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• W2073602994 abstract "FP prostanoid receptors have been identified as two isoforms named FPA and FPB. We have shown that the FPB isoform, but not the FPA, activates β-catenin-mediated transcription. We now report that the mechanism of this FPB-specific activation of β-catenin signaling occurs in two steps. The first is a conditioning step that involves an agonist-independent association of the FPBreceptor with phosphatidylinositol 3-kinase followed by constitutive internalization of a receptor complex containing E-cadherin and β-catenin. This constitutive internalization conditions the cell for subsequent β-catenin signaling by increasing the cellular content of cytosolic β-catenin. The second step involves agonist-dependent activation of Rho followed by cell rounding. Because of the conditioning step, this agonist-dependent step results in a stabilization of β-catenin and activation of transcription. Although stimulation of the FPA isoform activates Rho and induces cellular shape change, it does not activate β-catenin signaling, because the FPA does not undergo constitutive internalization and does not condition the cell for β-catenin signaling. The cellular conditioning described here for the FPB illustrates the potential of the receptor to alter the signaling environment of a cell even in the absence of agonist and has general significance for understanding G-protein-coupled receptor signaling. FP prostanoid receptors have been identified as two isoforms named FPA and FPB. We have shown that the FPB isoform, but not the FPA, activates β-catenin-mediated transcription. We now report that the mechanism of this FPB-specific activation of β-catenin signaling occurs in two steps. The first is a conditioning step that involves an agonist-independent association of the FPBreceptor with phosphatidylinositol 3-kinase followed by constitutive internalization of a receptor complex containing E-cadherin and β-catenin. This constitutive internalization conditions the cell for subsequent β-catenin signaling by increasing the cellular content of cytosolic β-catenin. The second step involves agonist-dependent activation of Rho followed by cell rounding. Because of the conditioning step, this agonist-dependent step results in a stabilization of β-catenin and activation of transcription. Although stimulation of the FPA isoform activates Rho and induces cellular shape change, it does not activate β-catenin signaling, because the FPA does not undergo constitutive internalization and does not condition the cell for β-catenin signaling. The cellular conditioning described here for the FPB illustrates the potential of the receptor to alter the signaling environment of a cell even in the absence of agonist and has general significance for understanding G-protein-coupled receptor signaling. Prostaglandin F2α(PGF2α) 1The abbreviations used are: PGF2α, prostaglandin F2α; Tcf, T-cell factor; GPCR, G-protein-coupled receptor; PI3K, phosphatidylinositol 3-kinase; GSK-3, glycogen synthase kinase-3; IP, immunoprecipitation; MES, 4-morpholineethanesulfonic acid; DAPI, 4′,6-diamidino-2-phenylindole. is an important autacoid that regulates a variety of physiological processes such as inflammation, cardiac hypertrophy, intraocular pressure, and regression of corpus luteum. PGF2α is synthesized from arachidonic acid by the cyclooxygenases and binds to FP prostanoid receptors to initiate its signaling cascade. FP receptors are in the family of prostanoid receptors, which in turn are in the superfamily of G-protein-coupled receptors (GPCRs). Other prostanoid receptors include the EP, DP, IP, and TP receptors, which mediate, respectively, the actions of prostaglandin E2, prostaglandin D2, prostacyclin, and thromboxane. The prostanoid receptor family also includes additional