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Structural and Functional Requirements for Agonist-induced Internalization of the Human Platelet-activating Factor Receptor

1997; Elsevier BV; Volume: 272; Issue: 34 Linguagem: Inglês

10.1074/jbc.272.34.21289

ISSN

1083-351X

Autores

Christian Le Gouill, Jean‐Luc Parent, Marek Rola‐Pleszczynski, Jana Staňková,

Tópico(s)

Protein Kinase Regulation and GTPase Signaling

Resumo

The receptor for platelet-activating factor (PAF) is a member of the G-protein-coupled receptor family. To study the structural elements and mechanisms involved in the internalization of human PAF receptor (hPAFR), we used the following mutants: a truncated mutant in the C-terminal tail of the receptor (Cys317→ Stop) and mutations in the (D/N)P(X)2,3Y motif (Asp289 → Asn,Ala and Tyr293 → Phe,Ala). Chinese hamster ovary cells expressing the Cys317→ Stop mutant exhibited a marked reduction in their capacity to internalize PAF, suggesting the existence of determinants important for endocytosis in the last 26 amino acids of the cytoplasmic tail. Substitution of Asp289 to alanine abolished both internalization and G-protein coupling, whereas substitution of Tyr293 to alanine abolished coupling but not internalization. Inhibition or activation of protein kinase C did not significantly affect the internalization process. Receptor sequestration and ligand uptake was, at least in part, blocked by concanavalin A and blockers of endocytosis mediated by clathrin-coated pits. Our data suggest that the internalization of a G-protein-coupled receptor and coupling to a G-protein can be two independent events. Moreover, the C terminus tail of hPAFR, but not the putative internalization motifs, may be involved in the internalization of hPAFR. The receptor for platelet-activating factor (PAF) is a member of the G-protein-coupled receptor family. To study the structural elements and mechanisms involved in the internalization of human PAF receptor (hPAFR), we used the following mutants: a truncated mutant in the C-terminal tail of the receptor (Cys317→ Stop) and mutations in the (D/N)P(X)2,3Y motif (Asp289 → Asn,Ala and Tyr293 → Phe,Ala). Chinese hamster ovary cells expressing the Cys317→ Stop mutant exhibited a marked reduction in their capacity to internalize PAF, suggesting the existence of determinants important for endocytosis in the last 26 amino acids of the cytoplasmic tail. Substitution of Asp289 to alanine abolished both internalization and G-protein coupling, whereas substitution of Tyr293 to alanine abolished coupling but not internalization. Inhibition or activation of protein kinase C did not significantly affect the internalization process. Receptor sequestration and ligand uptake was, at least in part, blocked by concanavalin A and blockers of endocytosis mediated by clathrin-coated pits. Our data suggest that the internalization of a G-protein-coupled receptor and coupling to a G-protein can be two independent events. Moreover, the C terminus tail of hPAFR, but not the putative internalization motifs, may be involved in the internalization of hPAFR. Platelet-activating factor (PAF) 1The abbreviations used are: PAF, platelet-activating factor; PAFR, PAF receptor; BSA, bovine serum albumin; G-protein, GTP-binding regulatory protein; GRK, G-protein-coupled receptor kinase; hPAFR, human platelet-activating factor receptor; PBS, phosphate-buffered saline; PKC, protein kinase C; PLC, phospholipase C; WT, wild type; CHO, Chinese hamster ovary; IP, inositol phosphate; RT, room temperature; PMA, phorbol 12-myristate 13-acetate.1The abbreviations used are: PAF, platelet-activating factor; PAFR, PAF receptor; BSA, bovine serum albumin; G-protein, GTP-binding regulatory protein; GRK, G-protein-coupled receptor kinase; hPAFR, human platelet-activating factor receptor; PBS, phosphate-buffered saline; PKC, protein kinase C; PLC, phospholipase C; WT, wild type; CHO, Chinese hamster ovary; IP, inositol phosphate; RT, room temperature; PMA, phorbol 12-myristate 13-acetate. is a potent phospholipid mediator that produces a wide range of biological responses through activation of a specific receptor on target cell surface (1Braquet P. Rola-Pleszczynski M. Prostaglandins. 