Agonist-induced Internalization of the Platelet-activating Factor Receptor Is Dependent on Arrestins but Independent of G-protein Activation
2002; Elsevier BV; Volume: 277; Issue: 9 Linguagem: Inglês
10.1074/jbc.m110058200
ISSN1083-351X
AutoresZhangguo Chen, Denis J. Dupré, Christian Le Gouill, Marek Rola‐Pleszczynski, Jana Staňková,
Tópico(s)Protein Kinase Regulation and GTPase Signaling
ResumoAs with most G-protein-coupled receptors, repeated agonist stimulation of the platelet-activating factor receptor (PAFR) results in its desensitization, sequestration, and internalization. In this report, we show that agonist-induced PAFR internalization is independent of G-protein activation but is dependent on arrestins and involves the interaction of arrestins with a limited region of the PAFR C terminus. In cotransfected COS-7 cells, both arrestin-2 and arrestin-3 could be coimmunoprecipitated with PAFR, and agonist stimulation of PAFR induced the translocation of both arrestin-2 and arrestin-3. Furthermore, coexpression of arrestin-2 with PAFR potentiated receptor internalization, whereas agonist-induced PAFR internalization was inhibited by a dominant negative mutant of arrestin-2. The coexpression of a minigene encoding the C-terminal segment of the receptor abolished PAF-induced arrestin translocation and inhibited PAFR internalization. Using C terminus deletion mutants, we determined that the association of arrestin-2 with the receptor was dependent on the region between threonine 305 and valine 330 because arrestin-2 could be immunoprecipitated with the mutant PAFRstop330 but not PAFRstop305. Consistently, stop330 could mediate agonist-induced arrestin-2 translocation, whereas stop305 could not. Two other deletion mutants with slightly longer regions of the C terminus, PAFRstop311 and PAFRstop317, also failed to induce arrestin-2 translocation. Finally, the PAFR mutant Y293A, containing a single substitution in the putative internalization motif DPXXY in the seventh transmembrane domain (which we had shown to be able to internalize but not to couple to G-proteins) could efficiently induce arrestin translocation. Taken together, our results indicate that ligand-induced PAFR internalization is dependent on arrestins, that PAFR can associate with both arrestin-2 and -3, and that their translocation involves interaction with the region of residues 318–330 in the PAFR C terminus but is independent of G-protein activation. As with most G-protein-coupled receptors, repeated agonist stimulation of the platelet-activating factor receptor (PAFR) results in its desensitization, sequestration, and internalization. In this report, we show that agonist-induced PAFR internalization is independent of G-protein activation but is dependent on arrestins and involves the interaction of arrestins with a limited region of the PAFR C terminus. In cotransfected COS-7 cells, both arrestin-2 and arrestin-3 could be coimmunoprecipitated with PAFR, and agonist stimulation of PAFR induced the translocation of both arrestin-2 and arrestin-3. Furthermore, coexpression of arrestin-2 with PAFR potentiated receptor internalization, whereas agonist-induced PAFR internalization was inhibited by a dominant negative mutant of arrestin-2. The coexpression of a minigene encoding the C-terminal segment of the receptor abolished PAF-induced arrestin translocation and inhibited PAFR internalization. Using C terminus deletion mutants, we determined that the association of arrestin-2 with the receptor was dependent on the region between threonine 305 and valine 330 because arrestin-2 could be immunoprecipitated with the mutant PAFRstop330 but not PAFRstop305. Consistently, stop330 could mediate agonist-induced arrestin-2 