EP receptor subtypes (EP1, EP2, EP3, and EP4) and alternative mRNA splice variants of the EP1, EP3, TP, and FP receptors. The FP receptor splice variants are designated FPA and FPB and are identical except for their intracellular carboxyl-terminal domains. Thus, the FPB isoform is basically a truncated version of the FPA isoform, which lacks the last 46 carboxyl-terminal amino acids. Both receptor isoforms are coupled to Gq and can activate phosphatidylinositol signaling followed by the activation of protein kinase C. In addition both isoforms activate Rho, a member of the Ras family of small GTPases. The activation of Rho by FPA and FPB receptor isoforms leads to tyrosine phosphorylation of p125 focal adhesion kinase and the induction of cellular shape change involving the retraction of filopodia, cell rounding, and aggregation (1Pierce K.L. Fujino H. Srinivasan D. Regan J.W. J. Biol. Chem. 1999; 274: 35944-35949Google Scholar). Unexpectedly the reversal of this Rho-mediated shape change was found to differ markedly between the two isoforms following the removal of agonist (2Fujino H. Pierce K.L. Srinivasan D. Protzman C.E. Krauss A.H. Woodward D.F. Regan J.W. J. Biol. Chem. 2000; 275: 29907-29914Google Scholar). Thus, 1 h after the removal of PGF2α, FPA-expressing cells return to their original cellular morphology, whereas FPB-expressing cells remain rounded and still show evidence of Rho-mediated signaling, including the presence of actin stress fibers and tyrosine phosphorylation of p125 focal adhesion kinase. Even 16 h after the removal of PGF2α, FPB-expressing cells are still rounded. In addition we have found that stimulation of FPB-expressing cells with PGF2α produces a marked activation of Tcf/β-catenin-mediated transcriptional activation, which is not observed in FPA-expressing cells (3Fujino H. Regan J.W. J. Biol. Chem. 2001; 276: 12489-12492Google Scholar). We have recently documented another fundamental difference between these FP receptor isoforms (4Srinivasan D. Fujino H. Regan J.W. J. Pharmacol. Exp. Ther. 2002; 302: 219-224Google Scholar). We have found that the FPAisoform undergoes a classic agonist-induced and clathrin-dependent internalization, whereas the FPB isoform undergoes an agonist-independent constitutive internalization that does not involve clathrin. We now report a molecular mechanism that we believe links the constitutive agonist-independent internalization of the FPB isoform with the selective activation of Tcf/β-catenin signaling by the FPB isoform. A key observation is that agonist-induced Tcf/β-catenin transcriptional activation by the FPBisoform was blocked by inhibition of Rho-mediated cellular shape change. Furthermore, we have found an association of the FPB isoform, but not the FPA isoform, with phosphatidylinositol 3-kinase (PI3K), which may explain its agonist-independent constitutive internalization. We hypothesize that constitutive internalization of the FPB isoform involving PI3K conditions FPB-expressing cells to subsequent agonist-induced Tcf/β-catenin signaling by increasing the cellular content of cytosolic β-catenin. HEK-293 cells stably expressing FPA and FPB prostanoid receptor isoforms (1Pierce K.L. Fujino H. Srinivasan D. Regan J.W. J. Biol. Chem. 1999; 274: 35944-35949Google Scholar), as well as FLAG-tagged FPA- and FPB-expressing cell lines (5Fujino H. Srinivasan D. Pierce K.L. Regan J.W. Mol. Pharmacol. 