were collected and washed three times in the extraction buffer, then once in the buffer without detergent, and then boiled in 0.5 ml of 0.5% SDS followed by addition of 5.2 ml of Ecolite+ (MP Biomedicals, Irvine, CA). The samples were analyzed directly by scintillation spectrometry. Counts obtained with nonimmune serum, representing nonspecifically bound radiolabel and generally in the range of 50 to 200 cpm, were subtracted before any portrayal of the data.

Miscellaneous. Western blots were performed by SDS-polyacrylamide gel (12% acrylamide) electrophoresis followed by transfer of protein to Immobilon-P membranes (Millipore, Billerica, MA); block of nonspecific binding sites with 5% nonfat milk in 0.1 M Tris-HCl, pH 7.5, 0.9% NaCl, and 0.1% Tween 20; incubation with primary antisera or antibodies, generally at 1:1000 dilutions; washing in the same buffer but without nonfat milk; incubation with horseradish peroxidase-conjugated secondary antibodies; washing; and visualization with enhanced chemiluminescence Western blotting detection reagents (GE Healthcare, Little Chalfont, Buckinghamshire, UK). [³H]SQ29548 and [³⁵S]GTPS binding were evaluated by nonlinear regression using Prism (GraphPad Software Inc., San Diego, CA). Statistical differences were determined using Student's t test, with p < 0.05 signifying a difference, or using 95% confidence intervals, with nonoverlapping intervals signifying a difference.

	Results

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To investigate the properties of transduction through members of the G₁₂ family of heterotrimeric G proteins, we chose to focus on the signaling effected through the thromboxane A₂ receptor TP.TP couples to both G₁₂ and G₁₃ and has been studied extensively in the context of platelet and vascular smooth muscle cell function. We examined first the activation of G₁₃, which provided more easily discerned signals for all the ligands tested (see below).

We explored the expression and functionality of a TP-G₁₃ fusion protein introduced into Sf9 cells. Receptor-G fusion proteins fix the ratio of receptor and G at 1:1 and have been well documented for receptors communicating with G_s and G_i (Siefert et al., 1999; Milligan, 2000). Sf9 cells contain few, if any, receptors that interfere with the analysis of those introduced by means of recombinant baculoviruses (Windh and Manning, 2002). Analysis by Western blotting of membranes from cells with an antibody directed toward the N-terminal HA epitope as well as with an antibody directed toward the C-terminal 10 residues of G₁₃ confirmed expression of the protein after infection (Fig. 1). The fusion protein had an electrophoretic mobility corresponding to 80 kDa, close to the predicted molecular mass of 82.7 kDa. Saturation binding with the radiolabeled antagonist [³H]SQ29548 revealed a B_max of 2 to 4 pmol/mg of membrane protein and a K_d of 57 ± 11 nM (n = 3). The K_d was similar to that observed when the receptor was expressed without the appended G (33 ± 2 nM; n = 3; not shown) or when it was overexpressed in HEK293 cells (35 nM) (Wilson et al., 2004). No binding was observed in the membranes without introduction of the fusion protein.

View larger version (17K): [in this window] [in a new window]   Fig. 1. Expression of TP-G13 in Sf9 cells. A, membranes were prepared from Sf9 cells that were, or were not, infected with a recombinant baculovirus encoding TP-G13. The membranes (30 µg of protein) were analyzed by Western blotting (W.B.) with a monoclonal antibody against the HA epitope N terminal to the receptor or with a rabbit antiserum generated against the C-terminal 10 residues of G13. The fusion protein has an apparent molecular mass of approximately 80 kDa, calculated from its migration relative to standards. The more rapidly migrating bands observed with the G antiserum were evident after infection with irrelevant baculoviruses as well. B, membranes (20 µg of protein) from Sf9 cells expressing TP-G13 were incubated in triplicate for 30 min at 30°C with the indicated concentrations of [3H]SQ29548. Bound radiolabel was determined after filtration. Nonspecific binding (less than 10% at Kd) was determined with 250 µM SQ29548 and was subtracted from total binding. Each of the experiments was representative of several others.