translocation, whereas stop305 could not. Two other deletion mutants with slightly longer regions of the C terminus, PAFRstop311 and PAFRstop317, also failed to induce arrestin-2 translocation. Finally, the PAFR mutant Y293A, containing a single substitution in the putative internalization motif DPXXY in the seventh transmembrane domain (which we had shown to be able to internalize but not to couple to G-proteins) could efficiently induce arrestin translocation. Taken together, our results indicate that ligand-induced PAFR internalization is dependent on arrestins, that PAFR can associate with both arrestin-2 and -3, and that their translocation involves interaction with the region of residues 318–330 in the PAFR C terminus but is independent of G-protein activation. Cell responsiveness to agonists of G-protein-coupled receptors (GPCRs) 1GPCRG-protein-coupled receptorGFPgreen fluorescent proteinGRKsGPCR-specific kinasesPAFplatelet-activating factorPAFRplatelet-activating factor receptorCHOChinese hamster ovaryWTwild typeIPinositol phosphatePBSphosphate-buffered salineRIPAradioimmune precipitation bufferSTATsignal transducers and activators of transcriptionMAPmitogen-activated proteinMAPKMAP kinaseDMEMDulbecco's modified Eagle's mediumAEBSF4-(2-aminoethyl)-benzenesulfonyl fluorideTLCKNα-p-tosyl-lysine chloromethyl ketone is usually characterized by a rapid desensitization to subsequent exposures, followed by a resensitization in the absence of stimulation (1Goldstein J.L. Brown M.S. Anderson R.G. Russell D.W. Schneider W.J. Annu. Rev. Cell Biol. 1985; 1: 1-39Crossref PubMed Scopus (1195) Google Scholar, 2Pearse B.M. Robinson M.S. Annu. Rev. Cell Biol. 1990; 6: 151-171Crossref PubMed Scopus (561) Google Scholar, 3Trowbridge I.S. Curr. Opin. Cell Biol. 1991; 3: 634-641Crossref PubMed Scopus (130) Google Scholar, 4Smythe E. Warren G. Eur. J. Biochem. 1991; 202: 689-699Crossref PubMed Scopus (140) Google Scholar). The internalization of GPCRs is believed to be responsible, at least in part, for desensitization and/or for resensitization (5Pippig S. Andexinger S. Lohse M.J. Mol. Pharmacol. 1995; 47: 666-676PubMed Google Scholar, 6Krueger K.M. Daaka Y. Pitcher J.A. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 5-8Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar, 7Zhang J. Barak L.S. Winkler K.E. Caron M.G. Ferguson S.S. J. Biol. Chem. 1997; 272: 27005-27014Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar, 8Volga O. Bogatkewitsch G.S. Wriske C. Krummenerl P. Jakobs K.H. van Koppen C.J. J. Biol. Chem. 1998; 273: 12155-12160Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 9Ishii I. Saito E. Izumi T., Ui, M. Shimizu T. J. Biol. Chem. 1998; 273: 9878-9885Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 10Oakley R.H. Laporte S.A. Holt J.A. Barak L.S. Caron M.G. J. Biol. Chem. 1999; 274: 32248-32257Abstract Full Text Full Text PDF PubMed Scopus (468) Google Scholar) of many GPCRs, the prototype being the β2-adrenoreceptor (5Pippig S. Andexinger S. Lohse M.J. Mol. Pharmacol. 1995; 47: 666-676PubMed Google Scholar, 6Krueger K.M. Daaka Y. Pitcher J.A. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 5-8Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar,11Yu S.S. Lefkowitz R.J. Hausdorff W.P. J. Biol. Chem. 1993; 268: 337-341Abstract Full Text PDF PubMed Google Scholar, 12von Zastrow M. Kobilka B.K. J. Biol. Chem. 1992; 267: 3530-3538Abstract Full Text PDF PubMed Google Scholar, 13von Zastrow M. Kobilka B.K. J. Biol. Chem. 1994; 269: 18448-18452Abstract Full Text PDF PubMed Google Scholar, 14Ferguson S.S. Menard L. Barak L.S. Koch W.J. Colapietro A.M. Caron M.G. J. Biol. Chem. 1995; 270: 24782-24789Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). Members