2000; 57: 353-358Google Scholar), were generated and cultured as described previously. Cells were pretreated with either vehicle (0.1% Me2SO or water), or 100 nm wortmannin (Sigma) for 15 min, or 40 μg/ml C3-toxin for 48 h at 37 °C. Then cells were incubated at 37 °C with either vehicle (sodium carbonate, 0.002% final) or 1 μm PGF2α(Cayman Chemical) for the times indicated in the figures. Cells were scraped and sonicated in a lysis buffer as described previously (3Fujino H. Regan J.W. J. Biol. Chem. 2001; 276: 12489-12492Google Scholar). Samples were centrifuged, the supernatant (cytosolic fraction) was removed, and the pellet (particulate fraction) was solubilized with lysis buffer containing 0.2% Triton X-100 (Bio-Rad) and then centrifuged again to remove insoluble debris. For immunoprecipitation (IP), samples (200–300 μg of protein) were rotated at 4 °C with anti-FLAG M2 affinity gel (Sigma) for 2 h, or with anti-E-cadherin antibody (BD Transduction Laboratories) for 16 h, or with anti-β-catenin antibody (BD Transduction Laboratories) for 2 h, all at a dilution of 1:100. For the E-cadherin and β-catenin IPs, 10 μl of a 1:1 slurry of protein G-Sepharose (Amersham Biosciences) was added and rotated for another hour. Samples were electrophoresed, transferred to nitrocellulose membranes, and incubated in 3% nonfat milk with 1:1,000 dilutions of either anti-PI3K p85 antibody (Upstate Biotechnology Inc.) for 2 h or a mixture of mouse monoclonal antibodies to phospho-serine (Sigma) and phospho-threonine (Sigma) for 16 h (3Fujino H. Regan J.W. J. Biol. Chem. 2001; 276: 12489-12492Google Scholar). The membranes were washed and incubated for 1 h at room temperature in 3% nonfat milk containing 1:10,000 dilutions of horseradish peroxidase-conjugated goat anti-rabbit (Sigma) or anti-mouse antibodies (Sigma). The immunoblots were then visualized by enhanced chemiluminescence (SuperSignal, Pierce). To verify amounts of immunoprecipitated proteins, the membranes were stripped and reprobed with 1:10,000 dilutions of either anti-FLAG M2 antibody (Sigma), anti-E-cadherin, or anti-β-catenin antibody for 16 h in 3% nonfat milk under the same conditions as described above. For the immunoprecipitations of phospho-GSK-3β, cells were scraped into a lysis buffer as described previously (6Fujino H. West K.A. Regan J.W. J. Biol. Chem. 2002; 277: 2614-2619Google Scholar), and 100 μg of protein was electrophoresed then transferred to nitrocellulose membranes. Membranes were incubated in 5% nonfat milk for 1 h and were washed and incubated for 16 h at 4 °C in 0.5% nonfat milk containing either anti-phospho-GSK-3β antibody (#9336, Cell Signaling) or anti-GSK-3β antibody (G22320, Transduction Laboratories) as previously described (6Fujino H. West K.A. Regan J.W. J. Biol. Chem. 2002; 277: 2614-2619Google Scholar). All antibodies were used at a dilution of 1:1,000. Membranes were washed three times and incubated for 1 h at room temperature in 0.5% nonfat milk for GSK-3β antibodies and then with a 1:10,000 dilution of the corresponding secondary antibodies conjugated with horseradish peroxidase. To ensure equal loading of proteins, the membranes were stripped and reprobed with anti-GSK-3β antibodies under the same conditions as described above. The resulting films were scanned, and quantitation was performed as described previously (3Fujino H. Regan J.W. J. Biol. Chem. 2001; 276: 12489-12492Google Scholar). Cells were pretreated with either vehicle (0.1% Me2SO) or 100 nmwortmannin for 15 min at 37 °C followed by stimulation with either vehicle (0.002% sodium carbonate) or 1 μmPGF2α for 1 h. Cells were then scraped in a lysis buffer consisting of 20 mm Tris-HCl (pH 7.5), 137 mm NaCl, 1 mm MgCl2, 1 mm CaCl2, 10% (v/v) glycerol, 1% Nonidet P-40, 1 mm sodium orthovanadate, and 1 mmphenylmethylsulfonyl fluoride and rotated for 20 min at 4 °C. For immunoprecipitation, samples were rotated with 500 μg of protein with anti-PI3K p85 for 2 h followed by the addition of 10 μl of 1:1 slurry of protein A-Sepharose (Sigma) for an additional hour. Samples were then washed twice with lysis buffer and twice with wash buffer consisting of 10 mm Tris-HCl (pH 7.5), 100 mmNaCl, and 1 mm EDTA followed by incubation with 0.1 mg/mll-α-phosphatidylinositol (Avanti