View larger version (14K): [in this window] [in a new window]   Fig. 2. Activation of G by U46619 [GenBank] . A, membranes from Sf9 cells expressing TP-G13 were incubated for 1 to 20 min at 30°C with [35S]GTPS with or without 100 µM U46619 [GenBank] . The membranes were solubilized, immunoprecipitation was carried out with an antiserum directed toward the C terminus of G13, and bound radioactivity was evaluated by scintillation spectrometry. The graph portrays ligand-dependent binding (i.e., the binding obtained with U46619 [GenBank] minus that obtained without U46619 [GenBank] ) [U46619 (Net)]. B, an individual experiment, in which membranes from Sf9 cells expressing TP-G13 were incubated in duplicate for 1 min at 30°C with [35S]GTPS and the indicated concentrations of U46619 [GenBank] . The membranes were processed as in A for determination of bound radioactivity. C, U46619 [GenBank] -effected binding was evaluated as in B and normalized by setting agonist-independent binding to 0% and maximum U46619 [GenBank] -effected binding to 100% (n = 3 independent experiments).

View this table: [in this window] [in a new window]   TABLE 1 Potency and efficacy of ligands in promoting [35S]GTPS binding to TP·G12 and TP·G13 EC50 values were calculated by nonlinear regression performed on concentration-response data for TP·G fusion proteins expressed in Sf9 cells and are provided here as means and 95% confidence intervals (CI). The maximal effect of a ligand on [35S]GTPS-binding (Emax) was also determined by nonlinear regression and is expressed relative to binding promoted by 100 µM U46619 [GenBank] ± 1 S.E. (n = 3); values for U44069 [GenBank] , isoprostanes, and PTA2 differ statistically (P < 0.05) from 1.0. Concentration-response experiments used assay times of 1 min (G13) and 5 or 10 min (G12), except for TP expressed endogenously in HEK293 cells, for which the assay time was 10 min.

A question of interest, in part bearing on the functionality of the fusion protein, is the extent to which G₁₃ can report differences in potencies and efficacies among ligands that operate through TP, because the properties of such ligands are commonly referenced to events, carried out through TP or not, where G_q instead plays a prominent role. We evaluated five compounds in addition to U46619 [GenBank] : U44069 [GenBank] , considered like U46619 [GenBank] , a full agonist; the isoprostanes 8-iso-PGE₂ and 8-iso-PGF₂, whose efficacy, if operating through TP at all, is uncertain; and PTA₂ and SQ29548, reportedly antagonists. Using again TP-G₁₃ in Sf9 cells and [³⁵S]GTPS binding, we found that all ligands but SQ29548 promoted activation of G₁₃ (Fig. 3). Differences in maximal binding were evident. No agonist exhibited a maximal activity (E_max) equal to that of U46619 [GenBank] (Table 1). Those of U44069 [GenBank] and 8-iso-PGE₂ were approximately 80%, and those of 8-iso-PGF₂ and PTA₂ were approximately 60%, the E_max of U46619 [GenBank] . The rank order of potencies was U44069 [GenBank] > U46619 [GenBank] PTA₂ > 8-iso-PGE₂ > 8-iso-PGF₂. Thus, G₁₃ can register differences in both efficacy and potency. The data also attest, for the first time, to the properties of isoprostanes as agonists for TP as visualized through activation of G₁₃ and point, unexpectedly, as well to the property of PTA₂ as an agonist.

View larger version (23K): [in this window] [in a new window]   Fig. 3. Actions of several ligands on G13 activity through TP. A, membranes from Sf9 cells expressing TP-G13 were incubated for 1 min at 30°C with [35S]GTPS and the indicated concentrations of U44069 [GenBank] , 8-iso-PGE2, 8-iso-PGF2, or PTA2. The membranes were solubilized, and immunoprecipitation was carried out with the G13-directed antiserum for quantitation of bound radiolabel. Binding was referenced to that obtained in the same experiments with 100 µM U46619 [GenBank] , which was assigned an activity of 1.0, after subtraction of ligand-independent activity. B, membranes were incubated with 10 to 100 µM SQ29548 or 10 µM U46619 [GenBank] for 10 min. Activity is expressed as a percentage of ligand-independent [35S]GTPS binding; the percentage of increase with U46619 [GenBank] is lower than in other experiments because of the time point chosen. The changes from ligand-independent activity with SQ29548 and U46619 [GenBank] are significant (p < 0.01). n = 3; bars represent 1 S.E.M.

