Phosphorylation of the Prostacyclin Receptor during Homologous Desensitization
1998; Elsevier BV; Volume: 273; Issue: 36 Linguagem: Inglês
10.1074/jbc.273.36.23258
ISSN1083-351X
AutoresEmer M. Smyth, W. Hong Li, Garret A. FitzGerald,
Tópico(s)Neuropeptides and Animal Physiology
ResumoAgonist-induced phosphorylation of an epitope-tagged prostacyclin receptor (HAhIP) is mediated primarily by PKC (Smyth, E. M., Nestor, P. V., and FitzGerald G. A. (1996) J. Biol. Chem. 271, 33698–33704). Based on the two consensus sites for protein kinase C (PKC) phosphorylation in the C-terminal region mutant HAhIPs were generated: S328A and S374A, in which an alanine replaced Ser-328 or Ser-374, respectively, S328A/S374A and C-DEL, in which the C-terminal portion was truncated at amino acid 313. Mutant receptors, stably expressed in HEK293 cells, coupled normally to cAMP production. Substantially less coupling to inositol phosphate was apparent with S328A, S328A/S374A, and C-DEL compared with HAhIP or S374A. Point mutants resolved by SDS-polyacrylamide gel electrophoresis as a broad band with a molecular mass of 44–62, indicating that the receptors are glycosylated, and immunofluoresence staining demonstrated their membrane localization. C-DEL demonstrated a substantial reduction in glycosylation; bands with molecular masses of 38–54 (glycosylated), 30, and 27 kDa (unglycosylated) were apparent. Although membrane localization was evident, cellular localization was more diffuse. HAhIP and S374A underwent iloprost- and PMA-induced phosphorylation (1 and 5 μm, respectively, for 10 min). S328A and S328A/S374A showed a markedly less iloprost- and no PMA-induced phosphorylation. Phosphorylation of C-DEL was completely absent with either agonist. Electrospray mass spectrometry indicated that a peptide, including Ser-328, was phosphorylated in vitro by PKC, whereas one including Ser-374 was not.Iloprost (1 μm, 10 min) desensitized HAhIP- and S374A-mediated adenylyl cyclase activation. A less impressive desensitization was evident with S328A and S328A/S374A, and no desensitization of C-DEL coupling was apparent. Exposure of transfected cells to iloprost (1 μm) for increasing times induced a rapid desensitization of subsequent iloprost-induced (1 μm) HAhIP and S374A adenylyl cyclase coupling. In contrast, no significant time-dependent desensitization of S328A, S328A/S374A, or C-DEL coupling was evident. These results indicate that PKC-dependent phosphorylation is of critical importance to homologous regulation of hIP. Ser-328 is a primary site for PKC phosphorylation of hIP. Agonist-induced phosphorylation of an epitope-tagged prostacyclin receptor (HAhIP) is mediated primarily by PKC (Smyth, E. M., Nestor, P. V., and FitzGerald G. A. (1996) J. Biol. Chem. 271, 33698–33704). Based on the two consensus sites for protein kinase C (PKC) phosphorylation in the C-terminal region mutant HAhIPs were generated: S328A and S374A, in which an alanine replaced Ser-328 or Ser-374, respectively, S328A/S374A and C-DEL, in which the C-terminal portion was truncated at amino acid 313. Mutant receptors, stably expressed in HEK293 cells, coupled normally to cAMP production. Substantially less coupling to inositol phosphate was apparent with S328A, S328A/S374A, and C-DEL compared with HAhIP or S374A. Point mutants resolved by SDS-polyacrylamide gel electrophoresis as a broad band with a molecular mass of 44–62, indicating that the receptors are glycosylated, and immunofluoresence staining demonstrated their membrane localization. C-DEL demonstrated a substantial reduction in glycosylation; bands with molecular masses of 38–54 (glycosylated), 30, and 27 kDa (unglycosylated) were apparent. Although membrane localization