of the arrestin family play an important role in the process of GPCR endocytosis (15Laporte S.A. Oakley R.H. Zhang J. Holt J.A. Ferguson S.S. Caron M.G. Barak L.S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3712-3717Crossref PubMed Scopus (543) Google Scholar, 16Laporte S.A. Oakley R.H. Holt J.A. Barak L.S. Caron M.G. J. Biol. Chem. 2000; 275: 23120-23126Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar, 17Luttrell L.M. Ferguson S.S. Daaka Y. Miller W.E. Maudsley S. Della Rocca G.J. Lin F. Kawakatsu H. Owada K. Luttrell D.K. Caron M.G. Lefkowitz R.J. Science. 1999; 283: 655-661Crossref PubMed Scopus (1286) Google Scholar). After agonist-activation, the receptors are phosphorylated by GPCR-specific kinases (GRKs) and second messenger-dependent kinases (18Hausdorff W.P. Caron M.G. Lefkowitz R.J. Faseb J. 1990; 4: 2881-2889Crossref PubMed Scopus (1092) Google Scholar). For the β2-adrenoreceptor and other GPCRs, agonist stimulation leads to arrestin recruitment to the plasma membrane and binding with the phosphorylated receptor. Phosphorylation and arrestins prevent coupling of the receptor to the heterotrimeric G-protein and are believed to be the key elements of a rapid desensitization (19Lin F.T. Krueger K.M. Kendall H.E. Daaka Y. Fredericks Z.L. Pitcher J.A. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 31051-31057Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar, 20Barak L.S. Ferguson S.S. Zhang J. Caron M.G. J. Biol. Chem. 1997; 272: 27497-27500Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar, 21Freedman N.J. Lefkowitz R.J. Recent Prog. Horm. Res. 1996; 51: 319-351PubMed Google Scholar). Arrestins play an adaptor role by targeting the receptor to clathrin-coated pits and by binding the heavy chain of clathrin as well as the adaptor molecule AP2 (15Laporte S.A. Oakley R.H. Zhang J. Holt J.A. Ferguson S.S. Caron M.G. Barak L.S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3712-3717Crossref PubMed Scopus (543) Google Scholar, 16Laporte S.A. Oakley R.H. Holt J.A. Barak L.S. Caron M.G. J. Biol. Chem. 2000; 275: 23120-23126Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar, 22Goodman O.B., Jr. Krupnick J.G. Santini F. Gurevich V.V. Penn R.B. Gagnon A.W. Keen J.H. Benovic J.L. Nature. 1996; 383: 447-450Crossref PubMed Scopus (1190) Google Scholar). In addition, arrestins may facilitate the action of dynamin by recruiting c-Src (17Luttrell L.M. Ferguson S.S. Daaka Y. Miller W.E. Maudsley S. Della Rocca G.J. Lin F. Kawakatsu H. Owada K. Luttrell D.K. Caron M.G. Lefkowitz R.J. Science. 1999; 283: 655-661Crossref PubMed Scopus (1286) Google Scholar, 23Miller W.E. Maudsley S. Ahn S. Khan K.D. Luttrell L.M. Lefkowitz R.J. J. Biol. Chem. 2000; 275: 11312-11319Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar), which is necessary for its phosphorylation (23Miller W.E. Maudsley S. Ahn S. Khan K.D. Luttrell L.M. Lefkowitz R.J. J. Biol. Chem. 2000; 275: 11312-11319Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, 24Ahn S. Maudsley S. Luttrell L.M. Lefkowitz R.J. Daaka Y. J. Biol. Chem. 1999; 274: 1185-1188Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar). The internalized receptor is subsequently either dephosphorylated and recycled to the plasma membrane or targeted for degradation. G-protein-coupled receptor green fluorescent protein GPCR-specific kinases platelet-activating factor platelet-activating factor receptor Chinese hamster ovary wild type inositol phosphate phosphate-buffered saline radioimmune precipitation buffer signal transducers and activators of transcription mitogen-activated protein MAP kinase Dulbecco's modified Eagle's medium 4-(2-aminoethyl)-benzenesulfonyl fluoride Nα-p-tosyl-lysine chloromethyl ketone Platelet-activating factor (PAF) is a potent proinflammatory phospholipid mediator involved in the physiology and pathology of a variety of systems, including central nervous, reproductive, respiratory, and immune responses (25Braquet P. Rola-Pleszczynski M. Prostaglandins. 