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Accelerated Communication

EP₄ Prostanoid Receptor Coupling to a Pertussis Toxin-Sensitive Inhibitory G Protein

Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson, Arizona

Received August 5, 2005; accepted October 4, 2005

	Abstract

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The EP₂ and EP₄ prostanoid receptor subtypes are G-protein-coupled receptors for prostaglandin E₂ (PGE₂). Both receptor subtypes are known to couple to the stimulatory guanine nucleotide binding protein (G_s) and, after stimulation with PGE₂, can increase the formation of intracellular cAMP. In addition, PGE₂ stimulation of the EP₄ receptor can activate phosphatidylinositol 3-kinase (PI3K) leading to phosphorylation of the extracellular signal-regulated kinases (ERKs) and induction of early growth response factor-1 (EGR-1) (J Biol Chem 278: 12151–12156, 2003). We now report that the PGE₂-mediated phosphorylation of the ERKs and induction of EGR-1 can be blocked by pretreatment of EP₄-expressing cells with pertussis toxin (PTX). Furthermore, pretreatment with PTX increased the amount of PGE₂-stimulated intracellular cAMP formation in EP₄-expressing cells but not in EP₂-expressing cells. These data indicate that the EP₄ prostanoid receptor subtype, but not the EP₂, couples to a PTX-sensitive inhibitory G-protein (G_i) that can inhibit cAMP-dependent signaling and activate PI3K/ERK-dependent signaling.

The EP receptor subtypes interact with several of the subfamilies of G-proteins to activate their respective signaling pathways. PGE₂ stimulation of the human EP₁ receptor increases the concentration of free intracellular Ca²⁺ (Funk et al., 1993) and stimulates inositol phosphate formation (J. W. Regan, unpublished observations), suggesting coupling to members of the G_q subfamily. The EP₃ receptors are traditionally thought to couple to G_i to inhibit adenylyl cyclase. However, the EP₃ receptors actually consist of multiple isoforms that are generated by alternative mRNA splicing, and their coupling to G-proteins is complex (Kotani et al., 1995). For example, in humans, there are eight isoforms, and at least two of these isoforms, the EP_3-II and EP_3-IV, seem to couple to G_s to stimulate adenylyl cyclase. The human EP_3-I and EP_3-II can also couple to G_q to stimulate inositol phosphate formation.

Stimulation of the human EP₂ and EP₄ receptors with PGE₂ increases intracellular cAMP formation, indicating that both of these isoforms can couple to G_s to stimulate adenylyl cyclase (Regan, 2003). However, functional coupling to the cAMP signaling pathway seems to be more efficient for the human EP₂ receptor subtype than for the EP₄ subtype. Thus, when stably expressed in HEK cells at similar levels of receptor expression, the maximal stimulation of intracellular cAMP formation by the EP₄ subtype is only 20 to 50% of that achieved by the EP₂ subtype (Fujino et al., 2002, 2005). It has also been found recently that the human EP₄ receptor subtype, but not the human EP₂ subtype, can activate a phosphatidylinositol 3-kinase (PI3K) signaling pathway by a mechanism that is independent of the activation of the cAMP/protein kinase A (PKA) pathway (Fujino et al., 2002, 2003, 2005). PGE₂-mediated activation of this PI3K signaling pathway by the human EP₄ receptor leads to the induction of functional expression of early growth response factor-1 (EGR-1) (Fujino et al., 2003) and to the inhibition of the activity of PKA (Fujino et al., 2005). We now report that activation of the PI3K signaling pathway by the human EP₄ receptor involves the coupling of this receptor to a PTX-sensitive, cAMP-inhibitory G-protein (G_i). Coupling of the EP₄ receptor to G_i explains, in part, the less efficient coupling of the EP₄ receptor to the cAMP/PKA signaling pathway compared with the EP₂ receptor subtype.