was evident, cellular localization was more diffuse. HAhIP and S374A underwent iloprost- and PMA-induced phosphorylation (1 and 5 μm, respectively, for 10 min). S328A and S328A/S374A showed a markedly less iloprost- and no PMA-induced phosphorylation. Phosphorylation of C-DEL was completely absent with either agonist. Electrospray mass spectrometry indicated that a peptide, including Ser-328, was phosphorylated in vitro by PKC, whereas one including Ser-374 was not. Iloprost (1 μm, 10 min) desensitized HAhIP- and S374A-mediated adenylyl cyclase activation. A less impressive desensitization was evident with S328A and S328A/S374A, and no desensitization of C-DEL coupling was apparent. Exposure of transfected cells to iloprost (1 μm) for increasing times induced a rapid desensitization of subsequent iloprost-induced (1 μm) HAhIP and S374A adenylyl cyclase coupling. In contrast, no significant time-dependent desensitization of S328A, S328A/S374A, or C-DEL coupling was evident. These results indicate that PKC-dependent phosphorylation is of critical importance to homologous regulation of hIP. Ser-328 is a primary site for PKC phosphorylation of hIP. human prostacyclin receptor prostacyclin receptor phospholipase C G protein-coupled receptor protein kinase C GPCR kinase hemagglutinin HAhIPS328A HAhIPS374A HAhIPS328A/S374A HAhIPCDEL Dulbecco's modified Eagle's medium polyacrylamide gel electrophoresis Tris-buffered saline TBS-Tween phorbol 12-myristate 13-acetate. The human prostacyclin receptor (hIP),1 thought to mediate the potent anti-platelet, vasodilator, and anti-inflammatory actions of prostacyclin, is a member of the G protein-coupled receptor (GPCR) superfamily. Studies have demonstrated that the IP is coupled to stimulation of both adenylyl cyclase and phospholipase C (PLC) (1Boie Y. Rushmore T.H. Darmon-Goodwin A. Grygorczyk R. Sliptez D.M. Metters K.M. Abramovitz M. J. Biol. Chem. 1994; 269: 12173-12178Abstract Full Text PDF PubMed Google Scholar, 2Namba T. Oida H. Sugimoto Y. Kakizuka A. Negishi M. Ichikawa A. Narumiya S. J. Biol. Chem. 1994; 269: 9986-9992Abstract Full Text PDF PubMed Google Scholar, 3Smyth E.M. Nestor P.V. FitzGerald G.A. J. Biol. Chem. 1996; 271: 33698-33704Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). In general, the response to GPCRs tends to be tightly regulated by desensitization. This involves rapid receptor phosphorylation, which results in uncoupling of receptor-G protein interactions (4Freedman N.J. Lefkowitz R.J. Recent Prog. Horm. Res. 1996; 51: 319-353PubMed Google Scholar) and subsequent receptor internalization. These events result in a diminished response to agonist. Thereafter, GPCRs may be degraded and/or recycled (5Koenig J.A. Edwards J.M. Trends Pharmacol. Sci. 1997; 18: 276-278Abstract Full Text PDF PubMed Scopus (299) Google Scholar). Generally, at least two classes of kinases may participate in GPCR phosphorylation: second messenger-dependent kinases, such as PKC and protein kinase A, and the GPCR kinases (GRKs) (4Freedman N.J. Lefkowitz R.J. Recent Prog. Horm. Res. 1996; 51: 319-353PubMed Google Scholar). For example, β-adrenoreceptors may be phosphorylated and desensitized by the sequential action of protein kinase A and GRKs 2 and 3 (6Freedman N.J. Liggett S.B. Drachman D.E. Pei G. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1995; 270: 17953-17961Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar, 7Hausdorff W.P. Lohse M.J. Bouvier M. Liggett S.B. Caron M.G. Lefkowitz R.J. Symp. Soc. Exp. Biol. 1990; 44: 225-240PubMed Google Scholar). Similarly, the thrombin (8Ishii K. Chen J. Ishii M. Koch W.J. Freedman N.J. Lefkowitz R.J. Coughlin S.R. J. Biol. Chem. 1994; 269: 1125-1130Abstract Full Text PDF PubMed Google Scholar) and angiotensin II-1A receptors (9Opperman M. Freedman N.J. Alexander R.W. Lefkowitz R.J. J. Biol. Chem. 1996; 271: 13266-13272Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar) are phosphorylated in turn by PKC and GRKs. The pathways involved in eicosanoid receptor regulation are not well understood, although stimulation-dependent phosphorylation and/or desensitization has been reported in the case of some prostaglandin E2 receptors (10Katoh H. Watabe A. Sugimoto Y. Ichikawa I. Megishi M. Biochim. Biophys. Acta. 1995; 1244: 41-48Crossref PubMed Scopus (72) Google Scholar, 11Bastepe M. Ashby B. Mol. Pharmacol. 1997; 51: 343-349Crossref PubMed Scopus (42) Google Scholar) and the thromboxane receptor (12Kinsella B.T. O'Mahony D.J. FitzGerald G.A. J. Biol. Chem. 1997; 269: 29914-29919Abstract Full Text PDF Google Scholar, 13Habib A. Vezza R. Creminon C. Maclouf J. FitzGerald G.A. J. Biol. Chem. 1997; 272: 7191-7200Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). We have demonstrated that this is also true of hIP (3Smyth E.M. Nestor P.V. FitzGerald G.A. J. Biol. Chem. 1996; 271: 33698-33704Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Phosphorylation of the hIP is primarily PKC-dependent, when it is overexpressed in vitro. In this study, we investigated further the pathways involved in hIP regulation and their implications for regulation of receptor function. Mutants of the hemagglutinin (HA)-tagged hIP were used to investigate the role of phosphorylation in desensitization of the hIP. We identified a single site of PKC-dependent phosphorylation as a critical determinant for desensitization of this biologically important (14Murata T. Ushikubi F. Matsuoka T. Hirata M. Tamaska A. Sugimoto Y. Ichikawa A. Aze Y. Tanaka T. Yoshida N. Uono A. Oh-Ishi S. Narumiya S. Nature (Lond.). 1997; 388: 678-682Crossref PubMed Scopus (676) Google Scholar) receptor. Iloprost, the cAMP radioimmunoassay and the enhanced chemiluminesence kits, as well as all radiochemicals, were purchased from Amersham Pharmacia Biotech. Monoclonal anti-HA (16B12 clone) was obtained from the Berkley Antibody Company (Richmond, CA). A Transformer site-directed mutagenesis kit was purchased fromCLONTECH (Palo Alto, CA) and the TA cloning kit and pcDNA III were obtained from Invitrogen (San Diego, CA). Deoxycholic acid, isobutylmethylxanthine, ATP, cAMP, GTP, alumina, phosphatidylcholine, and phosphatidylserine were all purchased from Sigma. All cell culture reagents, G418, and Albumax were obtained from Life Technologies, Inc. Protein G-Sepharose was purchased from Amersham Pharmacia Biotech and complete protease inhibitor tablets were obtained from Boehringer Mannheim. AG 1-X8 resin (formate form) and AG W-X4 resin (hydrogen form) were purchased from Bio-Rad. The hIP cDNA was generously donated by Dr. Kathleen Metters (Merck Frosst, Quebec, Canada). Peptides and oligonucleotides were from Genosys (The Woodlands, TX). HEK 293 cells (American Type Tissue Culture Collection, Rockville, MD) were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum, 50 units/ml penicillin, 50 μg/ml streptomycin, 25 mm HEPES, and 2 mm glutamine. Cells were seeded at 1.5 × 106 cells/100-mm dish and transfected with 100 μg/dish DNA by liposome-mediated transfer (DOTAP; Boehringer Mannheim) according to the manufacturer's instructions the next day. The media were replaced after approximately 6 h with fresh DMEM containing 1.5 mg/ml G418. Several resistant clones, arising from single cells, were selected within 15–20 days and expanded. The G418 concentration was maintained at 1 mg/ml in the cell culture medium. Targeted mutagenesis of the cDNA encoding HAhIP, in the mammalian expression vector pcDNAIII (HAhIPpcDNAIII; Ref. 3Smyth E.M. Nestor P.V. FitzGerald G.A. J. Biol. Chem. 1996; 271: 33698-33704Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar), was performed using the Transformer site-directed mutagenesis kit with appropriate oligonucleotides. We replaced Ser-328 and Ser-374 within the putative consensus sites for PKC phosphorylation in the C-terminal tail with alanines either alone or in combination. This resulted in the following mutants: HAhIPS328A (S328A) containing the serine 328 to alanine substitution, HAhIPS374A (S374A) containing the serine 374 to alanine substitution and HAhIPS328A/S374A (S328A/S374A) containing both substitutions. The C-terminal region of HAhIP was deleted by the introduction of a stop codon (TGA) at residue 313 using the polymerase chain reaction. The 5′-oligonucleotide (5′-CCTGGCGTCCATGCTCATCC-3′) contained 20 bases, which were just upstream of a uniqueEco47III site in the hIP cDNA sequence (1Boie Y. Rushmore T.H. Darmon-Goodwin A. Grygorczyk R. Sliptez D.M. Metters K.M. Abramovitz M. J. Biol. Chem. 1994; 269: 12173-12178Abstract Full Text PDF PubMed Google Scholar). The 3′-oligonucleotide (5′-TTAGGATCCATCAGAGGCACAGGCAGCAGAC-3′) contained 3 miscellaneous bases, a BamHI site, 1 miscellaneous base, 3 bases complimentary to a stop codon, and 18 bases complimentary to hIP amino acids 312 to 307. Polymerase chain reaction was carried out using the HAhIP cDNA as a template. Following cloning into pCR3.1, using the TA cloning kit, the EcoN47III-BamHI fragment was purified and ligated into HAhIPpcDNAIII, cut with the same enzymes, to give rise to a mutant termed HAhIPCDEL (C-DEL). Generation of the desired mutation(s) (Fig. 1) was confirmed by sequencing the region of interest. Membranes were prepared from confluent 100-mm dishes as follows. Cells were washed once with Dulbecco's phosphate-buffered saline and scraped into 20 mm Tris pH 7.4, containing 1 tablet/50 ml complete protease inhibitor mixture. Cells were lysed for 15 s with a Tissue Tearor (Biospec Products, Inc, Bartlesville, OK) on ice, and unlysed cells were removed by centrifugation 500 × g at 4 °C. Membrane fractions collected at 115,000 × g (1 h at 4 °C). The resulting pellet was resuspended in the same buffer and stored at −80 °C for further use. Cells grown in slide chamber assemblies (Nunc, Napierville, IL) were fixed and permeabilized with ice-cold 70% methanol, 30% acetone for 10 min at −20 °C and 5 min at room temperature. Fixed sections were treated with anti-HA (1/2000) in phosphate-buffered saline for 1 h at room temperature, and after being washed three times, each for 10 min, in phosphate-buffered saline, antigen-antibody complexes were visualized with a fluorescein isothiocyanate-labeled anti-mouse IgG antibody (1/500; Jackson Immunology, Westgrove, PA). Slides were mounted in Vectashield (Vector Labs, Burlingame, CA) and examined by fluorescence or confocal microscopy. Cells grown to confluence in 24-well plates were treated with iloprost (10 min at 37 °C). Reactions were terminated by aspiration and cAMP extracted with ice-cold 65% ethanol for 30 min. Samples were dried under vacuum, reconstituted in assay buffer, and cAMP was quantified by radioimmunoassay (Amersham Pharmacia Biotech) according to the manufacturer's instructions. An exception was that half the recommended amounts of binding protein and3H-cAMP tracer were used in the samples and standards. Adenylyl cyclase activity was assayed by a modification of the method of Solomon et al.(15Solomon T. Londos C. Rodbell M. Anal. Biochem. 