In contrast to the other ligands, SQ29548 did not exhibit the activity of an agonist (Fig. 3, bottom). Indeed, SQ29548 suppressed the small, but consistently obtained, degree of ligand-independent G₁₃ activation. Whether this effect represented neutral antagonism in the face of an endogenous agonist or inverse agonism could not be established. However, inhibition of the synthesis of cyclooxygenase-derived metabolites with aspirin did not modify activity (data not shown), ruling out involvement of endogenous thromboxane A₂. The data for SQ29548 demonstrate that ligand-independent activity was not entirely because of guanine nucleotide exchange intrinsic to G₁₃, but rather to some form of receptor-effected activation of the G subunit.

Given the activity observed with PTA₂, purportedly an antagonist, and with 8-iso-PGF₂, whose interaction with TP has been subject to debate—and also given the novelty of the receptor-G₁₃ fusion protein itself—we felt obliged to evaluate a different mode of receptor and G₁₃ expression altogether. We turned to HEK293 cells in which TP was (over)expressed stably. Here, the binding of [³⁵S]GTPS was measured for G₁₃ endogenous to the cells. We found that the rate of binding effected by U46619 [GenBank] through overexpressed TP was approximately the same as that noted for TP-G₁₃ (data not shown). The -fold increase in binding was also high (approximately 10-fold; Fig. 4A); however, the EC₅₀, 80 nM (Fig. 4B; Table 1), was less. As before, 8-iso-PGF₂ and PTA₂ exhibited significant activity (Fig. 4C). In fact, the activity of 8-iso-PGF₂ approached that of U46619 [GenBank] .

View larger version (28K): [in this window] [in a new window]   Fig. 4. Activation of G13 in HEK293 cells. Membranes from HEK293 cells (A-C) stably expressing heterologous TP (approximately 5 pmol of receptor/mg of membrane protein) or wild-type HEK293 cells (D-F) were incubated with [35S]GTPS and U46619 [GenBank] at the indicated concentrations, 300 µM 8-iso-PGF2, or 100 µM PTA2. Assay times were 1 (A-C) or 10 (D-F) min The membranes were solubilized, and immunoprecipitation of endogenous G13 was carried out with the G13-directed antiserum followed by quantitation of bound radiolabel. A and D represent single experiments in duplicate, and B and E represent combined data from three to five experiments expressed as a fraction of binding achieved with 100 µM U46619 [GenBank] . C and F represent binding promoted by 8-iso-PGF2 and PTA2 as fraction of that promoted by 100 µM U46619 [GenBank] in paired experiments. In F, the binding promoted by PTA2 is not statistically significant when evaluated for the stated concentration alone (p < 0.05 for a two-tailed t test), but it is statistically significant when concentration-response data are evaluated by nonlinear regression, wherein the 95% confidence interval for Emax is above 0.