1987; 34: 143-148Crossref PubMed Scopus (35) Google Scholar, 2Braquet P. Touqui L. Shen T.Y. Vargaftig B.B. Pharmacol. Rev. 1987; 39: 97-145PubMed Google Scholar). The PAF receptor is part of the large family of seven transmembrane domain receptors (3Sugimoto T. Tsuchimochi H. McGregor C.G.A. Mutoh H. Shimizu T. Kurachi Y. Biochem. Biophys. Res. Commun. 1992; 189: 617-624Crossref PubMed Scopus (83) Google Scholar, 4Nakamura 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, 5Kunz D. Gerard N.P. Gerard C. J. Biol. Chem. 1992; 267: 9101-9106Abstract Full Text PDF PubMed Google Scholar, 6Ye R.D. Prossnitz E.R. Zou A. Cochrane C.G. Biochem. Biophys. Res. Commun. 1991; 180: 105-111Crossref PubMed Scopus (166) Google Scholar, 7Chase P.B. Halonen M. Regan J.W. Am. J. Resp. Cell Mol. Biol. 1993; 8: 240-244Crossref PubMed Scopus (40) Google Scholar). These receptors, when exposed to an agonist, activate a heterotrimeric G-protein that in turn activates and regulates events leading to cellular responses. Following repeated or prolonged agonist stimulation, their signal transduction potential becomes modulated and attenuated, a phenomenon termed desensitization (8Dohlman H.G. Thorner J. Caron M.G. Lefkowitz R.J. Annu. Rev. Biochem. 1991; 60: 653-688Crossref PubMed Scopus (1133) Google Scholar). Three distinct events participate in the process of desensitization as follows: the functional uncoupling of the receptor from the G-protein, internalization or sequestration of the receptor from the cell surface toward the intracellular compartments, and finally, the down-regulation of receptor numbers, involving the degradation of the protein in lysosomes (9Nathanson N.M. Brown J.H. The Muscarinic Receptors. Humana Press Inc., Clifton Park, NY1989: 419-454Crossref Google Scholar). The role of phosphorylation in receptor desensitization has been widely studied. It seems clear, at this time, that phosphorylation by second messenger kinases (PKC, PKA, PKG) and the GRKs (G-protein-coupled receptor kinases) helps to maintain the receptors uncoupled from the G-protein (10Hargrave P. Hamm H. Sibley D.R. Housley M.D. Regulation of Signal Transduction Pathways by Desensitization and Amplification. John Wiley & Sons, Inc., New York1994: 25-67Google Scholar, 11Liggett S.B. Lefkowitz R.J. Sibley D.R. Housley M.D. Regulation of Signal Transduction Pathways by Desensitization and Amplification. John Wiley & Sons, Inc., New York1994: 71-98Google Scholar, 12Hausdorff W.P. Bouvier M. O'Dowd B.F. Irons G.P. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1989; 264: 12657-12665Abstract Full Text PDF PubMed Google Scholar, 13Rockman H.A. Choi D.