1994; 58: 541-548Crossref Scopus (3367) Google Scholar). Assays were carried out in 50 mm Tris containing 3 mm MgCl2, 1.5 mm EDTA, 0.15 mm ATP, 0.05 mm GTP, 0.1 mm cAMP, 2.8 mm phosphoenolpyruvate, and 0.1 mmisobutylmethylxanthine. Each reaction contained 1 unit of myokinase, 1 unit of pyruvate kinase, and 2 μCi [α32-P]ATP (30 Ci/mmol). Reactions were started by the addition of membranes (5 μg per assay tube), prepared as outlined above, and after 30 min at 30 °C, were quenched by the addition of 1 ml of 5% trichloroacetic acid containing 30,000 CPM [3H]cAMP (41 Ci/mmol) . Samples were subjected to sequential chromatography through Dowex (AG W-X4, hydrogen form) and alumina (WN-6, neutral) columns as described (15Solomon T. Londos C. Rodbell M. Anal. Biochem. 1994; 58: 541-548Crossref Scopus (3367) Google Scholar). Briefly, samples were eluted onto alumina with 3 ml of H2O following application to Dowex columns and one wash with 2.5 ml of H2O. Cyclic AMP was eluted from the alumina columns with 4 ml of 0.1 mm imidazole (pH 7.5).32P and 3H in the eluates were estimated by scintillation counting. Recovery of cAMP from the columns was monitored by measuring the level of [3H]cAMP in each sample. Cells grown to 70–80% confluence in 12-well plates coated with 0.2% gelatin were labeled overnight with 2 μCi/ml myo-[3H]inositol in DMEM (without inositol) containing 0.5% albumax, 50 units/ml penicillin, and 50 μg/ml streptomycin. 30 min before stimulation, cells were treated with 20 mm LiCl at 37 °C. After stimulation for 10 min at 37 °C, the reactions were terminated by aspiration. Total inositol phosphates were extracted with 750 μl of 10 mm formic acid for 30 min at room temperature. Samples were neutralized (final pH 8–9) with 3 ml 10 mm ammonia. Total inositol phosphates were recovered by anion exchange using Dowex 1-X8 AG anion exchange resin (formate form). Samples were applied to the resin, washed with 40 mm formic acid/ammonium formate (pH 5), and finally the inositol phosphates were eluted with 2m formic acid/ammonium formate (pH 5) (16Seuwen K. Lagarde A. Pouyssegur J. EMBO J. 1998; 7: 161-168Crossref Scopus (101) Google Scholar). Membrane proteins were prepared as outlined above. They were resolved (50 μg per lane) on 10% sodium dodecyl sulfate polyacrylamide gels (SDS-PAGE) and transferred to nitrocellulose. HA-tagged receptors were visualized by treating immunoblots with anti-HA (1:1500 dilution) in 5% milk in Tris-buffered saline (TBS; 50 mm Tris, 250 mmNaCl, pH 7.6) containing 1% Tween 20 (TBS-T) for 60 min at room temperature followed by horseradish peroxidase-conjugated anti-mouse IgG (1:5000 dilution; Jackson Immunology), after first blocking with 5% nonfat milk in TBS-T for 2 h at room temperature. Antigen-antibody complexes were visualized by enhanced chemiluminescence. All immunoprecipitation procedures were carried out at 4 °C. The cells were lysed (RIPA: 50 mm Tris, 5 mm EDTA, pH 8.0, containing 150 mm NaCl, 1% Nonidet P-40, 0.1% SDS, 0.5% deoxycholic acid, 1 tablet/50 ml complete protease inhibitor mixture, 10 mm sodium fluoride, and 10 mmNaH2P2O7), drawn though a 23-gauge needle 6 times and centrifuged at 14,000 rpm. The resulting supernatants were precleared by adding 100 μl of 10% (w/v) protein G-Sepharose to each tube and rotating for 60 min. Anti-HA-protein G-Sepharose was prepared by adding 1 μl anti-HA ascites per lysate to 10% protein G-Sepharose and rotating it for 60 min. The HAhIP was immunoprecipitated from precleared lysates by adding 100 μl of the anti-HA-protein G-Sepharose to each lysate and rotating for 2 h. Protein G was precipitated at 14,000 rpm for 1 min, washed three times with RIPA buffer, and finally resuspended in 60 μl of SDS-PAGE sample buffer (60 mm Tris, pH 6.8, 2% SDS, 10% glycerol, 0.002% bromphenol blue, 100 mm dithiothreitol). Samples were then boiled for 10 min and subjected to electrophoresis as outlined above. Gels were dried for phosphorimaging. Cells were plated in 60-mm dishes and grown to 70–80% confluence. 