1987; 34: 143-148Crossref PubMed Scopus (35) Google Scholar, 26Braquet P. Touqui L. Shen T.Y. Vargaftig B.B. Pharmacol. Rev. 1987; 39: 97-145PubMed Google Scholar). PAF binds to a specific receptor (PAFR), which is a member of the GPCR family (25Braquet P. Rola-Pleszczynski M. Prostaglandins. 1987; 34: 143-148Crossref PubMed Scopus (35) Google Scholar, 26Braquet P. Touqui L. Shen T.Y. Vargaftig B.B. Pharmacol. Rev. 1987; 39: 97-145PubMed Google Scholar, 27Sugimoto T. Tsuchimochi H. McGregor C.G. Mutoh H. Shimizu T. Kurachi Y. Biochem. Biophys. Res. Commun. 1992; 189: 617-624Crossref PubMed Scopus (85) Google Scholar, 28Nakamura M. Honda Z. Izumi T. Sakanaka C. Mutoh H. Minami M. Bito H. Seyama Y. Matsumoto T. Noma M. Shimizu T. J. Biol. Chem. 1991; 266: 20400-20405Abstract Full Text PDF PubMed Google Scholar, 29Kunz D. Gerard N.P. Gerard C. J. Biol. Chem. 1992; 267: 9101-9106Abstract Full Text PDF PubMed Google Scholar, 30Ye R.D. Prossnitz E.R. Zou A.H. Cochrane C.G. Biochem. Biophys. Res. Commun. 1991; 180: 105-111Crossref PubMed Scopus (171) Google Scholar, 31Chase P.B. Halonen M. Regan J.W. Am. J. Respir. Cell Mol. Biol. 1993; 8: 240-244Crossref PubMed Scopus (40) Google Scholar). PAFR couples to several second messenger systems that include phospholipid turnover through phospholipase (PL) Cγ and PLCβ activation (32Honda Z. Takano T. Gotoh Y. Nishida E. Ito K. Shimizu T. J. Biol. Chem. 1994; 269: 2307-2315Abstract Full Text PDF PubMed Google Scholar, 33Takano T. Honda Z. Sakanaka C. Izumi T. Kameyama K. Haga K. Haga T. Kurokawa K. Shimizu T. J. Biol. Chem. 1994; 269: 22453-22458Abstract Full Text PDF PubMed Google Scholar, 34Izumi T. Shimizu T. Biochim. Biophys. Acta. 1995; 1259: 317-333Crossref PubMed Scopus (208) Google Scholar, 35Chao W. Olson M.S. Biochem. J. 1993; 292: 617-629Crossref PubMed Scopus (425) Google Scholar), MAP kinase activation (32Honda Z. Takano T. Gotoh Y. Nishida E. Ito K. Shimizu T. J. Biol. Chem. 1994; 269: 2307-2315Abstract Full Text PDF PubMed Google Scholar, 36Nick J.A. Avdi N.J. Young S.K. Knall C. Gerwins P. Johnson G.L. Worthen G.S. J. Clin. Invest. 1997; 99: 975-986Crossref PubMed Scopus (281) Google Scholar), and the JAK/STAT pathway (37Lukashova V. Asselin C. Krolewski J.J. Rola-Pleszczynski M. Stankova J. J. Biol. Chem. 2001; 276: 24113-24121Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). By using the techniques of ligand binding, immunostaining, inositol phosphate (IP) production, and intracellular calcium concentration measurement, we and others have demonstrated that PAFR is also subject to desensitization, internalization, and resensitization (9Ishii I. Saito E. Izumi T., Ui, M. Shimizu T. J. Biol. Chem. 1998; 273: 9878-9885Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 33Takano T. Honda Z. Sakanaka C. Izumi T. Kameyama K. Haga K. Haga T. Kurokawa K. Shimizu T. J. Biol. Chem. 1994; 269: 22453-22458Abstract Full Text PDF PubMed Google Scholar, 38Ali H. Fisher I. Haribabu B. Richardson R.M. Snyderman R. J. Biol. Chem. 1997; 272: 11706-11709Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 39Le Gouill C. Parent J.L. Rola-Pleszczynski M. Stankova J. J. Biol. Chem. 1997; 272: 21289-21295Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Furthermore, it has been shown by us and others (9Ishii I. Saito E. Izumi T., Ui, M. Shimizu T. J. Biol. Chem. 1998; 273: 9878-9885Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar,39Le Gouill C. Parent J.L. Rola-Pleszczynski