	Materials and Methods

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Cell Culture. Cell lines stably expressing the EP₂ or EP₄ receptors were prepared using HEK-293–Epstein-Barr virus nuclear antigen cells and the mammalian expression vector pCEP₄ (Invitrogen, Carlsbad, CA) as described previously (Fujino et al., 2002). Cells were maintained in Dulbecco's modified Eagle's medium (Invitrogen) containing 10% fetal bovine serum, 250 µg/ml geneticin, 100 µg/ml gentamicin, and 200 µg/ml hygromycin B.

cAMP Assay. Cells were cultured in 12-well plates; 16 h before the immunoblotting experiments, cells were switched from their regular culture medium to Opti-MEM (Invitrogen) containing 250 µg/ml G-418 (Geneticin) and 100 µg/ml gentamicin. Cells were pretreated with either vehicle (water) or 100 ng/ml PTX (Calbiochem, San Diego, CA) for 16 h at 37°C. Cells were then treated with 0.1 mg/ml 3-isobutyl-1-methylxanthine (Sigma, St. Louis, MO) for 15 min followed by treatment with either vehicle (0.1% dimethyl sulfoxide), 1 µM PGE₂ (Cayman Chemical, Ann Arbor, MI), or 1 µM PGE₁-alcohol (PGE₁-OH; Cayman) for 10 min at 37°C. In experiments using forskolin, 3 µM forskolin (Calbiochem) was added for an additional 15 min after the initial treatments with PTX or drugs. Experiments were terminated by the removing the media and placing the cells on ice. Two hundred microliters of Tris/EDTA buffer (50 mM Tris-HCl and 4 mM EDTA, pH 7.5) was added, and the cells were scraped off and transferred to microcentrifuge tubes. The samples were boiled for 8 min, placed on ice, and centrifuged for 1 min at 14,000 rpm in a microcentrifuge. Five microliters of the supernatants (representing 5 x 10⁴ cells) was added to new tubes containing 50 µl of [³H]cAMP (PerkinElmer Life and Analytical Sciences, Boston, MA) and 100 µl of 0.06 mg/ml PKA (Sigma). The mixture was vortexed and incubated on ice for 2 h, followed by the addition of 100 µl of Tris/EDTA buffer containing 2% bovine serum albumin and 26 mg/ml powdered charcoal. After vortexing and centrifugation for 1 min at 14,000 rpm, 100-µl aliquots of the supernatants were removed, and radioactivity was measured by liquid scintillation counting. The amount of cAMP present was calculated from a standard curve prepared using nonradioactive cAMP and was expressed as picomoles per 5 x 10⁴ cells.

Western Blotting. Sixteen hours before the immunoblotting experiments, cells were switched from their regular culture medium to Opti-MEM (Invitrogen) containing 250 µg/ml G-418 and 100 µg/ml gentamicin. Cells were pretreated with either vehicle (water) or 100 ng/ml PTX for 16 h at 37°C. Cells were then treated with either vehicle (0.1% dimethyl sulfoxide), 1 µM PGE₂ for 10 min (phospho-ERKs), or 60 min (EGR-1) at 37°C. Cells were scraped into a lysis buffer (consisting of 150 mM NaCl, 50 mM Tris-HCl, pH 8.0, 5 mM EDTA, pH 8.0, 1% Nonidet P-40, 0.5% sodium deoxycholate, 10 mM sodium fluoride, 10 mM disodium pyrophosphate, 0.1% SDS, 0.1 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, 10 µg/ml leupeptin, and 10 µg/ml aprotinin) and transferred to microcentrifuge tubes. The samples were rotated for 30 min at 4°C and were centrifuged at 16,000g for 15 min. Aliquots of the supernatants containing 20100 µg of protein were electrophoresed on 10% SDS-polyacrylamide gels and transferred to nitrocellulose membranes as described previously (Fujino et al., 2003). Membranes were incubated in 5% nonfat milk for 1 h and were then washed and incubated for 16 h at 4°C with primary antibodies using the following conditions. For the ERKs, incubations were done in 3% nonfat milk containing either a 1:1000 dilution of antiphospho-ERK1/2 antibody (Cell Signaling Technology Inc., Beverly, MA) or mixture of a 1:500 dilution of anti-ERK1 antibody and a 1:10,000 dilution of anti-ERK2 antibody (Santa Cruz Biotechnology, Santa Cruz, CA). For EGR-1, incubations were done in 3% nonfat milk containing a 1:1000 dilution of anti-EGR-1 antibody (Santa Cruz Biotechnology). After incubating with the primary antibody, membranes were washed three times and incubated for 1 h at room temperature with a 1:10,000 dilution of the appropriate secondary antibodies conjugated with horseradish peroxidase using the same conditions as described above for each of the primary antibodies. After washing three times, immunoreactivity was detected by chemiluminescence as described previously (Fujino et al., 2003). To ensure equal loading of proteins, the membranes were stripped and re-probed with appropriate antibodies under the same conditions as described above.