32P-labeling was carried out using 150–200 μCi/ml [32P]orthophosphate in phosphate-free DMEM containing 0.5% bovine serum albumin (Fisher, Malvern, PA) for 60 min at 37 °C. Labeled cells were treated with the stimulant. Dishes were placed on ice, and the overlying medium was removed at the end of the reaction. Cells were lysed with RIPA, following one wash with ice-cold phosphate-buffered saline, and the HAhIP was immunoprecipitated as outlined above. Following phosphorimaging of dried gels, rectangles of equal size were drawn around each of the bands observed, and the intensity quantified using ImageQuant (Molecular Dynamics, Sunnyvale, CA). Based on the PKC consensus site sequences found in hIP, peptides containing either serine 328 (hIPS328: LASGRRDPRAP) or serine 374 (hIPS372: AVGTSSKAEA) were synthesized. A third peptide, hIPT230 (LGPRPRTGEDEV), which does not contain a consensus site for PKC phosphorylation was also generated. PKC-α-pseudosubstrate peptide was used as a positive control. Using these as substrates, in vitro PKC phosphorylation assays were carried as described (17Vezza R. Habib A. Li H. Lawson J.A. FitzGerald G.A. J. Biol. Chem. 1996; 271: 30028-30033Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). Briefly, reactions were carried out in 50 mm Tris (pH 7.5) containing 250 μm bovine serum albumin, 10 μm CaCl2, 100 μg/ml phosphatidylcholine/phosphatidylserine, 1 μl of PKC (rat brain), 100 μm ATP, 7.5 mm magnesium acetate, and 1 μCi [γ-32P]ATP (6000 Ci/mmol) at 30 °C. Reactions were terminated by spotting half the reaction mixture on 1 inch squares of P81 cation exchange chromatography paper (Whatman, Maidstone, United Kingdom). Following three washes in 0.5% phosphoric acid, the squares were subjected to scintillation counting. Phosphorylation of the peptides was carried out as described above; at the end of the incubation period, samples were put on ice and then frozen at −80 °C until the analysis. The high pressure liquid chromatography pump was a Hewlett Packard series 1050 quaternary pump (Wilmington, DE). The mobile phase consisted of water (solvent A) and acetonitrile:water (90:10, solvent B), both with 0.05% (v:v) trifluoroacetic acid. A LC Packings (San Francisco, CA) 300 μm × 15 cm column with Vydec C18 5 μm 300 Å stationary phase was used to separate samples. The flow rate of the pump was controlled at 0.4 ml/min and split before the injector to 4 μl/min. An aliquot of solution containing 100 pmol of peptide was injected on column, a linear solvent gradient program starting at 5% solvent B and ramping to 40% solvent B in solution was injected on column, and the mobile phase was set at 3% solvent B. The high pressure liquid chromatography was connected online to a mass spectrometer (17Vezza R. Habib A. Li H. Lawson J.A. FitzGerald G.A. J. Biol. Chem. 1996; 271: 30028-30033Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). The capillary voltage was set to 3.5 kV, the sampling cone voltage to 40 V, and the source temperature to 65 °C. The mass analyzer was scanned continuously from m/z 300 to 1400 with a scan duration of 3 s. The relative amount of the peptide phosphorylation was calculated from the selected ion chromatography peak area obtained from the high pressure liquid chromatography/mass spectrometry experiments. Western blot analysis (Fig. 2) of membranes prepared from HEK 293 cells transfected with HAhIP or its mutants demonstrated that, similar to the nonmutated receptor (HAhIP), each of the point mutants resolved as a broad band with a molecular mass from 44 to 66 kDa. The mass of C-DEL was shifted