M. Stankova J. J. Biol. Chem. 1997; 272: 21289-21295Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar) that the intracellular C terminus of PAFR plays an important role in receptor internalization, but its association with arrestins has not been shown. The molecular mechanism through which PAFR is desensitized, internalized, and resensitized has not yet been fully elucidated. The aim of the current study is to demonstrate whether nonvisual arrestins interact with PAFR and participate in receptor internalization after agonist stimulation. Our results indicated that PAFR internalization was regulated by arrestin-2 and -3 and that the C terminus of PAFR contained arrestin-2 or -3 binding sites and contributed to arrestin translocation after PAF stimulation. In addition, the DPXXY motif in the seventh transmembrane domain of PAFR may be involved in maintaining the proper receptor conformation responsible for arrestin translocation and colocalization with PAFR. Finally, agonist-induced PAFR internalization and arrestin recruitment were independent of G-protein signaling. Reagents were obtained from the following sources: oligonucleotides were synthesized at Invitrogen,Pwo polymerase was from Roche Molecular Biochemicals, restriction endonucleases and modifier enzymes were from Promega and Amersham Biosciences, bovine serum albumin and protein A-Sepharose from Sigma-Aldrich, FuGENE6 Transfection reagent from Roche, platelet-activating factor from Calbiochem, anti-GFP antibody from CLONTECH (Palo Alto, CA), anti-β-arrestin-1 from Transduction Laboratory (San Diego, CA), anti-c-Myc from ATCC (Manassas, VA), rhodamine-conjugated goat anti-mouse IgG from Bio Can Scientific (Mississauga, ON), and ECL Western blotting detection reagent kit was from Amersham Biosciences. Arrestin-2-GFP and arrestin-3-GFP expression vectors were kindly provided by Dr. J. Benovic (Jefferson University, Philadelphia). The PAFR-GFP construct was made by amplifying PAFR cDNA without a stop codon by PCR using the forward primer 5′-TCAAGCTTATGGAGCCACATGACTCC-3′ and reverse primer 5′-GAGGGCCCAATTTTTGAGGGAATTGCCA-3′, digesting the PCR product withHind-III and Apa-I, and replacing the entire sequence of arrestin-3 from the arrestin-3-GFP expression vector digested with the same restriction endonucleases. The PAFR stop mutants were generated as already described (39Le Gouill C. Parent J.L. Rola-Pleszczynski M. Stankova J. J. Biol. Chem. 1997; 272: 21289-21295Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). The oligonucleotides used for Stop305 were 5′-CCACATGACTCCTCCCACATG-3′ and 5′-CGGGATTCCTCAGAGGTGCTTGCGGAACTTC-3′. Stop311 was constructed by digestion with SphI. Constructs were sequenced at the University of Calgary, Alberta, Canada. COS-7, CHO, and HEK293 cells were grown in Dulbecco's modified Eagle's medium high glucose (DMEM), Dulbecco's modified Eagle's medium F12, and minimum essential medium (MEM), respectively, supplemented with 10% fetal bovine serum and 100 μg/ml garamycin. For confocal microscopy, cells were seeded in 6-well plates containing a coverslip at a density of 1 × 105COS-7/well, 2 × 105 CHO/well, or 3 × 105 HEK293/well. Cells were transfected following manufacturer's instructions using FuGENE6 transfection reagent kits. 1–1.5 μg of DNA per well was used. For each 100-mm Petri dish (for coimmunoprecipitation), the total amount of DNA was 6 μg. RBL-2H3 cells were grown in MEM supplemented with 15% fetal bovine serum and 100 μg of garamycin transfected with LipofectAMINE (Invitrogen), according to the manufacturer's instructions. Competition binding curves were done on COS-7 cells expressing the wild type (WT) and mutant PAFR. Cells were harvested and washed twice in Hepes-Tyrode's