polar lipids) in buffer containing 880 μm cold ATP with 30 μCi of [γ-32P]ATP (Amersham Biosciences), and 20 mm MgCl2 for 10 min at 30 °C in a final volume of 80 μl. To terminate the incubation, 20 μl of 8m HCl was added to the samples, and then the lipids were extracted with 160 μl of chloroform/methanol (1:1) and centrifuged (300 × g) for 15 min at 4 °C. The lower organic phases were removed and applied to an LK6D silica gel 60-Å TLC plate (Whatman). TLC plates were developed in chloroform/methanol/water/ammonium hydroxide (30:23.5:5.65:1, v/v) followed by drying and visualization by autoradiography using Hyperfilm MP (Amersham Biosciences). Cells were pretreated with either vehicle (0.1% Me2SO) or 100 nmwortmannin for 15 min at 37 °C followed by stimulation with either vehicle (0.002% sodium carbonate) or 1 μmPGF2α for 1 h. They were then trypsinized, centrifuged at 500 × g for 2 min, and resuspended at a concentration of 107 cells/ml in ice-cold MES buffer (2Fujino H. Pierce K.L. Srinivasan D. Protzman C.E. Krauss A.H. Woodward D.F. Regan J.W. J. Biol. Chem. 2000; 275: 29907-29914Google Scholar). [3H]PGF2α binding was performed using 2.5 nm [3H]PGF2α (AmershamBiosciences) as previously described (2Fujino H. Pierce K.L. Srinivasan D. Protzman C.E. Krauss A.H. Woodward D.F. Regan J.W. J. Biol. Chem. 2000; 275: 29907-29914Google Scholar). Samples were incubated for 1 h at room temperature, and the assays were terminated by filtration through Whatman GF/C glass filters using a cell harvester (M-24R, Brandel) (2Fujino H. Pierce K.L. Srinivasan D. Protzman C.E. Krauss A.H. Woodward D.F. Regan J.W. J. Biol. Chem. 2000; 275: 29907-29914Google Scholar). Cells were split and grown in six-well plates containing 22-mm round glass coverslips for 3–4 days. To evaluate agonist-dependent internalization, the cells were pretreated with either vehicle (0.1% Me2SO) or 100 nm wortmannin for 15 min at 37 °C followed by the addition of a 1:500 dilution of anti-FLAG M2 antibodies in vehicle (0.002% sodium carbonate) or a 1:500 dilution of anti-FLAG M2 antibodies in 1 μm PGF2α for 10 min at 37 °C. The cells were fixed, permeabilized, and labeled with fluorescein isothiocyanate-conjugated anti-mouse IgG secondary antibodies as previously described (4Srinivasan D. Fujino H. Regan J.W. J. Pharmacol. Exp. Ther. 2002; 302: 219-224Google Scholar). The cells were then examined by scanning confocal microscopy as described previously (4Srinivasan D. Fujino H. Regan J.W. J. Pharmacol. Exp. Ther. 2002; 302: 219-224Google Scholar). Cells were split and grown in six-well plates containing 22-mm round glass cover slips for 3–4 days. Cells were pretreated with either vehicle (0.1% Me2SO or water) or inhibitors, 100 nmwortmannin for 15 min, or 40 μg/ml C3-toxin for 48 h at 37 °C. Cells were then incubated at 37 °C with either vehicle (0.002% sodium carbonate) or 1 μm PGF2αfor 1 h and were rapidly washed and fixed with methanol/acetone (7:3, v/v). The fixed cells were incubated with a 1:1000 dilution of antibody to β-catenin in 3% bovine serum albumin and were then washed and incubated with a 1:10,000 dilution of fluorescein isothiocyanate-conjugated goat anti-mouse secondary antibody in 0.1% nonfat milk. Nuclei were stained with 0.05 nm/ml 4′,6-diamidino-2-phenylindole (DAPI, Sigma). Cells were visualized by phase-contrast microscopy, as described previously (3Fujino H. Regan J.W. J. Biol. Chem. 2001; 276: 12489-12492Google Scholar). Cells, grown in six-well plates, were transiently transfected using FuGENE 6 (Roche Molecular Biochemicals) with 1 μg/well of either the TOP flash or FOP flash reporter plasmids (Upstate Biotechnology Inc.) as described previously (3Fujino H. Regan J.W. J. Biol. Chem. 2001; 276: 12489-12492Google Scholar). Cells were pretreated with either vehicle (0.1% Me2SO or water), 100 nm wortmannin for 15 