2

Data for purified G₁₂ and G₁₃ suggest equivalent [³⁵S]GTPS-binding properties (Kozasa and Gilman, 1995); however, evidence for selectivity in activation by receptors is emerging (Riobo and Manning, 2005). We therefore wished to evaluate the activation of G₁₂ by TP. We returned to the fusion protein as a reporter, using now TP-G₁₂. Our intent was to compare the properties of G₁₂ to those of G₁₃ (i.e., TP-G₁₃) under equivalent conditions; G₁₂ is not expressed in HEK293 cells at detectable levels, nor would we have been able to factor the influence of slight differences in receptor/G ratios or the influence of other receptors in these cells. Expression of TP-G₁₂ in Sf9 cells was comparable with that of TP-G₁₃, as was recognition of the two subunits by their respective antisera in Western blots (Fig. 5A; not shown). U46619 [GenBank] effected [³⁵S]GTPS binding to TP-G₁₂,as it did to TP-G₁₃ (Fig. 5B). Of interest, the rate of binding for TP-G₁₂ was quite low, at most 5% of the maximum within 1 min. G₁₂ was activated, therefore, approximately 10-fold more slowly in response to U46619 [GenBank] than was G₁₃. The EC₅₀ for activation was slightly greater than that for activation of TP-G₁₃ (870 versus 520 nM; Fig. 5C; Table 1). Thus, although the response of G₁₂ and G₁₃ to U46619 [GenBank] -activated TP differs in terms of the kinetics of guanine nucleotide binding, the sensitivity of the two subunits toward the receptor is similar. TP-G₁₂ was less sensitive than TP-G₁₃ to 8-iso-PGF₂; activation did not approach saturation, so an EC₅₀ could not be calculated. It is remarkable that TP-G₁₂ was completely refractory to PTA₂. This observation suggested that TP-G₁₂, unlike TP-G₁₃, did not bind PTA₂ or could not respond to PTA₂ once the ligand was bound. PTA₂ antagonized the activation of TP-G₁₂ by U46619 [GenBank] (data not shown), indicating the latter possibility. The expression of activity on the part of 8-iso-PGF₂ and PTA₂ depends, therefore, on the nature of the G used as the reporter.

View larger version (18K): [in this window] [in a new window]   Fig. 5. Activation of G12 through TP. Membranes were prepared from Sf9 cells infected with recombinant baculovirus encoding TP-G12 (and, in A, TP-G13 also), or not infected, as noted. A, membranes (30 µg of protein/lane) were analyzed by Western blotting with a monoclonal antibody against the HA epitope or an antiserum against the C-terminal 10 residues of G12. B, U46619 [GenBank] -promoted binding of [35S]GTPS to TP-G12 as a function of time [U46619 (Net)], determined as binding obtained with 100 µM U46619 [GenBank] minus that obtained without U46619 [GenBank] (inset), using the G12-directed antiserum for immunoprecipitation here and in C. C, U46619 [GenBank] -, 8-iso-PGF2 -, and PTA2-effected binding of [35S]GTP S to TP-G12, normalized by setting agonist-independent binding to 0 and maximum U46619 [GenBank] -effected binding to 1 (n = 3-4; 10-min assay).

	Discussion

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2

The idea of evaluating R-G fusion proteins for G₁₂ and G₁₃ was based on successes achieved elsewhere for G_s and G_i (for review, see Siefert et al., 1999; Milligan, 2000). The invariant ratio of receptor to G subunit was attractive, because it theoretically obviates differences in expression that can complicate, ultimately, comparisons among different receptors and G proteins. Fusion proteins were used previously for G₁₂ and G₁₃ to help characterize the nature of Rac inhibition through the sphingosine 1-phosphate S1P₂ receptor (Sugimoto et al., 2003); however, activation of the G subunits was not measured directly and collateral activation of endogenous G₁₂ and G₁₃ was not precluded.

Ours is the first study to explicitly evaluate parameters of agonist action as translated by G₁₂ or G₁₃. The EC₅₀ for activation by U46619 [GenBank] of G₁₂ and G₁₃ fused to TP was approximately 0.5 µM, consistent with EC₅₀ values reported for a large number of U46619 [GenBank] -effected phenomena and also consistent with the activation of G₁₃ through TP endogenous to HEK293 cells (0.3 µM). The EC₅₀ is higher than that reported for changes in platelet shape (EC₅₀ 15 nM; Ohkubo et al., 1996), a response unquestionably linked to G₁₃ and not in some way achieved through G_q or a combination of G_q and G₁₃ (Moers et al., 2003). The difference is conceivably due to the existence of spare receptors in platelets with respect to actin polymerization. Overexpression of TP results in a greater potency of U46619 [GenBank] toward G₁₃ activation itself, and it is not too speculative to assume limiting steps exist downstream of the G protein as well. The rank ordering of potencies for ligands as monitored through activation of at least G₁₃ fused to TP in Sf9 cells was in general consistent with that of K_i values for displacement of [³H]SQ29548 in several types of cells (Fukunaga et al., 1993a,b; Dorn et al., 1997; Cao et al., 2004; Wilson et al., 2004). There is no evidence, therefore, of selectivity among ligands in G₁₃ activation apart from what can be inferred through competitive binding.