	Results

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Pertussis Toxin Potentiates PGE₂-Stimulated cAMP Formation in HEK Cells Stably Expressing the Human EP₄ Prostanoid Receptor. We have reported previously that the maximal level of PGE₂-stimulated cAMP formation is significantly lower in HEK cells stably expressing the human EP₄ prostanoid receptor compared with HEK cells stably expressing the human EP₂ receptor, even though the levels of receptor expression were very similar (Fujino et al., 2002). We have also found that the EP₄ receptor can activate a PI3K/ERKs signaling pathway to induce the expression of EGR-1, whereas the EP₂ receptor subtype does not (Fujino et al., 2003). We had hypothesized previously that the EP₄ receptor was less efficiently coupled to G_s, but recently we considered the possibility that the EP₄ receptor might be additionally coupled to G_i, as has been shown for cardiac ₂-adrenergic receptors (Xiao et al., 1999a,b). To test this hypothesis we pretreated cells with PTX, which catalyzes the transfer of ADP-ribose from NAD to G_i and thereby blocks the ability of G_i to inhibit the activity of adenylyl cyclase (Ui, 1984). Thus, untransfected HEK cells and HEK cells stably expressing either the human EP₂ or EP₄ receptors were pretreated for 16 h with PTX and were then treated for 10 min with various concentrations of PGE₂. As shown in Fig. 1, there was no appreciable accumulation of cAMP in untransfected HEK cells with or without PTX pretreatment. In the absence of PTX pretreatment, the maximal stimulation of cAMP formation in HEK cells expressing the EP₂ receptor was approximately twice that obtained in HEK cells expressing the EP₄ receptor (36 pmol versus 19 pmol, respectively). Pretreatment with PTX resulted in a significant 33% increase in maximal PGE₂-stimulated cAMP formation in HEK cells expressing the EP₄ receptor, whereas, in EP₂-expressing cells, pretreatment with PTX essentially had no effect. The EC₅₀ for PGE₂ stimulation of cAMP formation was approximately 4-fold lower for EP₄-expressing cells compared with EP₂-expressing cells (0.4 nM versus 1.7 nM, respectively), and it was not affected by pretreatment with PTX. These data clearly support the hypothesis that the human EP₄ prostanoid receptor, but not the EP₂ receptor, can functionally couple to G_i in addition to coupling to G_s.

View larger version (16K): [in this window] [in a new window]   Fig. 1. The effects of PTX on PGE2-stimulated cAMP formation in untransfected HEK cells and in HEK cells transfected with human EP2 or EP4 prostanoid receptors. Cells were pretreated with vehicle () or 100 ng/ml PTX () for 16 h, followed by treatment with the indicated concentrations of PGE2 for 10 min. cAMP formation was determined as described under Materials and Methods. Data are the means ± S.E.M. of three independent experiments, each performed in duplicate. *, p < 0.01, analysis of variance, followed by Bonferroni post testing.