-J. Rahman N.U. Akhter S.A. Lefkowitz R.J. Koch W.J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9954-9959Crossref PubMed Scopus (180) Google Scholar). However, the role of phosphorylation in receptor sequestration and down-regulation is less defined and seems to vary from receptor to receptor (14Eason M.G. Moreira S.P. Ligget S.B. J. Biol. Chem. 1995; 270: 4681-4688Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 15Pals-Rylaarsdam R. Xu Y. Witt-Enderby P. Benovic J.L. Hosey M.M. J. Biol. Chem. 1995; 270: 29004-29011Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). Receptor internalization permits ligand entry and seems to participate in resensitization as well as desensitization (16Goldstein J.L. Brown M.S. Anderson R.G.W. Schneider W.J. Annu. Rev. Cell. Biol. 1985; 1: 1-39Crossref PubMed Scopus (1111) Google Scholar, 17Pearse B.M. Robinson M.S. Annu. Rev. Cell. Biol. 1990; 6: 151-171Crossref PubMed Scopus (535) Google Scholar, 18Trowbridge I.S. Curr. Opin. Cell. Biol. 1991; 3: 634-641Crossref PubMed Scopus (128) Google Scholar, 19Smythe E. Waren G. Eur. J. Biochem. 1991; 202: 689-699Crossref PubMed Scopus (138) Google Scholar, 20Vaux D. Trends Cell Biol. 1992; 2: 189-192Abstract Full Text PDF PubMed Scopus (37) Google Scholar). The internalization process can be mediated by clathrin-coated vesicles for some receptors (17Pearse B.M. Robinson M.S. Annu. Rev. Cell. Biol. 1990; 6: 151-171Crossref PubMed Scopus (535) Google Scholar, 21Ghinea N. Vu Hai M.T. Groyer-Picard M.T. Schoevaert D. Milgrom E. J. Cell Biol. 1992; 118: 1347-1358Crossref PubMed Scopus (87) Google Scholar), but not for others (22Raposo G. Dunia I. Delavier-Klutchko C. Kaveri Strosberg A.D. Benedetti E.L. J. Cell Biol. 1989; 50: 340-352Google Scholar, 23Raposo G. Dunia I. Marullo S. Andre C. Guillet J.G. Strosberg A.D. Benedetti E.L. Hoebeke J. Biol. Cell. 1987; 60: 117-123Crossref PubMed Scopus (72) Google Scholar), and a phosphorylation step may or may not be essential (24Hausdorff W.P. Cambell P.T. Ostrowski J. Yu S.S. Caron M.G. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2979-2983Crossref PubMed Scopus (139) Google Scholar). The structural elements involved in this phenomenon can be found in the intracellular loops (25Van Koppen C.J. Lenz W. Nunes J.P.L. Zhang C. Schmidt M. Jakobs K.H. Eur. J. Biochem. 1995; 234: 536-541Crossref PubMed Scopus (10) Google Scholar, 26Jockers R. Da Silva A. Strosberg A.D. Bouvier M. Marullo S. J. Biol. Chem. 1996; 271: 9355-9362Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 27Lameh J. Philip M. Sharma Y.K. Moro O. Ramachandran J. Sadee W. J. Biol. Chem. 1992; 267: 13406-13412Abstract Full Text PDF PubMed Google Scholar) as well as the C-terminal tail (28Nussenveig D.R. Heinflink M. Gershengorn M.C. J. Biol. Chem. 1993; 268: 2389-2392Abstract Full Text PDF PubMed Google Scholar, 29Rodriguez M.C. Xie Y.-B. Wang H. Collison K. Segaloff D.L. Mol. Endocrinol. 1992; 6: 327-336PubMed Google Scholar, 30Hermans E. Octave J.-N. Maloteaux J.-M. Mol. Pharmacol. 1996; 49: 365-372PubMed Google Scholar, 31Thomas G.W. Baker K.M. Motel T.J. Thekkumkara T.J. J. Biol. Chem. 1995; 270: 22153-22159Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 32Benya R.V. Fathi Z. Battey J.F. Jensen R.T. J. Biol. Chem. 1993; 268: 20285-20290Abstract Full Text PDF PubMed Google Scholar) of the individual receptors. No consensus motif has as yet been found, although both positive (28Nussenveig D.R. Heinflink M. Gershengorn M.C. J. Biol. Chem. 1993; 268: 2389-2392Abstract Full Text PDF PubMed Google Scholar, 30Hermans E. Octave J.-N. Maloteaux J.-M. Mol. Pharmacol. 1996; 49: 365-372PubMed Google Scholar, 31Thomas G.W. Baker K.M. Motel T.J. Thekkumkara T.J. J. Biol. Chem. 1995; 270: 22153-22159Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 32Benya R.V. Fathi Z. Battey J.F. Jensen R.T. J. Biol. Chem. 1993; 268: 20285-20290Abstract Full Text PDF PubMed Google Scholar) and negative (29Rodriguez M.C. Xie Y.-B. Wang H. Collison K. Segaloff D.L. Mol. Endocrinol. 