Differences in maximal effects on G₁₃ among some of ligands were evident. In TP-G₁₃, the ligands 8-iso-PGF₂ and PTA₂ exhibited approximately half the activity of U46619 [GenBank] , whereas U44069 [GenBank] and 8-iso-PGE₂ exhibited approximately 80% the activity. Insofar as examined, relative differences were maintained in HEK293 cells, both those overexpressing TP and those for which agonists worked through endogenous TP. G₁₃ can therefore resolve ligands according to efficacy. The higher activity of partial agonists in the case of overexpressed TP relative to the same agonists using endogenous TP in HEK293 cells is likely due to spare receptors in the former instance. In contrast to the other ligands, SQ29548 did not activate G₁₃ but instead suppressed the small amount of ligand-independent activity evident for the fusion protein. A definitive demonstration of inverse agonism will require identifying a neutral antagonist; however, the possibility of inverse agonism for SQ29548 raises the spectre of receptor selection in a widely used radioligand binding assay, wherein [³H]SQ29548 would prefer the noncoupled form of receptor.

Obtaining an EC₅₀ value that reflects best a ligand's actions through TP requires recognition of how quickly G₁₃ can be activated. At high concentrations of U46619 [GenBank] (10-100 µM), the binding of [³⁵S]GTPS for TP-G₁₃ and for G₁₃ activated through overexpressed TP in HEK293 cells departed from linearity within 2 min. The use of longer times in our assay, such as those used previously (Windh and Manning, 2002), resulted in a substantial decrease in EC₅₀ because of lower concentrations of agonist having time to achieve saturation. This was not the case with TP-G₁₂. [³⁵S]GTPS binding for G₁₂ was far slower, and assay times up to 20 min provided satisfactory results in the evaluation of EC₅₀ values. The nature of the difference in binding rates is unknown. Previous data with purified G₁₂ and G₁₃ provide no indication of differences in intrinsic GDP/[³⁵S]GTPS exchange activity (Kozasa and Gilman, 1995). We think the difference in rates is likely to be a property of the G protein response to TP. This difference, in the intact cell, may imply that G₁₃ engages downstream pathways more rapidly than G₁₂ in response to activation of the receptor. The difference may also imply a heightened sensitivity of G₁₃ toward low concentrations of agonists or partial agonists, if the rate of activation is sufficiently high relative to the rate of deactivation (GTP hydrolysis) to cause a summation of activation events.

Differences in the kinetics of [³⁵S]GTPS binding notwith-standing, G₁₂ and G₁₃ were similarly sensitive to activation by U46619 [GenBank] through TP, as manifested in similar EC₅₀ values. Of interest, the two G protein subunits perceived PTA₂ quite differently. PTA₂ is a potent agonist when viewed through G₁₃ but has no activity whatsoever when viewed through G₁₂. Our data suggest that the PTA₂-bound conformation of receptor is not recognized by G₁₂. The data, therefore, identify the potential for differential receptivity of G subunits toward ligands.