View larger version (14K): [in this window] [in a new window]   Fig. 2. The effects of PTX on forskolin-stimulated cAMP formation in the presence of the EP3/EP4-selective agonist PGE1-OH in untransfected HEK cells and in HEK cells transfected with either the human EP2 or EP4 prostanoid receptors. Cells were pretreated with either vehicle or 100 ng/ml PTX for 16 h, followed by treatment with 1 µM PGE1-OH for 10 min, followed by an additional incubation with 3 µM forskolin for 15 min. cAMP formation was determined as described under Materials and Methods. Data were normalized to the vehicle-treated controls for each group. Data are the means ± S.E.M. of three independent experiments each performed in duplicate. *, p < 0.05, t test.

Coupling of the Human EP₄ Prostanoid Receptor to G_i Mediates PGE₂-Stimulated ERK Phosphorylation and Induction of EGR-1 Expression. We have shown previously that PGE₂ stimulation of the human EP₄ receptor, but not the human EP₂ receptor, can induce the functional expression of EGR-1 through the activation of the PI3K and ERK signaling pathways (Fujino et al., 2003). It has also been reported that the ₂-adrenergic receptor can activate a PI3K signaling pathway by coupling through G_i (Jo et al., 2002). We therefore decided to examine whether the PGE₂-mediated activation of PI3K/ERKs signaling and induction of EGR-1 expression occurs through a mechanism involving coupling of the EP₄ receptor to G_i. For these experiments, cells were either untreated or pretreated with PTX for 16 h and were then incubated with either vehicle or 1 µM PGE₂. The expression of the phospho-ERKs, total ERKs, and EGR-1 were then examined by immunoblot analysis. Figure 3A, top, shows that in the absence of PTX pretreatment, PGE₂-stimulated ERK phosphorylation in EP₄-expressing cells, but not in EP₂-expressing cells, and that pretreatment with PTX completely abolished this effect. Likewise, Fig. 3B, top, shows that in the absence of PTX pretreatment, PGE₂ stimulated the expression of EGR-1 in EP₄-expressing cells, but not in EP₂-expressing cells, and that pretreatment with PTX also blocked this action. In addition, Fig. 3, A and B, bottom, show that nearly identical amounts of ERKs 1 and 2 were present under all conditions and in both cell lines. These data support the conclusion that the activation of ERK signaling and induction of EGR-1 by PGE₂ is mediated by coupling of the human EP₄ prostanoid receptor to a PTX-sensitive G-protein.

View larger version (57K): [in this window] [in a new window]   Fig. 3. The effects of PTX on PGE2-stimulated phosphorylation of the extracellular signal-regulated kinases (pERK 1/2) (A) and on the expression of early growth response factor-1 (EGR-1) (B) in HEK cells transfected with either the human EP2 or EP4 prostanoid receptors. Cells were pretreated with either vehicle or 100 ng/ml PTX for 16 h, followed by treatment with either vehicle (v) or 1 µM PGE2 for 10 min (A) or for 60 min (B). Cells were then subjected to immunoblot analysis as described under Materials and Methods. A, top, immunoblotting with antibodies against phospho-ERKs 1 and 2 (pERK1/2). A, bottom, the blots shown in A, top, were stripped and re-probed with antibodies against ERKs 1 and 2 (ERK1/2). B, top, immunoblotting with antibodies against EGR-1 (EGR1). B, bottom, the blots shown in B, top, were stripped and re-probed with antibodies against ERKs 1 and 2 (ERK1/2). Results are representative of three independent experiments with each antibody and condition.