to a 38–54-kDa band, indicating the loss of 74 amino acids from the C-terminal tail. Two lower molecular weight species (30 and 27), not seen with any of the other receptors, were evident in membranes prepared from C-DEL-transfected cells. The level of expression of the mature receptor was similar in HAhIP-, S328A-, S374A-, and S328A/S374A- and apparently lower in C-DEL- transfected cells. Immunofluoresence staining of transfected cells showed the expected pattern of membrane localization of all mutant receptors (Fig. 3 A). This was similar to HAhIP-transfected cells for all point mutant receptors (S328A, S374A, and S328A/S374A), whereas somewhat more diffuse staining was apparent in cells transfected with C-DEL. Analysis of HAhIP and C-DEL localization by confocal microscopy confirmed the membrane localization of both receptors, although cytosolic localization of the latter was also apparent (Fig. 3 B). We have previously shown that activation of HAhIP in this system leads to an increase in both intracellular cAMP and inositol phosphates (3Smyth E.M. Nestor P.V. FitzGerald G.A. J. Biol. Chem. 1996; 271: 33698-33704Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). As demonstrated in Fig. 4 A, treatment of transfected cells with increasing concentrations of iloprost caused an increase in cAMP levels. Coupling of HAhIP and mutant receptors to cAMP production was comparable (Fig. 4, B–E). Basal levels (0.7–2.2 pmol cAMP/106 cells) and EC50 values for iloprost (Table I) were comparable between each of the cell lines. Thus, all receptors were functional, and the mutations did not substantially affect receptor coupling to the Gs-cAMP pathway.Table IEC50 values (nm) for iloprost-stimulated cAMP and inositol phosphate production in cells transfected with HAhIP or the mutant receptorscAMP productionInositol phosphate productionHAhIP0.10 ± 0.03 (n = 3)78.4 ± 19.4 (n = 4)S374A0.12 ± 0.07 (n = 3)78.8 ± 10.8 (n = 5)S328A0.22 ± 0.09 (n = 4)142.98 ± 36.8 (n = 4)S328A/S374A0.11 ± 0.04 (n = 3)192.3 ± 11.9 (n = 3)C-DEL0.27 ± 0.14 (n = 3)169.0 ± 35.1 (n = 4)Data are the mean ± S.E. from the indicated number of experiments (n), each performed in duplicate. Open table in a new tab Data are the mean ± S.E. from the indicated number of experiments (n), each performed in duplicate. Iloprost stimulated inositol phosphate production in HAhIP- and S374A-transfected cells with similar potency and efficacy (Fig. 5, A and B and Table I). However, stimulation of inositol phosphate production reached only 2–3-fold over basal in cells transfected with S328A, S328A/S374A, and C-DEL (Fig. 5, C–E) and EC50 values (Table I) were slightly higher. Similar changes in coupling to inositol phosphate production were seen with a second prostacyclin analog, cicaprost (data not shown). Previously, we reported a rapid iloprost-stimulated phosphorylation of HAhIP (3Smyth E.M. Nestor P.V. FitzGerald G.A. J. Biol. Chem. 1996; 271: 33698-33704Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). A similar pattern of rapid phosphorylation was evident only with cells transfected with the S374A mutant (Fig. 6 A). Both HAhIP- and S374A-transfected cells demonstrated a 4–6-fold increase in phosphorylated receptor following treatment with iloprost for increasing time intervals. The minor dip evident in the S374A time course is probably a result of loading errors because this was not a consistent observation. Furthermore, PMA-induced phosphorylation also occurred in these cells (Fig. 6 B), indicating that PKC-mediated phosphorylation occurs despite the substitution of serine 374. In contrast, both S328A- and S328A/S374A-transfected cells showed a marked reduction of iloprost-induced phosphorylation. Although iloprost-induced