Agonist-independent Desensitization and Internalization of the Human Platelet-activating Factor Receptor by Coumermycin-Gyrase B-induced Dimerization
2003; Elsevier BV; Volume: 278; Issue: 30 Linguagem: Inglês
10.1074/jbc.m212302200
ISSN1083-351X
AutoresAmélie Perron, Zhangguo Chen, Denis Gingras, Denis J. Dupré, Jana Staňková, Marek Rola‐Pleszczynski,
Tópico(s)Protein Kinase Regulation and GTPase Signaling
ResumoPlatelet-activating factor (PAF) is a phospholipid with potent and diverse physiological actions, particularly as a mediator of inflammation. We have reported previously that mutant G protein-coupled receptors (GPCRs) affect the functional properties of coexpressed wild-type human PAF receptor (hPAFR) (Le Gouill, C., Parent, J. L., Caron, C. A., Gaudreau, R., Volkov, L., Rola-Pleszczynski, M., and Stankova, J. (1999) J. Biol. Chem. 274, 12548–12554). Increasing evidence suggests that dimerization of GPCRs may play an important role in the regulation of their biological activity. Additional data have also suggested that dimerization may be important in the subsequent internalization of the δ-opioid receptor. To investigate the specific role of dimerization in the internalization process of GPCRs, we generated a fusion protein of hPAFR and bacterial DNA gyrase B (GyrB), dimerized through the addition of coumermycin. We found that dimerization potentiates PAF-induced internalization of hPAFR-GyrB in Chinese hamster ovary cells stably expressing c-Myc-hPAFR-GyrB. Coumermycin-driven dimerization was also sufficient to induce an agonist-independent sequestration process in an arrestin- and clathrin-independent manner. Moreover, the protein kinase C inhibitors staurosporine and GF109203X blocked the coumermycin-induced desensitization of hPAFR-GyrB, suggesting the implication of protein kinase C in the molecular mechanism mediating the agonist-independent desensitization of the receptor. Taken together, these findings suggest a novel mechanism of GPCR desensitization and internalization triggered by dimerization. Platelet-activating factor (PAF) is a phospholipid with potent and diverse physiological actions, particularly as a mediator of inflammation. We have reported previously that mutant G protein-coupled receptors (GPCRs) affect the functional properties of coexpressed wild-type human PAF receptor (hPAFR) (Le Gouill, C., Parent, J. L., Caron, C. A., Gaudreau, R., Volkov, L., Rola-Pleszczynski, M., and Stankova, J. (1999) J. Biol. Chem. 274, 12548–12554). Increasing evidence suggests that dimerization of GPCRs may play an important role in the regulation of their biological activity. Additional data have also suggested that dimerization may be important in the subsequent internalization of the δ-opioid receptor. To investigate the specific role of dimerization in the internalization process of GPCRs, we generated a fusion protein of hPAFR and bacterial DNA gyrase B (GyrB), dimerized through the addition of coumermycin. We found that dimerization potentiates PAF-induced internalization of hPAFR-GyrB in Chinese hamster ovary cells stably expressing c-Myc-hPAFR-GyrB. Coumermycin-driven dimerization was also sufficient to induce an agonist-independent sequestration process in an arrestin- and clathrin-independent manner. Moreover, the protein kinase C inhibitors staurosporine and GF109203X blocked the coumermycin-induced desensitization of hPAFR-GyrB, suggesting the implication of protein kinase C in the molecular mechanism mediating the agonist-independent desensitization of the receptor. Taken together, these findings suggest a novel mechanism of GPCR desensitization and internalization triggered by dimerization. Platelet-activating factor (PAF) 1The abbreviations used are: PAF, platelet-activating factor; Ab(s), antibody(ies); arr, arrestin; BSA, bovine serum albumin; CHO, Chinese hamster ovary; GFP, green fluorescent protein; GPCR(s), G protein-coupled receptor(s); GyrB, bacterial DNA gyrase B; h, human; HA, hemagglutinin; IP, inositol phosphate(s); PAFR, PAF receptor; PBS, phosphate-buffered saline; PKC, protein kinase C; PMA, phorbol 12-myristate, 13-acetate.1The abbreviations used are: PAF, platelet-activating factor; Ab(s), antibody(ies); arr, arrestin; BSA, bovine serum albumin; CHO, Chinese hamster ovary; GFP, green fluorescent protein; GPCR(s), G protein-coupled receptor(s); GyrB, bacterial DNA gyrase B; h, human; HA, hemagglutinin; IP, inositol phosphate(s); PAFR, PAF receptor; PBS, phosphate-buffered saline; PKC, protein kinase C; PMA, phorbol 12-myristate, 13-acetate. is a phospholipid with potent chemoattractant and leukocyte activating properties (1Snyder F. Am. J. Physiol. 