min, or 40 μg/ml C3-toxin for 48 h at 37 °C. The cells were then incubated at 37 °C with either vehicle (0.002% sodium carbonate) or 1 μm PGF2α for 1 h and were rapidly washed three times each with 1 ml/well Opti-MEM and then incubated for 16 h at 37 °C in 2 ml of Opti-MEM containing 250 μg/ml Geneticin, 100 μg/ml gentamicin. Cell extracts were prepared using the Luciferase assay system (Promega). Luciferase activity was measured using a Turner TD-20/20 luminometer as described previously (3Fujino H. Regan J.W. J. Biol. Chem. 2001; 276: 12489-12492Google Scholar) using 10 μg of protein per sample. Measurements were corrected for background activity by subtraction of the FOP flash values from the corresponding TOP flash values. Cells were cultured in 6-cm plates in Dulbecco's modified Eagle's medium with 10% fetal bovine serum and pretreated either with vehicle (water) or 40 μg/ml C3-toxin for 48 h at 37 °C followed by preincubation with 3 μCi/ml myo-[2-3H]inositol (AmershamBiosciences) for 16 h. Cells were then incubated at 37 °C with either vehicle (0.002% sodium carbonate) or 1 μmPGF2α for 1 h at 37 °C in culture media containing 10 mm LiCl. Assays were terminated by the addition of 1 ml of methanol, and cells were scraped and added to 1.5 ml of chloroform/water (1:0.5, v/v). Total [3H]inositol phosphates were then determined by anion-exchange chromatography as described previously (5Fujino H. Srinivasan D. Pierce K.L. Regan J.W. Mol. Pharmacol. 2000; 57: 353-358Google Scholar). We have previously shown that PGF2α can activate Tcf/β-catenin signaling in FPB-expressing cells but not in FPA-expressing cells (3Fujino H. Regan J.W. J. Biol. Chem. 2001; 276: 12489-12492Google Scholar). This selective activation of Tcf/β-catenin signaling by the FPB isoform was associated with an agonist-mediated increase in cytoplasmic β-catenin and a decrease in its phosphorylation status. Decreased phosphorylation stabilizes β-catenin levels by preventing its degradation and thereby promotes nuclear translocation and Tcf transcriptional activation. However, the molecular mechanisms leading to the decrease in β-catenin phosphorylation following stimulation of the FPB receptor were unclear. One possible mechanism leading to decreased phosphorylation of β-catenin would involve the sequential activation of PI3K and Akt resulting in the phosphorylation of glycogen synthase kinase-3 (GSK-3). Phosphorylation of GSK-3 inhibits its kinase activity and decreases β-catenin phosphorylation. The potential involvement of PI3K with FPB-mediated signaling was, therefore, explored in HEK cells stably expressing FLAG-tagged FPA and FPBreceptors that were treated with 1 μm PGF2αand then examined by a combination of immunoblotting with antibodies to p85 PI3K (Fig. 1, A andB) and measurement of PI3K activity (Fig. 1C). FLAG-tagged FP-expressing cell lines were selected as reported previously (5Fujino H. Srinivasan D. Pierce K.L. Regan J.W. Mol. Pharmacol. 2000; 57: 353-358Google Scholar) that had comparable levels of expression based on agonist-stimulated IP formation and on the radioligand binding (e.g. FPA, 3.56 ± 0.04 pmol/mg of protein; FPB, 2.15 ± 0.17 pmol/mg of protein). For the immunoblotting experiments the particulate fractions of cell lysates were first immunoprecipitated with monoclonal antibodies (M2) to the FLAG epitope. As shown in the upper panels of Fig. 1A, the p85 subunit of PI3K co-immunoprecipitated with the FPB receptor isoform but not with the FPAisoform. Interestingly the association of PI3K with the FPBisoform was disrupted by treatment with PGF2α indicating a preferential interaction with the unstimulated receptor. Thelower panels of Fig. 1A show that similar amounts of the FLAG-tagged FPA and FPB receptors were expressed in these cells and could be immunoprecipitated with the anti-FLAG M2 antibodies. We