1992; 6: 327-336PubMed Google Scholar, 33Parker E.M. Swigart P. Nunnally M.H. Perkins J.P. Ross E.M. J. Biol. Chem. 1995; 270: 6482-6487Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar) regulatory elements have been identified for some receptors. The motif (D/N)P(X)2,3Y is found at the end of the 7th transmembrane domain of most receptors of this family and seems involved in the internalization process of some (17Pearse B.M. Robinson M.S. Annu. Rev. Cell. Biol. 1990; 6: 151-171Crossref PubMed Scopus (535) Google Scholar, 34Barak L.S. Tiberi M. Freedman N.J. Kwatra M.M. Lefkowitz R.J. Caron M.G. J. Biol. Chem. 1994; 269: 2790-2795Abstract Full Text PDF PubMed Google Scholar) but not all (35Slice L.W. Wong H.C. Sternini C. Grady E.F. Bunnett N.W. Walsh J.H. J. Biol. Chem. 1994; 269: 21755-21762Abstract Full Text PDF PubMed Google Scholar, 36Hunyady L. Bor M. Baukal A.J. Balla T. Catt K.J. J. Biol. Chem. 1995; 270: 16602-16609Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 37Laporte S.A. Servant G. Richard D.E. Escher E. Guillemette G. Leduc R. Mol. Pharmacol. 1996; 49: 89-95PubMed Google Scholar) receptors. In addition, the substitution of residues in this motif was found to affect other functions of the receptor, such as G-protein coupling, affinity for the ligand (37Laporte S.A. Servant G. Richard D.E. Escher E. Guillemette G. Leduc R. Mol. Pharmacol. 1996; 49: 89-95PubMed Google Scholar, 38Barak L.S. Ménard L. Fergusson S.G. Colapietro A.-M. Caron M.G. Biochemistry. 1995; 34: 15407-15414Crossref PubMed Scopus (139) Google Scholar), and even the phosphorylation level of the receptor. These results suggest a global role for this region in the conservation of adequate receptor conformation. It has also been shown that the medium chains (μ1 and μ2) of clathrin-associated protein complexes AP-1 and AP-2 can specifically interact with a tyrosine-based signal YXXΦ (Φ is an amino acid with a bulky hydrophobic side chain) motif (39Ohno H. Stewart J. Fournier M.-C. Bosshart H. Rhee I. Miyatake S. Saito T. Gallusser A. Kirchhausen T. Bonifacino J.S. Science. 1995; 269: 1872-1875Crossref PubMed Scopus (824) Google Scholar). Both these motifs are found in the sequenceDPVIYCFL present in the hPAFR. It has been shown that the PAFR undergoes a ligand-specific, temperature-dependent internalization in transfected cells (40Ye R.D. Prossnitz E.R. Zou A. Cochrane C.G. Biochem. Biophys. Res. Commun. 1991; 180: 105-111Crossref PubMed Scopus (164) Google Scholar). The aim of our study was to identify the structural elements and mechanisms involved in the internalization of the hPAFR. The role of the two motifs (D/N)P(X)2,3Y and YXXΦ was evaluated with the mutant receptors Asp289 → Ala,Asn (41Parent J.-L. Le Gouill C. Escher E. Rola-Pleszczynski M. Stankova J. J. Biol. Chem. 1996; 271: 23298-23303Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar) and Tyr293 → Ala,Phe (this study). The presence of regulatory elements in the cytoplasmic tail was verified with the mutant Cys317 → Stop that does not contain the last 26 amino acids of the C-terminal tail. The involvement of clathrin-coated vesicles in hPAFR internalization was addressed with internalization blockers. Finally, to determine whether signalization was an important step in the initiation or regulation of the hPAFR internalization process, certain inhibitors and an activator of PKC, in addition to uncoupled mutants of the receptor, D63N (43Parent J.L. Le Gouill C. Rola-Pleszczynski M. Stankova J. Biochem. Biophys. Res. Commun. 1996; 219: 968-975Crossref PubMed Scopus (24) Google Scholar), A230E (44Parent J.-L. Le Gouill C. de Brum-Fernandes A.J. Rola-Pleszczynski M. Stankova J. J. Biol. Chem. 1996; 271: 7949-7955Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar), D289A (41Parent J.