Isoprostanes arise through cyclooxygenase-independent, free radical-induced peroxidation of membrane lipids, and the F₂ isoprostanes in particular are likely to be pathological mediators of oxidative injury. Whether the isoprostanes are viewed to work through TP has been complicated by inconsistent blockade with TP antagonists, inconsistent mimicry of U46619 [GenBank] , and the possible existence of other receptors that mediate their actions. Our data emphasize the importance of context in sorting these possibilities out. 8-iso-PGF₂ un- equivocally signals through G₁₃. The EC₅₀ for 8-iso-PGF₂ (approximately 45 µM through TP-G₁₃), however, is quite high. We suspect that, in vivo, the actions of 8-iso-PGF₂ through TP would require significant receptor reserve or the aforementioned accumulation of activated G₁₃ as a result of activation outpacing deactivation. It is interesting to note that dynamic changes in TP levels, perhaps influencing responsiveness to 8-iso-PGF₂, have been argued to occur in pathophysiological states, including oxidative stress (Valentin et al., 2004). Signaling for 8-iso-PGF₂ through the G₁₂ family of G proteins may not be evident in cells where G₁₂ predominates as a transducer. The sensitivity of a cell to 8-iso-PGF₂ will also depend on how well it activates, through TP receptors, G_q and whether 8-iso-PGF₂ might use other receptors more or less effectively as well.

Receptors that couple to G₁₂ and G₁₃ invariably couple to other G proteins additionally (Riobo and Manning, 2005) and would therefore seem to be especially relevant prospects for agonist-directed trafficking. The interplay between the G_q and G₁₂ families, for example, at the level of protein kinase D (Yuan et al., 2001) and serum response factor activation (Sagi et al., 2001), has received considerable attention. The capacity to monitor potency and efficacy as reported by G₁₂ and G₁₃, combined with the extension of these methods to other G proteins, provides an important step in the pursuit of trafficking.

	Acknowledgements

 
We thank Natalia Riobo for comments regarding the manuscript and for input throughout the work.

	Footnotes

 
This work was supported by National Institutes of Health grants GM066892 (to D. R. M.) and HL066233 (to E.M.S.).

ABBREVIATIONS: 8-iso-PGE₂, 8-iso-prostaglandin E₂; 8-iso-PGF₂, 8-iso-prostaglandin F₂; SQ29548, [1S-[1,2(Z),3,4]]-7-[3-[[2-[(phenyl amino)carbonyl]hydrazine]methyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid; U46619 [GenBank] , 9,11-dideoxy-9, 11-methanoepoxy-prosta-5Z,13E-dien-1-oic acid; PTA₂, pinane-thromboxane A₂; U44069 [GenBank] , 9,11-dideoxy-9, 11-epoxymethano-prosta-5Z,13E-dien-1-oic acid; GTPS, guanosine 5'-(3-O-thio)triphosphate; HA, the sequence YPYDVDPDYA from hemagglutinin; HEK, human embryonic kidney; TP, thromboxane A₂ receptor.

Address correspondence to: Dr. David R. Manning, Department of Pharmacology, University of Pennsylvania School of Medicine, 3620 Hamilton Walk, Philadelphia, PA 19104-6084. E-mail: manning{at}pharm.med.upenn.edu

	References

Top Abstract Materials and Methods Results Discussion References

 
Butkerait P, Zheng Y, Hallak H, Graham TE, Miller HA, Burris KD, Molinoff PB, and Manning DR (1995) Expression of the human 5-hydroxytryptamine receptor in Sf9 cells. _1A J Biol Chem 270: 18691-18699.[Abstract/Free Full Text]

Cao J, Wakatsuki A, Yoshida M, Kitazawa T, and Taneike T (2004) Thromboxane A₂ (TP) receptor in the non-pregnant porcine myometrium and its role in regulation of spontaneous contractile activity. Eur J Pharmacol 485: 317-327.[CrossRef][Medline]

Cheng Y, Austin SC, Rocca B, Koller BH, Coffman TM, Grosser T, Lawson JA, and FitzGerald GA (2002) Role of prostacyclin in the cardiovascular response to thromboxane A₂. Science (Wash DC) 296: 539-541.[Abstract/Free Full Text]

Dorn GW 2nd, Davis MG, and D'Angelo DD (1997) Structural determinants for agonist binding affinity to thromboxane/prostaglandin endoperoxide (TP) receptors: analysis of chimeric rat/human TP receptors. J Biol Chem 272: 12399-12405.[Abstract/Free Full Text]