	Discussion

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The regulation of intracellular cAMP by E-type prostaglandins has been known for nearly forty years (Butcher and Baird, 1968). Thus, PGE₁ was found to lower intracellular cAMP in isolated fat pads but to increase it in several other cell types. Direct evidence for the existence of specific receptors for the E-type prostaglandins was initially obtained in radioligand binding studies with [³H]PGE₁ (Kuehl and Humes, 1972), which were also used to show that the binding of [³H]PGE₁ could be modulated by guanine nucleotides (Moore and Wolff, 1973). This was among the first evidence that E-type prostaglandin receptors, together with the glucagon and catecholamine receptors, interacted with G-proteins and that this interaction might constitute a general mechanism for signaling between cell surface receptors and adenylyl cyclase (Rodbell, 1980). Extensive physiological, pharmacological and molecular biological studies later defined the receptors for the E-type prostaglandins as EP receptors and classified them into the EP₁, EP₂, EP₃, and EP₄ subtypes (Coleman et al., 1994; Regan, 2003; Hata and Breyer, 2004). As reviewed in the Introduction, the EP₁ and EP₃ receptors have been generally regarded as coupling to G_q and G_i, respectively, whereas the EP₂ and EP₄ receptors have been considered to be exclusively coupled to G_s. The present findings now show for the first time that in addition to coupling to G_s, EP₄ receptors can also couple to a PTX-sensitive G-protein to inhibit intracellular cAMP formation and activate PI3K and ERKs signaling cascades. Furthermore, the inhibition of cAMP formation by the EP₄ receptor suggests specific coupling to G_i.

We have reported previously that PGE₂ stimulation of human EP₂ and EP₄ receptors can activate Tcf/Lef signaling but that EP₂ receptors do this primarily through a cAMP/PKA pathway, whereas EP₄ receptors mainly use a PI3K pathway (Fujino et al., 2002). We have also reported that PGE₂ stimulation of human EP₄ receptors, but not EP₂ receptors, results in the functional expression of EGR-1 through the activation of PI3K and MAP kinase signaling (Fujino et al., 2003). As for the present study, these previous studies were conducted exclusively with a recombinant cell system consisting of HEK cells stably transfected with either the human EP₂ or EP₄ receptors. There is increasing evidence, however, that such observations will eventually be extended to endogenous EP₂ and EP₄ receptors in native cell systems. For example, Sheng et al. (2001) reported that PGE₂ stimulation of endogenous EP₄ receptors in human colorectal cancer cells increased cell growth and motility through the activation of PI3K and Akt. Likewise, Pozzi et al. (2004) found that PGE₂ stimulation of endogenous EP₄ receptors in mouse colon adenocarcinoma cells increased cellular proliferation by a mechanism that was independent of any measurable effect on cAMP and that involved the activation of the Akt and MAP kinases. Reno and Cannas (2005) have reported that PGE₂ stimulation of endogenous EP₂ or EP₄ receptors in human myeloid leukemia cells increased PMA-induced macrophage differentiation by a mechanism that was independent of the activation of a cAMP/PKA pathway and that involved the activation of PI3K and MAP kinase signaling. Similar findings were also obtained by Caristi et al. (2005), who found that endogenous EP₄ receptors in human T lymphocytes mediate interleukin-8 gene transcription by a mechanism that is PKA-independent and involves the activation of PI3K signaling. Thus, there are endogenous EP₄ receptors in native cell systems that can activate PI3K signaling by mechanisms that seem to be independent of coupling to G_s.

It is well established that GPCRs can activate PI3K and Akt signaling through the interaction of G subunits with either the p110 or p110 subunits of PI3K (Yart et al., 2002). In most cases in which it has been examined, the activation of PI3K and Akt signaling involves G_I-coupled receptors (Kim et al., 2004). Given the present findings, it is likely that the PTX-sensitive activation of PI3K and ERK signaling by the EP₄ receptor reflects specific coupling to G_i as opposed to G_o.