buffer (140 mm NaCl, 2.7 mm KCl, 1 mmCaCl2, 12 mm NaHCO3, 5.6 mmd-glucose, 0.49 mmMgCl2, 0.37 mmNaH2PO4,and 25 mm Hepes, pH 7.4) containing 0.1% (w/v) bovine serum albumin. Binding reactions were carried out on 5 × 104 cells in a total volume of 0.25 ml in the same buffer with 10 nm[3H]WEB2086 and increasing concentrations of nonradioactive WEB2086 or PAF for 90 min at 25 °C. Reactions were stopped by centrifugation. The cell-associated radioactivity was measured by liquid scintillation. Confocal microscopy analysis was performed as previously described with some modifications (36Nick J.A. Avdi N.J. Young S.K. Knall C. Gerwins P. Johnson G.L. Worthen G.S. J. Clin. Invest. 1997; 99: 975-986Crossref PubMed Scopus (281) Google Scholar). The cells were grown on coverslips (22 mm), 40 h post-transfection, treated with PAF 10−7m at 37 °C for different times, and fixed with 2% paraformaldehyde (15 min at room temperature). The coverslips were placed in 0.1% saponin in PBS for 20 min and then sequentially incubated with 5% dry milk and 0.01m glycine at room temperature for 20 min each. The cells were next incubated with an anti-c-Myc monoclonal antibody, followed with rhodamine-conjugated goat anti-mouse IgG antibodies. For live cell visualization, the cells grown on 22-mm coverslips were pretreated with 20 μg/ml cycloheximide for 30 min, and the coverslip was placed in a tissue culture chamber. The medium with cycloheximide, without fetal bovine serum, was added, and the temperature was maintained at 37 °C. 10−7m PAF was added after the first image was taken. The cells were analyzed on a Molecular Dynamics (Sunnyvale, CA) Multi-Probe 2001 confocal argon laser scanning system equipped with a Nikon Diaphot epifluorescence inverted microscope. Scanned images were transferred onto a Silicon Graphics Indy 4000 work station equipped with Molecular Dynamics Imagespace analysis software. 48 h after transfection, cells grown on 100-mm dishes were washed twice with PBS, treated with 10−7m PAF at 37 °C for the indicated time, and then lysed with 0.5 ml of radioimmune precipitation buffer (50 mm Tris, pH 7.5, 5 mm EDTA, 150 mmNaCl, 0.5% sodium deoxycholate, 1% IGEPAL, 0.1% SDS, 2 μg/ml aprotinin, 1 μm/ml leupeptin, 10 μg/ml soybean trypsin inhibitor, 100 μg/ml AESBF, and 40 μg/ml TLCK). The lysate was solubilized by incubation at 4 °C for 30 min, precleared with 50 μl of protein A-Sepharose beads at 4 °C for 1 h, and clarified by centrifugation at 14,000 rpm for 30 min. The concentration of soluble protein was determined with the BCA protein assay kit (Pierce). Equal amounts of protein were used for all subsequent immunoprecipitations. The precleared lysate was incubated with anti-c-Myc for 4 h, and then 50 μl of protein A-Sepharose beads were added and incubated for 3 h. After extensive washing with radioimmune precipitation buffer, the immunoprecipitated proteins were eluted from beads with 2× SDS sample buffer, resolved by SDS-PAGE, and subjected to Western blot analysis. COS-7 cells transfected with correspondent plasmids were harvested 40 h post-transfection and subjected to flow cytometry analysis using anti-c-Myc antibody and fluorescein isothiocyanate-conjugated goat anti-mouse antibody (BD Biosciences, Mississauga, ON), as described previously (40Parent J.L. Gouill C.L. Escher E. Rola-Pleszczynski M. Stankova J. J. Biol. Chem. 1996; 271: 23298-23303Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). Analysis was performed on a FACScan flow cytometer (BD Biosciences). COS-7 cells transfected with indicated cDNAs were labeled for 18–24 h with [3H]myo-inositol (Amersham Biosciences) at 3 μCi/ml in DMEM (high glucose, without inositol, from Invitrogen). After labeling, cells were washed