-L. Le Gouill C. Escher E. Rola-Pleszczynski M. Stankova J. J. Biol. Chem. 1996; 271: 23298-23303Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar), and Y293A (this study), were used. Reagents were obtained from the following sources: oligonucleotides were synthesized at Life Technologies, Inc., Pwo polymerase was from Boehringer Mannheim, restriction endonucleases and T4 DNA ligase were from Promega and Pharmacia Biotech Inc., bovine serum albumin, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, pertussis toxin, phenylarsine oxide, and concanavalin A were from Sigma, staurosporine and calphostin C were from BIOMOL Research Laboratories, Inc., NH4Cl and sucrose were from Fisher, lipid-free bovine serum albumin and hexadecyl-PAF were from Calbiochem, AG1-X8 resin (in formate form, 100–200 mesh) was from Bio-Rad, cell culture media and LipofectAMINE were from Life Technologies, Inc., [3H]hexadecyl-PAF andmyo-[2-3H]inositol were from Amersham Corp., and 3H-WEB2086 was purchased from NEN Life Science Products. The pJ3M expression vector (45Sells M.A. Chernoff J. Gene ( Amst .). 1995; 152: 187-189Crossref PubMed Scopus (65) Google Scholar) and the clone Kp132 containing the hPAFR cDNA (40Ye R.D. Prossnitz E.R. Zou A. Cochrane C.G. Biochem. Biophys. Res. Commun. 1991; 180: 105-111Crossref PubMed Scopus (164) Google Scholar) were kindly provided by Dr. J. Chernoff (Fox Chase Cancer Center, Philadelphia, PA) and Dr. Richard Ye (The Scripps Research Institute, La Jolla, CA), respectively. A tagged hPAFR cDNA was generated by polymerase chain reaction (46Higuchi R. Innis M.A. Gelfand D.H. Sninski J.J. White T.J. PCR Protocols. Academic Press, New York1990: 177-183Google Scholar) from Kp132 using the oligonucleotide 5′-CCACATGACTCCTCCCACATG-3′ and the M13 sequencing primer (5′-GTAAAACGACGGCCAGT-3′). The resulting fragment was then digested with Acc65I and subcloned into theEco1CR1-Acc65I sites of the pJ3M vector. In this construction, the N-terminal initiator methionine was replaced by the peptide sequence MEQKLISEEDLSRGSPG, resulting in a c-mycepitope-tagged PAF receptor coding sequence. Mutated receptors were constructed by polymerase chain reaction using Kp132 as template. Tyr293 → Phe and Tyr293 → Ala substitutions were created using the oligonucleotides 5′-TGTTATCTTCTGTTTCC-3′ and 5′-CCTGTTATCGCCTGTTTCC-3′ with their reverse complements, respectively. The point mutation was then introduced in the epitope-tagged receptor coding sequence using BstEII andAcc65I restriction enzymes. Truncated forms, Cys317 → Stop and Lys298 → Stop, of the receptor were also constructed. To construct these mutants the polymerase chain reaction product generated with the oligonucleotides 5′-CCACATGACTCCTCCCACATG-3′ and 5′-GCCCGGGATCATTTCCGG-3′ (Cys317 → Stop) or 5′-CCAATTCTAGGTGAGGAAAC-3′ (Lys298 → Stop) were subcloned in the siteEco1CR1 of pJ3M vector. Mutations and the integrity of the coding sequence were confirmed by dideoxy sequencing (University of Alberta, Alberta, Canada). COS-7 and CHO cells were grown in Dulbecco's modified Eagle's medium high glucose and Dulbecco's modified Eagle's medium F12, respectively, supplemented with 10% fetal bovine serum. Cells were plated in 30-mm dishes (2.0 × 105 COS-7 cells/dish or 3.0 × 105 CHO cells/dish), transiently transfected with the constructions encoding the WT and the mutant receptors using 4 μl of LipofectAMINE and 1 μg of DNA per dish and harvested 48 h after transfection. For studies of biphasic isotherms, 100-mm dishes with 3 × 106 CHO cells/dish were transfected with constructions encoding the WT and Y293A mutant using 30 μl of LipofectAMINE and 4 μg of DNA. Stably transfected CHO cells (44Parent J.