In many ways, the classification of the EP receptor subtypes and their pattern of G-protein coupling bears similarities to the adrenergic receptor subtypes. For example, the ₁- and ₂-adrenergic receptors are generally regarded as coupling to G_q and G_i, respectively, whereas the -adrenergic receptor subtypes were long considered to be exclusively coupled to G_s. It has become apparent, however, that the ₂-adrenergic receptor has additional coupling to G_i, which results, as in the EP₄ receptor, in the inhibition cAMP formation and activation of PI3K and ERK signaling cascades (Daaka et al., 1997; Chesley et al., 2000). This is of particular functional significance for the cardiac -receptors because it profoundly alters the consequences of persistent activation of these receptors. Thus, transgenic overexpression of ₁-adrenergic receptors in mice leads to cardiac hypertrophy, heart failure, and early death, whereas, overexpression of the ₂-adrenergic receptor actually improves cardiac function and does not adversely affect life span (Xiao et al., 1999b). Although cardiac ₂-adrenergic receptors can couple to G_s, it has been found that the protective effects of ₂-adrenergic receptor over expression depend upon coupling to G_i2 and G_i3 (Foerster et al., 2003). At present, the physiological and pathophysiological consequences of the unique signaling properties of the EP₄ receptor are unknown. However, like the ₁- and ₂-adrenergic receptor subtypes, the EP₂ and EP₄ prostanoid receptor subtypes are frequently coexpressed in the same tissues, and it is likely that there is a functional basis for this coexpression.

One possibility as it concerns the coexpression of the EP₂ and EP₄ receptor subtypes might be related to a cell or tissue's ability to respond to different concentrations of endogenous PGE₂. It has been clearly established that the binding affinity of PGE₂ is 10- to 20-fold higher for the EP₄ receptor compared with the EP₂ receptor (Kiriyama et al., 1997; Abramovitz et al., 2000; Fujino et al., 2002). Furthermore, this difference in affinity is reflected in functional measures of the activation of these receptors. For example, in one detailed study of the functional pharmacology of the human EP₂ and EP₄ receptor subtypes, the EC₅₀ for the stimulation of cAMP formation in cells expressing the EP₄ receptor was 0.05 nM, whereas for the EP₂ receptor, it was 30 nM (Wilson et al., 2004). Thus, cells expressing the EP₄ receptor are able to respond to lower concentrations of endogenous PGE₂. In addition, the pattern of intracellular signaling in cells expressing the EP₄ receptor will include the activation of both the G_s and G_i pathways.

The activation of a G_i signaling pathway by the EP₄ receptor provides an interesting potential mechanism for further amplification of the initial PGE₂ signal. As demonstrated in the present study, the EP₄ receptor-mediated activation of G_i signaling leads to the activation of the ERKs and induction of EGR-1 expression. It has been shown that EGR-1 can induce the expression of PGE₂ synthase (Naraba et al., 2002), which could be expected to increase the biosynthesis of PGE₂, perhaps to a level that would initiate the activation of EP₂ (and EP₁) receptors. This amplification of PGE₂ signaling would take place only in tissues or cells that express the EP₄ receptor subtype and would represent a mechanism for generating a differential response to low levels of endogenous PGE₂. PGE₂ is produced at low levels by a large number of cell types, and under various physiological and pathophysiological conditions, its biosynthesis is dramatically increased. This increase in PGE₂ biosynthesis is frequently correlated with the induction of COX-2, but the conditions and factors that regulate these events are unclear. Invasion of tissues by macrophages and up-regulation of their EP₄ receptors, which has been shown to occur in a mouse model of autoimmune inflammation (Akaogi et al., 2004), or up-regulation of EP₄ receptors by resident dendritic cells (Harizi et al., 2003), could provide a potential mechanism for inducing COX-2 and PGE₂ synthase expression and increasing the biosynthesis of PGE₂.

The present study further emphasizes the differences in the signaling potential of the EP₂ and EP₄ receptors and clarifies the mechanism of the activation of the PI3K and ERK signaling pathways by the EP₄ receptor. Thus far, human EP₂ prostanoid receptors seem to be exclusively coupled to G_s, and stimulation of these receptors by PGE₂ leads to a strong activation of the cAMP/PKA signaling pathway. On the other hand, PGE₂ stimulation of human EP₄ prostanoid receptors results in the activation of both G_s and G_i. Compared with the EP₂ receptor, the activation of the cAMP/PKA signaling pathway by the EP₄ receptor is significantly less, which is a consequence of two mechanisms. The first is that activation of G_i probably results in a direct inhibition of adenylyl cyclase, which offsets the stimulation of adenylyl cyclase through G_s. The second is that PGE₂-mediated activation of PI3K signaling by the EP₄ receptor inhibits the activity of PKA (Fujino et al., 2005). A similar inhibition of PKA activity has been reported after the activation of PI3K signaling by the ₂-adrenergic receptor (Jo et al., 2002). It is significant to note that even in the presence of PTX, the maximal cAMP response elicited by PGE₂ stimulation of the EP₄ receptor was less than that obtained with the EP₂ receptor (Fig. 1). This indicates that the efficiency of EP₄ receptor coupling to G_s-mediated signaling is less than that of the EP₂ receptor even in the absence of the activation of G_i mediated signaling.