1990; 259: 697-708Crossref PubMed Google Scholar, 2Venable M.E. Zimmerman G.A. McIntyre T.M. Prescott S.M. J. Lipid Res. 1993; 34: 691-702Abstract Full Text PDF PubMed Google Scholar). PAF is thought to be a mediator of cell-to-cell communication, which may function either as an intercellular or an intracellular messenger (3Chao W. Olson M.S. Biochem. J. 1993; 292: 617-629Crossref PubMed Scopus (414) Google Scholar). Its presence has been associated with a number of inflammatory and allergic responses that may result in pathologic events that affect the cardiovascular, cerebral, respiratory, gastrointestinal, and pancreatic systems, as well as transplanted tissues (4Evangelou A.M. Prostaglandins Leukotrienes Essent. Fatty Acids. 1994; 50: 1-28Abstract Full Text PDF PubMed Scopus (94) Google Scholar, 5Imaizumi T.A. Stafforini D.M. Yamada Y. McIntyre T.M. Prescott S.M. Zimmerman G.A. J. Intern. Med. 1995; 238: 5-20Crossref PubMed Scopus (139) Google Scholar). PAF drives its biological activity through a single type of GPCR, referred to as the PAF receptor (PAFR) (6Izumi T. Shimizu T. Biochim. Biophys. Acta. 1995; 1259: 317-333Crossref PubMed Scopus (208) Google Scholar). Upon binding to its receptor, PAF stimulates a number of transduction pathways including turnover of phosphatidylinositol, elevation in intracellular calcium concentration, and activation of protein kinases. The former involves protein kinase C (PKC), protein-tyrosine kinases, and G protein receptor kinase (7Ali H. Richardson R.M. Tomhave E.D. DuBose R.A. Haribabu B. Snyderman R. J. Biol. Chem. 1994; 269: 24557-24563Abstract Full Text PDF PubMed Google Scholar, 8Bito H. Shimizu T. Adv. Exp. Med. Biol. 1997; 400A: 215-221Crossref PubMed Scopus (9) Google Scholar, 9Ishii S. Shimizu T. Prog. Lipid Res. 2000; 39: 41-82Crossref PubMed Scopus (327) Google Scholar). Previous studies have shown that the human (h)PAFR undergoes a ligand-specific and clathrin-dependent internalization in transfected cells (10Ishii 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, 11Chen Z. Dupre D J. Le Gouill C. Rola-Pleszczynski M. Stankova J. J. Biol. Chem. 2002; 277: 7356-7362Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). However, we have reported that G protein coupling and signal transduction are not necessary for hPAFR internalization (12Le 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).Exposure of GPCRs to their agonists is usually followed by a rapid desensitization of signaling (13Bunemann M. Hosey M.M. J. Physiol. 1999; 517: 5-23Crossref PubMed Scopus (163) Google Scholar, 14Ferguson S.S. Pharmacol. Rev. 2001; 53: 1-24PubMed Google Scholar, 15Tsao P. von Zastrow M. Curr. Opin. Neurobiol. 2000; 10: 365-369Crossref PubMed Scopus (133) Google Scholar). A series of distinct events is known to participate in this mechanism, including a functional uncoupling of the receptor from its cognate G protein, sequestration of the receptors into intracellular compartments, and a net loss of receptors (down-regulation). GPCR internalization has become the subject of intensive investigation over the past several years, and a large volume of data has accumulated regarding the mechanisms regulating their endocytosis. The β2-adrenergic receptor and a large number of GPCRshavebeendemonstratedtointernalizethroughadynamin-dependent mechanism involving clathrin-coated pits (16Krupnick J.G. Benovic J.L. Annu. Rev. Pharmacol. Toxicol. 1998; 38: 289-319Crossref PubMed Scopus (855) Google Scholar, 17Lefkowitz R.J. J. Biol. Chem. 1998; 273: 18677-18680Abstract Full Text Full Text PDF PubMed Scopus (903) Google Scholar). There are exceptions to this generalization, however, because some receptors such the dopamine D2 receptor, the angiotensin 1A receptor, and the m2 muscarinic receptor can internalize by an unidentified mechanism that shows an atypical sensitivity to dynamin (18Vickery R.G. von Zastrow M. J. Cell Biol. 1999; 144: 31-43Crossref PubMed Scopus (197) Google Scholar, 19Pals-Rylaarsdam R. Gurevich V.V. Lee K.B. Ptasienski J.A. Benovic J.L. Hosey M.M. J. Biol. Chem. 1997; 272: 23682-23689Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 20Werbonat Y. Kleutges N. Jakobs K.H. van Koppen C.J. J. Biol. Chem. 2000; 275: 21969-21974Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 21Zhang J. Ferguson S.S. Barak L.S. Menard L. Caron M.G. J. Biol. Chem. 1996; 271: 18302-18305Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar). In addition, further analysis revealed that the N-formyl peptide receptor and the C5a chemoattractant receptor are internalized via an arrestin- and dynamin-independent pathway, which leads to questions about an alternative mechanism involved in mediating internalization of these receptors (22Gilbert T.L. Bennett T.A. Maestas D.C. Cimino D.F. Prossnitz E.R. Biochemistry. 