-L. Le Gouill C. de Brum-Fernandes A.J. Rola-Pleszczynski M. Stankova J. J. Biol. Chem. 1996; 271: 7949-7955Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar) were grown in Dulbecco's modified Eagle's medium F12 containing 400 μg/ml G418. Competition binding curves were done on COS-7 cells expressing the WT and mutant receptor species. 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 mm NaH2PO4, 25 mm Hepes, pH 7.4) containing 0.1% (w/v) bovine serum albumin (BSA) (47Honda Z. Takano T. Gotoh Y. Nishida E. Ito K. Shimizu T. J. Biol. Chem. 1994; 269: 2307-2315Abstract Full Text PDF PubMed Google Scholar). Binding reactions were carried out on 5 × 104 cells in a total volume of 0.25 ml in the same buffer with 10 nm3H-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. COS-7 cells were transfected as described above with the WT or mutant receptors and labeled the following day for 18–24 h withmyo-[3H]inositol at 5 μCi/ml in Dulbecco's modified Eagle's medium (high glucose, without inositol). After labeling, cells were washed once in phosphate-buffered saline (PBS), pretreated or not with the indicated inhibitors (concanavalin A (0.25 mg/ml, 20 min), sucrose (0.45 m, 20 min), NH4Cl (10 mm, 10 min), phenylarsine oxide (80 μm, 5 min with 30-min rest)) and preincubated 5 min in PBS at 37 °C. At the end of this preincubation period, the PBS was removed, and cells were incubated in pre-warmed Dulbecco's modified Eagle's medium (high glucose, without inositol) containing 0.1% BSA and 20 mmLiCl for 5 min. Cells were then stimulated for 30 s with indicated concentrations of PAF. The reactions were terminated with the addition of perchloric acid followed by a 30-min incubation on ice. Inositol phosphates were extracted (48Martin T.J.F. J. Biol. Chem. 1983; 258: 14816-14822Abstract Full Text PDF PubMed Google Scholar) and separated on Dowex AG1-X8 columns (49Berridge M.T. Dawson R.M.C. Downes C.P. Hyslop J.P. Irvine R.F. Biochem. J. 1983; 212: 473-482Crossref PubMed Scopus (1541) Google Scholar). Total labeled inositol phosphates were then counted by liquid scintillation. The evaluation of ligand internalization kinetics was done on CHO cells transiently transfected in 12-well dishes with constructions encoding mutant and WT receptors. 48 h after transfection, cells were pretreated or not with the indicated inhibitors (pertussis toxin (150 ng/ml, 20 h), concanavalin A (0.25 mg/ml, 20 min), sucrose (0.45 m, 20 min), NH4Cl (10 mm, 10 min), phenylarsine oxide (80 μm, 5 min with 30 min rest), calphostin C (2 μm, 20 min), staurosporine (3 μm, 20 min), and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (10 μm, 20 min]) or 4α-PMA (inactive) or 4β-PMA (active) (80 nm, 30 min)) and then incubated at 37 °C with 2 nm [3H]hexadecyl-PAF in a buffer containing 150 mm choline chloride, 10 mm Tris-HCl, pH 7.5, 10 mm MgCl2, and 0.25% lipid-free BSA (40Ye R.D. Prossnitz E.R. Zou A. Cochrane C.G. Biochem. Biophys. Res. Commun. 1991; 180: 105-111Crossref PubMed Scopus (164) Google Scholar) for 5, 10, 20, 30, 45, or 100 min. After the incubation period, cells were washed two times with 1 ml of the same buffer but containing 2% BSA. Cells were then lysed in 0.1 n NaOH, and internalized radioactivity was evaluated by liquid scintillation. Stably transfected CHO cells were grown on coverslips (25 mm). The cells were incubated in the presence or absence of sucrose and/or PAF (0.5 μm) for 20 min and fixed with 4% paraformaldehyde (15 min at RT). The coverslips were then placed in 0.1% Triton (20 min RT) and then sequentially incubated with 5% milk (30 min RT) and 0.1% glycine (60 min RT). The cells were then incubated with anti-PAFR antibodies (polyclonal, directed at the C-terminal tail; Cayman Chemical, Ann