The G_i-mediated activation of PI3K signaling further differentiates the signaling properties of the EP₄ receptor compared with the EP₂ receptor. Thus, we have shown previously that PGE₂ stimulation of the EP₄ receptor leads to the PI3K-dependent activation of ERK signaling pathways, which is not observed after PGE₂ stimulation of the EP₂ receptor (Fujino et al., 2003). Despite these differences, some of the downstream signaling consequences after PGE₂ stimulation of the EP₂ or EP₄ receptors seem to be quite similar. For example, PGE₂ stimulation of either receptor leads to an increase Tcf transcriptional activation (Fujino et al., 2002) and in the phosphorylation of the cAMP response element binding protein (Fujino et al., 2005). However, the increase in Tcf transcription activation and cAMP response element binding protein phosphorylation by the EP₄ receptor is mainly through a PI3K-dependent mechanism, whereas for the EP₂ receptor, it is mainly through a cAMP/PKA-dependent pathway. This means that the regulation of EP₂ and EP₄ receptor signaling by cross-talk through the activation of other of types of receptors has the potential to be quite different. For example, receptors whose activation can modulate PI3K signaling will have greater potential to influence signaling mediated by the EP₄ receptor as opposed to that mediated by the EP₂ receptor.

In summary, we have shown that human EP₄ receptors, but not EP₂ receptors, can couple to PTX-sensitive G-proteins when expressed heterologously in HEK cells. Coupling of EP₄ receptors to PTX-sensitive G-proteins decreases PGE₂-mediated cAMP accumulation, suggesting specific coupling to G_i rather than G_o. The activation of PI3K signaling by the EP₄ receptor probably occurs through the release of G subunits after coupling of the receptor to G_i. We have discussed studies showing that PGE₂ stimulation of endogenous EP₄ receptors in native cell systems can activate PI3K and ERK signaling by mechanisms that seem to be independent of coupling to G_s. These findings suggest the coupling of endogenous EP₄ receptors to G_i, but clearly this will need to be further investigated. In fact, we do not believe that EP₄ receptors will be shown to have universal coupling to G_i and PI3K/ERK signaling. For example, in an elegant study of prostanoid receptor-mediated signaling in human airway smooth muscle cells, Clarke et al. (2005) found that the effects of EP₄ receptor stimulation could be explained solely by activation of a cAMP/PKA-dependent pathway. We speculate that the specific signaling pathways used by more "promiscuous" GPCRs, such as EP₄ and ₂-adrenergic receptors, will be very cell-type–dependent compared with more dedicated "monogamous" receptors, such as EP₂ and ₁-adrenergic receptors.

	Footnotes

 
Support for this work was provided by National Institutes of Health grant EY11291 and by Allergan Inc.

Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.

doi:10.1124/mol.105.017749.

ABBREVIATIONS: PG, prostaglandin; COX, cyclooxygenase; PTX, pertussis toxin; Tcf, T-cell factor; HEK, human embryonic kidney; PI3K, phosphatidylinositol 3-kinase; PKA, protein kinase A; EGR-1, early growth response factor-1; PGE₁-OH, PGE₁-alcohol; ERK, extracellular signal-regulated kinase.

Address correspondence to: John W. Regan, Department of Pharmacology and Toxicology, College of Pharmacy, The University of Arizona, Tucson, AZ 85721-0207. E-mail: regan{at}pharmacy.arizona.edu

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