then used wortmannin, an inhibitor of PI3K, to see if the association of the p85 subunit with the FPB receptor was influenced by its kinase activity. As shown in Fig. 1B, pretreatment of FPB-expressing cells with 100 nm wortmannin for 15 min significantly decreased the co-immunoprecipitation of the p85 subunit with the FPBreceptor in the absence of PGF2α treatment. As in Fig. 1A, treatment with 1 μm PGF2αfor 1 h decreased the association of PI3K with the FPBreceptor both under control conditions and following pretreatment with wortmannin. PI3K activity was then determined under the same conditions as those used for the experiments in Fig. 1B, and the results are shown in Fig. 1C. PI3K activity was greater in FPB-expressing cells as compared with FPA-expressing cells and was decreased ∼50% in FPB-expressing cells after treatment with 1 μm PGF2α for 1 h. As expected, pretreatment with wortmannin decreased the PI3K activity in both vehicle-treated FPB cells and in cells treated with 1 μm PGF2α for 1 h. In summary, it appears that PI3K activity is required for an interaction with the FPB receptor and may, in fact, be induced by this interaction. This is supported by the observation that treatment of FPB-expressing cells with PGF2α decreases PI3K activity while at the same time decreasing its interaction with the receptor. It has been reported that the endocytosis of E-cadherin is regulated by Rac1 and that this may involve the activation of PI3K (7Nakagawa M. Fukata M. Yamaga M. Itoh N. Kaibuchi K. J. Cell Sci. 2001; 114: 1829-1838Google Scholar, 8Kovacs E.M. Ali R.G. McCormack A.J. Yap A.S. J. Biol. Chem. 2002; 277: 6708-6718Google Scholar). Furthermore, it appears that the endocytosis of E-cadherin involves a clathrin-independent mechanism (9Akhtar N. Hotchin N.A. Mol. Biol. Cell. 2001; 12: 847-862Google Scholar). Recently we have found that the FPB receptor isoform undergoes an agonist-independent constitutive internalization that is also clathrin-independent, whereas, the FPA isoform undergoes agonist-induced internalization that is clathrin-dependent (4Srinivasan D. Fujino H. Regan J.W. J. Pharmacol. Exp. Ther. 2002; 302: 219-224Google Scholar). Given the association of PI3K with the FPB isoform (shown in Fig. 1) we were interested in the potential role of PI3K with respect to the agonist-independent constitutive internalization of the FPB isoform. For these experiments HEK cells stably expressing the FPA and FPB receptor isoforms were pretreated with either vehicle or 100 nm wortmannin followed by treatment with either vehicle or 1 μm PGF2α. Cell lines expressing wild type FP receptors were selected as reported previously (5Fujino H. Srinivasan D. Pierce K.L. Regan J.W. Mol. Pharmacol. 2000; 57: 353-358Google Scholar) that had comparable levels of expression based on agonist-stimulated IP formation and on the radioligand binding (e.g. FPA, 3.55 ± 0.28 pmol/mg of protein; FPB, 4.09 ± 0.49 pmol/mg of protein). Receptor desensitization and internalization were then assessed, respectively, by the whole cell binding of [3H]PGF2α(Fig. 2A) and by immunofluorescence confocal microscopy with anti-FLAG M2 antibodies (Fig. 2B). As shown in Fig. 2A, treatment with PGF2αresulted in a 50% decrease in [3H]PGF2αbinding in both FPA- and FPB-expressing cells (2Fujino H. Pierce K.L. Srinivasan D. Protzman C.E. Krauss A.H. Woodward D.F. Regan J.W. J. Biol. Chem. 2000; 275: 29907-29914Google Scholar). Interestingly pretreatment with wortmannin, alone, increased [3H]PGF2α binding in FPB cells, but not in FPA cells. This sensitization of [3H]PGF2α binding following wortmannin pretreatment, although slight, was statistically significant and was obtained consistently and repeatedly in FPB cells, but not in FPA cells. Fig. 2B shows the results of immunofluorescence microscopy of the FLAG-tagged FPA and FPB