Arbor, MI) followed with rhodamine-conjugated goat anti-rabbit antibodies (Bio/Can Scientific, Mississauga, Ontario). The cells were then 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 workstation equipped with Molecular Dynamics' Imagespace analysis software. The mutants used in the present report are illustrated in Fig. 1. The deletion mutants, Cys317 → Stop and Lys298 → Stop, were made to analyze the involvement of the cytoplasmic tail in the internalization of the receptor. The tyrosine 293 putatively involved in the motifs (N/D)P(X)2,3Y and YXXΦ was substituted with phenylalanine and alanine. The role of receptor-mediated signal transduction in internalization was studied with the help of G-protein-uncoupled mutants that were distributed in distinct areas of the receptor (D63N, A230E, D289A, and Y293A). The binding characteristics of the WT receptor and the mutants, Cys317 → Stop, Y293A, and Y293F, were examined in transiently transfected COS-7 cells using the agonist (PAF) and an antagonist (WEB2086) (Fig. 2). The Lys298 → Stop mutant was not illustrated as it did not bind either of the ligands, despite comparable cell-surface expression to the WT construct, as examined by cytofluorimetry with an anti-c-myc antibody (results not shown). No significant difference was found in the affinities for WEB2086 (Fig. 2 A) or PAF (Fig. 2 B) between the different mutants and the WT receptor. Similar levels of expression were also obtained in COS-7 as well as in CHO cells (WT, 962,310 ± 163,592; Y293A, 961,945 ± 146,532; Y293F, 857,332 ± 155,432 receptors/CHO cell) except for Cys317 → Stop (342,578 ± 68,515 receptors/CHO cell) which was expressed at a lower level. To further characterize these receptors, the signaling response of the mutant (Cys317 → Stop, Y293A, and Y293F) and WT receptors was measured by IP accumulation (Fig. 3 A). In COS-7 cells, hPAFR-induced IP production would be mediated by PLCβ1 activation via Gq/11 as the IP production is not blocked by pertussis toxin or tyrosine kinase inhibitors (44Parent J.-L. Le Gouill C. de Brum-Fernandes A.J. Rola-Pleszczynski M. Stankova J. J. Biol. Chem. 1996; 271: 7949-7955Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Transiently transfected COS-7 cells were exposed for 30 s to graded concentrations of PAF, and total IP accumulation was measured. As shown, the Cys317→ Stop deletion of the cytoplasmic tail did not impair the response of the receptor. Since nearly half the potential phosphorylation sites of the cytoplasmic tail were eliminated by this deletion, the slightly augmented response of this mutant could reflect the role played by these residues in controlling the response to PAF. On the other hand, the Y293A mutant did not generate an IP response, except at very high concentrations of PAF (1 μm). In contrast, the substitution of Y293F had no significant effect on the response of the receptor. When conditions of transfection are such that low levels of hPAFR receptors are expressed (44Parent J.-L. Le Gouill C. de Brum-Fernandes A.J. Rola-Pleszczynski M. Stankova J. J. Biol. Chem. 1996; 271: 7949-7955Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar), receptors can be observed in both a G-protein-coupled state (high affinity) and uncoupled state (low affinity). As shown in Fig. 3 B, under these conditions of receptor expression, the mutant Y293A appears to exist only in its uncoupled, low affinity state, when compared with the WT, which might explain its inability to activate PLC after PAF stimulation. The effects of different inhibitors of internalization on receptor sequestration and endocytosis of 3H-PAF are illustrated in Fig. 4. After a 20-min stimulation of stably