and preincubated for 5 min in PBS at 37 °C. The PBS was removed, and cells were incubated in DMEM containing 0.1% bovine serum albumin and 20 mm LiCl for 5 min, then stimulated for 10 min with the 10−6m PAF. The reactions were terminated by the addition of perchloric acid. Inositol phosphates were extracted and separated on Dowex AG1-X8 (Bio-Rad) columns. Total labeled inositol phosphates were then counted by liquid scintillation. The mutants of PAFR used in this report are illustrated in Fig.1. The PAFR-GFP fusion protein was constructed in order to follow PAFR internalization in real time, and the C terminus deletion mutants were constructed in order to define the region of arrestin and GRK binding. The stop317, Asp-289, and Tyr-293 mutants have been described previously (39Le Gouill C. Parent J.L. Rola-Pleszczynski M. Stankova J. J. Biol. Chem. 1997; 272: 21289-21295Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 40Parent J.L. Gouill C.L. Escher E. Rola-Pleszczynski M. Stankova J. J. Biol. Chem. 1996; 271: 23298-23303Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). The binding characteristics of PAFR-GFP and stop311 were examined in transiently transfected COS-7 cells using WEB2086, a PAFR receptor antagonist, (Fig. 2) and showed that both mutant receptors had an affinity comparable with that of wild-type PAFR. Response to PAF as measured by inositol phosphate production was similar to WT in both PAFR-GFP and stop311 (results not illustrated). Stop305, on the other hand, showed significantly reduced binding and IP production, ∼10% of WT (results not illustrated). In this study, we used four different cell types (COS-7, CHO, HEK293, and RBL-2H3) to assure ourselves of the universality of the studied mechanisms. Experiments were done with all the cell types, but only one is illustrated for each experiment.Figure 2Competition binding isotherms of [3H]WEB2086 by WEB2086 in COS-7 cells.[3H]WEB2086 binding was measured as indicated under "Experimental Procedures" on cells transiently expressing the WT, stop311, and PAFR-GFP receptors. The results are representative of three independent experiments, each done in duplicate.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We had shown that the human PAFR is internalized via clathrin-coated vesicles (39Le Gouill C. Parent J.L. Rola-Pleszczynski M. Stankova J. J. Biol. Chem. 1997; 272: 21289-21295Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). We have now investigated whether internalization was associated with arrestins. Fig. 3 A shows that in COS-7 cells transfected with PAFR-GFP cDNA coexpression of arrestin-2 potentiated internalization after PAF stimulation, whereas a dominant negative mutant of arrestin-2 inhibited internalization. When cells were observed in real time using a temperature-controlled chamber, the overexpression of arrestin-2 in HEK293 cells also potentiated internalization. Fig. 3 B shows that in cells transfected with PAFR-GFP the receptor was partially internalized within 30 min, but when arrestin-2 was coexpressed the receptor was completely internalized within this time frame. In addition, the receptor was internalized in much larger vesicles than without arrestin coexpression. The same phenomena were observed when the cells were cotransfected with arrestin-3 (results not illustrated). We coexpressed WT PAFR and arrestin-2-GFP or arrestin-3-GFP in COS-7 cells, immunoprecipitated the receptor, and showed that both arrestin-2 and arrestin-3 could coimmunoprecipitate with the WT receptor (Fig.4 A). In CHO cells cotransfected with PAFR and arrestin-2-GFP cDNAs, PAF induced a redistribution of arrestin-2 from the cytosol to the cellular membrane within 5 min; by 30 min, both the receptor and