receptor isoforms using live cell labeling of cell surface receptors (4Srinivasan D. Fujino H. Regan J.W. J. Pharmacol. Exp. Ther. 2002; 302: 219-224Google Scholar). A comparison of panels a andb shows that the FPA isoform is localized primarily on the cell surface membrane in vehicle-treated FPA cells and that following treatment with PGF2α the receptor undergoes extensive internalization. A comparison of panels c and d shows that pretreatment of FPA cells with wortmannin did not affect this pattern of receptor localization. In contrast, examination ofpanels e and f shows that the FPBisoform is localized both intracellularly and on the cell surface regardless of PGF2α treatment. This, as we have previously reported, reflects constitutive agonist-independent internalization of the FPB isoform. Comparison ofpanels e and g further shows that pretreatment with wortmannin reduced the intracellular localization of the FPB isoform in vehicle-treated FPB-expressing cells; this suggests that inhibition of PI3K blocked the constitutive agonist-independent internalization of the FPB isoform. This is consistent with the sensitization of [3H]PGF2α binding observed above following pretreatment of the FPB cells with wortmannin. The interaction of E-cadherin with β-catenin is well established (10Daniels D.L. Spnik K.E. Weis W.I. Trends Biochem. Sci. 2001; 26: 672-678Google Scholar), and recent studies have shown additional interaction between E-cadherin and PI3K (8Kovacs E.M. Ali R.G. McCormack A.J. Yap A.S. J. Biol. Chem. 2002; 277: 6708-6718Google Scholar). Having established an association between PI3K and the FPBreceptor (Fig. 1) and an effect of wortmannin on FPBreceptor localization (Fig. 2), we sought to determine the effects of wortmannin on the localization of E-cadherin and β-catenin in FPB-expressing HEK cells. Fig. 3 shows representative immunoblots (A) and the pooled densitometric analyses (B) for the expression of membrane associated (particulate) E-cadherin and β-catenin and for the expression of cytosolic β-catenin following pretreatment of FPA- and FPB-expressing cells with 100 nm wortmannin for 15 min. As we have previously reported, the expression of β-catenin is higher in FPB-expressing cells as compared with FPA-expressing cells in both the particulate and cytosolic fractions (3Fujino H. Regan J.W. J. Biol. Chem. 2001; 276: 12489-12492Google Scholar). We now show that the expression of particulate E-cadherin is also higher in FPB-expressing cells. Fig. 3B shows that wortmannin pretreatment of vehicle-treated FPB cells slightly, but significantly, increased the expression of membrane-associated (particulate) E-cadherin and β-catenin, while simultaneously decreasing the expression of cytosolic β-catenin. These increases in membrane-associated E-cadherin and β-catenin are very similar to the sensitization of [3H]PGF2α binding to the FPBreceptors observed in Fig. 2A following pretreatment with the PI3K inhibitor, wortmannin. As noted above, PI3K has recently been found to bind E-cadherin (8Kovacs E.M. Ali R.G. McCormack A.J. Yap A.S. J. Biol. Chem. 2002; 277: 6708-6718Google Scholar). The immunoblot shown in Fig. 3C confirms this interaction by showing co-immunoprecipitation of the p85 subunit of PI3K with E-cadherin in both FPA- and FPB-expressing HEK cells. This figure also shows that PGF2α treatment of FPB-expressing cells increased the co-immunoprecipitation of the p85 subunit with E-cadherin suggesting that PGF2αtreatment, which we have shown in Fig. 1C to inhibit PI3K activity, increases its association with E-cadherin. This conclusion is corroborated by the effects of the wortmannin pretreatment of vehicle-treated FPB cells, which also increased the co-immunoprecipitation of the p85 subunit with E-cadherin. Notably, such corresponding changes were not observed in the FPA-expressing