Egan KM, Wang M, Lucitt MB, Zukas AM, Pure E, Lawson JA, and FitzGerald GA (2005) Cyclooxygenases, thromboxane and atherosclerosis: plaque destabilization by cyclooxygenase-2 inhibition combined with thromboxane receptor antagonism. Circulation 111: 334-342.[Abstract/Free Full Text]

Francois H, Athirakul K, Mao L, Rockman H, and Coffman TM (2004) Role for thromboxane receptors in angiotensin-II-induced hypertension. Hypertension 43: 364-369.[Abstract/Free Full Text]

Fukunaga M, Makita N, Roberts LJ, Morrow JD, Takahashi K, and Badr KF (1993a) Evidence for the existence of F₂-isoprostane receptors on rat vascular smooth muscle cells. Am J Physiol 264: C1619-C1624.[Medline]

Fukunaga M, Takahashi K, and Badr KF (1993b) Vascular smooth muscle actions and receptor interactions of 8-iso-prostaglandin E₂, an E₂-isoprostane. Biochem Biophys Res Commun 195: 507-515.[CrossRef][Medline]

Janssen LJ (2001) Isoprostanes: an overview and putative roles in pulmonary pathophysiology. Am J Physiol 280: L1067-L1082.

Kinsella BT, O'Mahony DJ, and FitzGerald GA (1997) The human thromboxane A receptor isoform (TP) functionally couples to the G proteins G_q and G₁₁ in vivo and is activated by the isoprostane 8-epi prostaglandin F _{q 11 2}. J Pharmacol Exp Ther 281: 957-964.[Abstract/Free Full Text]

Kobayashi T, Tahara Y, Matsumoto M, Iguchi M, Sano H, Murayama T, Arai H, Oida H, Yurugi-Kobayashi T, Yamashita JK, et al. (2004) Roles of thromboxane A₂ and prostacyclin in the development of atherosclerosis in apoE-deficient mice. J Clin Investig 114: 784-794.[CrossRef][Medline]

Kozasa T and Gilman AG (1995) Purification of recombinant G proteins from Sf9 cells by hexahistidine tagging of associated subunits. J Biol Chem 270: 1734-1741.[Abstract/Free Full Text]

Miggin SM and Kinsella BT (1998) Expression and tissue distribution of the mRNAs encoding the human thromboxane A2 receptor (TP) and isoforms. Biochim Biophys Acta 1425: 543-559.[Medline]

Milligan G (2000) Insights into ligand pharmacology using receptor—G-protein fusion proteins. Trends Pharmacol Sci 21: 24-28.[CrossRef][Medline]

Moers A, Nieswandt B, Massberg S, Wettschureck N, Gruner S, Konrad I, Schulte V, Aktas B, Gratacap MP, Simon MI, et al. (2003) G₁₃ is an essential mediator of platelet activation in hemostasis and thrombosis. Nat Med 9: 1418-1422.[CrossRef][Medline]

Montuschi P, Barnes PJ, and Roberts LJ (2004) Isoprostanes: markers and mediators of oxidative stress. FASEB J 18: 1791-1800.[Abstract/Free Full Text]

Morrow JD, Moore KP, Awad JA, Revenscraft MD, Marini G, Badr KF, Williams R, and Roberts LJ (1992) The F₂-isoprostane, 8-epi-prostaglandin F₂, a potent agonist of the vascular thromboxane/endoperoxide receptor, is a platelet thromboxane/endoperoxide receptor antagonist. Prostaglandins 44: 155-163.[CrossRef][Medline]

Offermanns S, Laugwitz KL, Spicher K, and Schultz G (1994) G proteins of the G₁₂ family are activated via thromboxane A₂ and thrombin receptors in human platelets. Proc Natl Acad Sci USA 91: 504-508.[Abstract/Free Full Text]

Ohkubo S, Nakahata N, and Ohizumi Y (1996) Thromboxane A₂-mediated shape change: independent of G_q-phospholipase C-Ca²⁺ pathway in rabbit platelets. Br J Pharmacol 117: 1095-1104.[Medline]