phosphorylation occurred over the same rapid time course, maximum increases of only 2–3-fold were achieved (Fig. 6 A). PMA-induced S328A and S328A/S374A phosphorylation was completely absent (Fig. 6 B). Cells transfected with C-DEL showed no phosphorylation with either agonist. These data indicate that serine 328 is the locus of iloprost-induced PKC-mediated rapid HAhIP phosphorylation. In vitro phosphorylation of hIP peptides corroborated this finding. Three hIP peptides were used in these experiments: hIPS328 and hIPS374, containing the consensus sites for PKC phosphorylation, and hIPT230, containing a threonine residue that is not in a PKC consensus sequence. hIPS328 was phosphorylated by PKC, but hIPS374 and hIPT230 were not (Fig. 7). A known PKC substrate, α-pseudosubstrate peptide (18Nakadate T. Jeng A.Y. Blumberg P.M. J. Biol. Chem. 1987; 262: 11507-11513Abstract Full Text PDF PubMed Google Scholar), was used as a positive control. Analysis of the peptides by mass spectrometry confirmed these findings (Fig. 8). In the case of the α-pseudosubstrate peptide, roughly 50% of the total peptide was phosphorylated. Less than 1% of the hIPS328 peptide, by contrast, was phosphorylated. However, no phosphorylation of the hIPS374 or hIPT230 peptides was detected.Figure 8High pressure liquid chromatography/electrospray mass spectrometry analysis.Electrospray mass spectra of the doubly charged ion of PKC-α-pseudosubstrate peptide (A), doubly charged ion of hIPS328 (B), singly charged ion of hIPS347 (C), and doubly charged ion of hIPT230 (D) analyzed alone (upper panels) or after preincubation (90 min, 30 °C) with PKC (lower panels). The x axis represents the mass to charge ration (Da/e), and they axis represents the relative intensity for each species. The two molecular species in panel C are the protonated and sodiated molecular species of this peptide. A molecular mass shift of m/z 80 for singly charged ions orm/z 40 for doubly charged ions is the evidence of phosphorylation.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Pretreatment with iloprost induced rapid desensitization of receptor coupling to adenylyl cyclase in membranes prepared from HAhIP-transfected cells (Fig. 9). Treatment of cells for 10 min with 1 μm iloprost, conditions which maximally phosphorylate the receptor (Fig. 6 and Ref. 3Smyth E.M. Nestor P.V. FitzGerald G.A. J. Biol. Chem. 1996; 271: 33698-33704Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar), induced a striking reduction in concentration-dependent iloprost-stimulated adenylyl cyclase activation (Fig. 9 A). This effect was rapid; it was evident after only a 15 s treatment (Fig. 9 B). Substitution of serine 374 with alanine had minimal impact on either desensitization of the concentration-dependent response of membrane adenylyl cyclase to iloprost (Fig. 10 A), or the time course of desensitization to 1 μm iloprost (Fig. 11 A). In contrast, truncation of the C-terminal tail completely prevented iloprost-induced desensitization (Figs. 10 D and 11 D). Substitution of serine 328 with alanine either alone or in combination with serine 374, resulted in a markedly reduced level of desensitization. Although pretreatment with 1 μm iloprost produced some abrogation of iloprost-stimulation of adenylyl cyclase in S328A- and S328A/S374A-transfected cells, this was only evident at lower agonist concentrations (Fig. 10, B and C). In addition, pretreatment of S328A- or S328A/S374A-transfected cells with 1 μm iloprost for increasing times did not produce a significant level of desensitization, in contrast to HAhIP and S374A-transfected cells (Fig. 11, B and C).Figure 10Desensitization of mutant
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