cells. One of the most pronounced effects of the treatment of FPB-expressing cells with PGF2α is a dramatic reorganization of the β-catenin as evidenced by immunofluorescence microscopy using antibodies to β-catenin (3Fujino H. Regan J.W. J. Biol. Chem. 2001; 276: 12489-12492Google Scholar). Fig. 4 shows this effect in which the localization of β-catenin is green and the nuclear staining of 4′,6-diamidino-2-phenylindole (DAPI) is blue. Thus, a comparison ofpanels e and f shows that treatment with PGF2α causes a marked increase in β-catenin immunofluorescence in regions of cell to cell contact. A comparison ofpanels a and b shows that a corresponding reorganization of β-catenin does not occur in FPA-expressing cells after treatment with PGF2α. Pretreatment with wortmannin was used to determine the possible influence of PI3K activity on this PGF2α-induced reorganization of β-catenin. Comparison of panels e and g shows that wortmannin pretreatment of vehicle-treated FPB cells produced a slight increase in β-catenin immunofluorescence in regions of cell to cell adhesion. However, as shown by comparing panels g andh, wortmannin pretreatment of FPB-expressing cells did not have any major effect on the PGF2α-induced reorganization of β-catenin. Stabilization of β-catenin expression and the promotion of Tcf transcriptional activation involves the decreased phosphorylation of β-catenin and is directly influenced by the activity of GSK-3β. The activity of GSK-3β, in turn, is a function of its state of phosphorylation, and it is known that phosphorylation of GSK-3β at serine 9 inhibits its kinase activity (11Cohen P. Frame S. Nat. Rev. Mol. Cell. Biol. 2001; 2: 769-776Google Scholar). Immunoblotting of phospho-GSK-3β and phospho-β-catenin (Fig. 5A) and direct measurement of Tcf transcriptional activation (Fig. 5B) following pretreatment of FPB-expressing cells with wortmannin were, therefore, used to assess the potential role of PI3K on the PGF2α-induced activation of Tcf/β-catenin signaling. As shown in Fig. 5A, treatment of FPB-expressing cells with 1 μm PGF2α for 1 h increased the phosphorylation of GSK-3β (panel a) and was accompanied by a marked reduction in phosphorylation of cytosolic β-catenin (panel c). On the other hand, in FPA-expressing cells treatment with PGF2α had no apparent effect on the phosphorylation of GSK-3β (panel a), and there was a marked increase in the phosphorylation of cytosolic β-catenin (panel c). It would be expected, therefore, that following treatment with PGF2α the degradation of β-catenin would be favored in FPA-expressing cells, whereas, the stabilization of β-catenin and potential activation of transcription would be favored in FPB-expressing cells. In fact, as shown in Fig. 5B, this is what was found when transcriptional activation was measured with the use of the Tcf-responsive luciferase reporter gene. Thus, luciferase activity was stimulated ∼3-fold in PGF2α-treated FPB-expressing cells, whereas it was essentially unaffected in FPA-expressing cells as shown previously (3Fuj" @default.
• W2073602994 created "2016-06-24" @default.
• W2073602994 creator A5014847392 @default.
• W2073602994 creator A5069663542 @default.
• W2073602994 creator A5089867564 @default.
• W2073602994 date "2002-12-01" @default.
• W2073602994 modified "2023-10-15" @default.
• W2073602994 title "Cellular Conditioning and Activation of β-Catenin Signaling by the FPB Prostanoid Receptor" @default.
• W2073602994 cites W1898333961 @default.
• W2073602994 cites W1991702914 @default.
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• W2073602994 doi "https://doi.org/10.1074/jbc.m209393200" @default.
• W2073602994 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/12368277" @default.
• W2073602994 hasPublicationYear "2002" @default.
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