Pratico D, Smyth EM, Violi F, and FitzGerald GA (1996) Local amplification of platelet function by 8-epi prostaglandin F₂ is not mediated by thromboxane receptor isoforms. J Biol Chem 271: 14916-14924.[Abstract/Free Full Text]

Raychowdhury M, Yukawa M, Collins L, McGrail S, Kent K, and Ware J (1994) Alternative splicing produces a divergent cytoplasmic tail in the human endothelial thromboxane A₂ receptor [published erratum appears in J Biol Chem 1995; 270:7011]. J Biol Chem 269: 19256-19261.[Abstract/Free Full Text]

Riobo NA and Manning DR (2005) Receptors coupled to heterotrimeric G proteins of the G₁₂ family. Trends Pharmacol Sci 26: 146-154.[CrossRef][Medline]

Rudic RD, Brinster D, Cheng Y, Fries S, Song W-L, Austin SC, Coffman TM, and FitzGerald GA (2005) COX-2-derived prostacyclin modulates vascular remodeling. Circ Res 96: 1240-1247.[Abstract/Free Full Text]

Sagi SA, Seasholtz TM, Kobiashvili M, Wilson BA, Toksoz D, and Brown JH (2001) Physical and functional interactions of G_q with Rho and its exchange factors. J Biol Chem 276: 15445-15452.[Abstract/Free Full Text]

Siefert R, Wenzel-Seifert K, and Kobilka BK (1999) GPCR-G fusion proteins: molecular analysis of receptor-G-protein coupling. Trends Pharmacol Sci 20: 383-389.[CrossRef][Medline]

Singer WD, Miller RT, and Sternweis PC (1994) Purification and characterization of the subunit of G₁₃. J Biol Chem 269: 19796-19802.[Abstract/Free Full Text]

Smith WL, DeWitt DL, and Garavito RM (2000) Cyclooxygenases: structural, cellular and molecular biology. Annu Rev Biochem 69: 145-182.[CrossRef][Medline]

Sugimoto N, Takuwa N, Okamoto H, Sakurada S, and Takuwa Y (2003) Inhibitory and stimulatory regulation of Rac and cell motility by the G_12/13-Rho and G_i pathways integrated downstream of a single G protein-coupled sphingosine-1-phosphate receptor isoform. Mol Cell Biol 23: 1534-1545.[Abstract/Free Full Text]

Valentin, Field MC, and Tippins JR (2004) The mechanism of oxidative stress stabilization of the thromboxane receptor in COS-7 cells. J Biol Chem 279: 8316-8324.[Abstract/Free Full Text]

Weber TJ and Markillie LM (2003) Regulation of activator protein-1 by 8-iso-prostaglandin E₂ in a thromboxane A₂ receptor-dependent and -independent manner. Mol Pharmacol 63: 1075-1081.[Abstract/Free Full Text]

Wilson SJ, Roche AM, Kostetskaia E, and Smyth EM (2004) Dimerization of the human receptors for prostacyclin and thromboxane facilitates thromboxane receptor-mediated cAMP generation. J Biol Chem 279: 53036-53047.[Abstract/Free Full Text]

Windh RT, Lee M-J, Hla T, An S, Barr AJ, and Manning DR (1999) Differential coupling of the sphingosine 1-phosphate receptors Edg-1, Edg-3 and H218/Edg-5 to the G_i, G_q, and G₁₂ families of heterotrimeric G proteins. J Biol Chem 274: 27351-27358.[Abstract/Free Full Text]

Windh RT and Manning DR (2002) Analysis of G protein activation in Sf9 and mammalian cells by agonist-promoted [³⁵S]GTPS binding. Methods Enzymol 344: 3-14.[Medline]

Yuan J, Slice LW, and Rozengurt E (2001) Activation of protein kinase D by signaling through Rho and the subunit of the heterotrimeric G protein G₁₃. J Biol Chem 276: 38619-38627.[Abstract/Free Full Text]

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