arrestin colocalized to discrete intracytoplasmic vesicles (Fig. 4 B). This contrasted with much more discrete internalization of the PAFR when cotransfected with only the GFP protein (Fig. 4 B,lower left panel). Similar results were seen with arrestin-3-GFP cotransfection (results not illustrated). Because our results indicated that arrestins were necessary for PAFR internalization, we used certain mutants of PAFR that showed different internalization properties to verify whether they could coimmunoprecipitate arrestin-2. COS-7 cells were cotransfected with arrestin-2 and either WT or mutant PAFR or pcDNA3. The cell lysates were subjected to immunoprecipitation with anti-c-Myc antibodies and immunoblotted with anti-arrestin-2 antibodies, followed by anti-PAFR polyclonal antibodies to verify the levels of PAFR. The total cell lysates were blotted with anti-arrestin-2 antibodies to verify equivalent arrestin expression in all samples. The results indicate that the 25 amino acids between residues 305 and 330 of the receptor are essential for arrestin association (Fig.5). Because we had shown that this region was also, at least in part, involved in the internalization of the receptor, we used a mutant receptor which does not internalize but has a mutation that is not in the C terminus. The mutant D289A, which is impaired in its internalization potential, failed to coimmunoprecipitate arrestin-2, whereas the mutant D289N (39Le Gouill C. Parent J.L. Rola-Pleszczynski M. Stankova J. J. Biol. Chem. 1997; 272: 21289-21295Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar), which internalizes normally, coimmunoprecipitated with arrestin-2 comparably to WT (Fig. 5). We next examined whether mutant PAFRs that did not internalize and/or were not coupled to G-proteins could induce the translocation of arrestins. Both mutants D289A and Y293A are not coupled to G-proteins but, unlike D289A, Y293A internalizes comparably to WT (39Le Gouill C. Parent J.L. Rola-Pleszczynski M. Stankova J. J. Biol. Chem. 1997; 272: 21289-21295Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). The conservative substitutions of these residues D289N and Y293F produce receptors that are both coupled to G-proteins and internalize. Fig. 6 shows that after PAF stimulation all mutants induced arrestin-2-GFP translocation to the membrane with the exception of D289A. These results support the hypothesis that arrestins are necessary for PAFR internalization but that G-protein coupling is not necessary for arrestin translocation. The role of the C terminus of PAFR in the redistribution of arrestins was examined with C terminus deletion mutants. COS-7 cells were cotransfected with arrestin-2-GFP and PAFR WT or stop mutant cDNAs, and redistribution of arrestin-2-GFP was examined after a 20 min stimulation with PAF. Fig. 7 shows that stop305, stop311, and stop317 did not induce membrane translocation of arrestins, in contrast to the stop330 mutant, which induced arrestin translocation similarly to the WT. These results indicate that the portion of the C terminus between residues 318 and 330 is necessary for PAFR association with and translocation of arrestins (Figs. 5 and 7, respectively). To study the role of the C terminus of PAFR without using truncated mutants, we constructed a minigene that expressed residues 300 to 342 from the C terminus. The C terminus blocked PAF-induced PAFR-GFP internalization when cotransfected with the receptor in RBL-2H3 cells (Fig. 8 A). In COS-7 cells, C terminus cotransfection prevented the PAF-induced cell-surface loss of receptors, both in the presence and absence of overexpressed arrestin-3 (Fig. 8 B) or arrestin-2 (results not illus
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