transfected hPAFR-CHO cells with 0.5 μm PAF, sequestration was studied by confocal microscopy with an antibody raised against a peptide sequence of the C-terminal portion of the hPAFR (Fig. 4 A). Cells were pretreated with medium (a and c) or with hyperosmolar concentration of sucrose (b and d) to block clathrin-mediated internalization before stimulation with PAF (c andd). PAF induced the translocation of receptors toward the intracellular compartment (c), whereas sucrose blocked the process of hPAFR sequestration (d). In addition, sucrose as well as other inhibitors of clathrin-mediated endocytosis significantly blocked the internalization of3H-PAF (Fig. 4 B), indicating that receptor-specific endocytosis of PAF is mediated via clathrin-coated vesicles. Fig. 4 C shows that inhibition of PAFR endocytosis was not a nonspecific effect of the inhibitors on ligand binding or receptor signal transduction. The level of IP accumulation was measured after PAF stimulation in the presence of the inhibitors, which, except for phenylarsine oxide, did not significantly inhibit PAFR response. In fact, the apparent decrease in PAF-induced response in cells pretreated with phenylarsine oxide was due to enhanced basal levels of IP production. The different mutants were compared with the WT receptor for their capacity to internalize 3H-PAF (Fig. 5). The Lys298 → Stop mutant was used as a control for non-receptor-mediated3H-PAF internalization since it did not detectably bind PAF. The results indicate that the majority of mutants that are uncoupled from G-proteins (Fig. 5 A) were impaired in their capacity to internalize PAF, except for the Y293A mutant. This mutant indicated that receptor signalization does not participate in ligand-mediated internalization of hPAFR. Although PAF-induced mitogen-activated protein kinase activation in guinea pig PAFR-transfected CHO cells was reported to be pertussis toxin-sensitive (47Honda Z. Takano T. Gotoh Y. Nishida E. Ito K. Shimizu T. J. Biol. Chem. 1994; 269: 2307-2315Abstract Full Text PDF PubMed Google Scholar), internalization of human PAFR in stably transfected CHO cells was not affected by pertussis toxin treatment (data not illustrated). In contrast, the Cys317 → Stop mutant was impaired in its capacity to mediate PAF internalization (Fig. 5 B), despite its efficient signal transduction, indicating that the last 26 amino acids of the C-terminal tail could be important for internalization. As Cys317 → Stop is expressed less than the WT, we expressed the WT at different levels and compared the internalization. When the WT is expressed at 30% maximal level (comparable to Cys317→ Stop expression), its internalization is only 25% less than maximal WT internalization, whereas Cys317 → Stop internalizes 90% less than the WT. In addition, our results indicate (Fig. 5 B) that the substitution of the residues Asp289 or Tyr293, within the putative internalization motifs (N/D)P(X)2,3Y and YXXΦ, by conserved residues (Asn and Phe, respectively) partially decreased but did not abolish the potential for internalization. Phosphorylation by PKC does not appear to be involved in the initiation or control of PAFR internalization. Modulation of PKC activity by inhibitors (calphostin C, staurosporine, and H7) or an activator, in its active 4β-PMA or inactive 4α-PMA form, did not affect PAF-induced internalization (Fig. 6). In agreement with data obtained by Takano et al. (50Takano 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), with a truncated guinea pig PAF receptor, our data indicate that the last 26 amino acids of the C-terminal end of the hPAFR are not required

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