2001; 40: 3467-3475Crossref PubMed Scopus (47) Google Scholar).An increasing number of studies have shown that many GPCRs form homodimers as well as heterodimers (23Bouvier M. Nat. Rev. Neurosci. 2001; 4: 274-286Crossref Scopus (578) Google Scholar). Subsequent reports demonstrated homodimerization among the β2-adrenergic receptor (24Hebert T.E. Moffett S. Morello J.P. Loisel T.P. Bichet D.G. Barret C. Bouvier M. J. Biol. Chem. 1996; 271: 16384-16392Abstract Full Text Full Text PDF PubMed Scopus (679) Google Scholar), the δ-opioid receptor (25Cvejic S. Devi L.A. J. Biol. Chem. 1997; 272: 26959-26964Abstract Full Text Full Text PDF PubMed Scopus (414) Google Scholar), the chemokine receptors CCR2b, CCR4, and CCR5 (26Rodriguez-Frade J.M. Vila-Coro A.J. de Ana A.M. Albar J.P. Martinez-A C. Mellado M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3628-3633Crossref PubMed Scopus (200) Google Scholar, 27Rodriguez-Frade J.M. Vila-Coro A.J. Martin A. Nieto M. Sanchez-Madrid F. Proudfoot A.E. Wells T.N. Martinez-A C. Mellado M. J. Cell Biol. 1999; 144: 755-765Crossref PubMed Scopus (109) Google Scholar), the Ca2+-sensing receptor (28Zhang Z. Sun S. Quinn S.J. Brown E.M. Bai M. J. Biol. Chem. 2001; 276: 5316-5322Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar), and the metabotropic glutamate receptor 5 (29Romano C. Yang W.L. O'Malley K.L. J. Biol. Chem. 1996; 271: 28612-28616Abstract Full Text Full Text PDF PubMed Scopus (440) Google Scholar). Agonists have also been shown to stabilize the dimeric form of several receptors including the β2-adrenergic receptor (24Hebert T.E. Moffett S. Morello J.P. Loisel T.P. Bichet D.G. Barret C. Bouvier M. J. Biol. Chem. 1996; 271: 16384-16392Abstract Full Text Full Text PDF PubMed Scopus (679) Google Scholar) as well as the chemokine receptors CCR2b, CCR4, and CCR5 (26Rodriguez-Frade J.M. Vila-Coro A.J. de Ana A.M. Albar J.P. Martinez-A C. Mellado M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3628-3633Crossref PubMed Scopus (200) Google Scholar, 27Rodriguez-Frade J.M. Vila-Coro A.J. Martin A. Nieto M. Sanchez-Madrid F. Proudfoot A.E. Wells T.N. Martinez-A C. Mellado M. J. Cell Biol. 1999; 144: 755-765Crossref PubMed Scopus (109) Google Scholar). This suggests that homodimerization might play a role either directly in the activation mechanism of receptors or in subsequent agonist-dependent internalization as shown for the δ-opioid receptor (25Cvejic S. Devi L.A. J. Biol. Chem. 1997; 272: 26959-26964Abstract Full Text Full Text PDF PubMed Scopus (414) Google Scholar).We have demonstrated previously that coexpression of certain mutant hPAFR receptors with wild-type hPAFR modified specific characteristics of the native receptor, such as basal level of activity, affinity for the ligand, and cell surface expression (30Le Gouill C. Parent J.L. Caron C.A. Gaudreau R. Volkov L. Rola-Pleszczynski M. Stankova J. J. Biol. Chem. 1999; 274: 12548-12554Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). To determine whether artificially induced oligomerization of hPAFR could modulate the internalization process of the receptor, we applied the coumermycin-induced dimerization system (31Farrar M.A. Alberola-Ila J. Perlmutter R.M. Nature. 1996; 383: 178-181Crossref PubMed Scopus (266) Google Scholar), which is based on the binding of coumermycin to the amino-terminal 24-kDa subdomain of the B subunit of bacterial DNA gyrase (GyrB) (32Gilbert E.J. Maxwell A. Mol. Microbiol. 1994; 12: 365-373Crossref PubMed Scopus (67) Google Scholar). A hPAFR-GyrB fusion protein was thus generated, with the GyrB moiety fused to the COOH-terminal tail of hPAFR. We report here that coumermycin treatment on CHO cells stably expressing c-Myc-hPAFR-GyrB led to the formation of dimers/oligomers, which is sufficient to induce the sequestration process by an unknown endocytic machinery. This coumermycin-driven desensitization of hPAFR-GyrB is regulated by PKC as well as a tyrosine kinase- and phospholipase C-independent mechanism. More broadly, these data suggest that signals determined by secondary structure/conformation of the receptor involved in the conversion between the monomeric and the dimeric form of GPCRs may be implicated in the heterologous desensitization process, which may be correlated with the receptor susceptibility to phosphorylation by second messenger-dependent kinases.MATERIALS AND METHODSChemical Reagents—Oligonucleotides, cell culture media, and LipofectAMINE were purchased from Invitrogen. Restriction endonucleases and T4 DNA ligase were from Promega and Amersham Biosciences. The hPAFR cDNA was a generous gift form Dr. R. Ye (University of Chicago), and pcDNA3-Raf-GyrB was kindly provided by Dr. Michael A. Farrar (Merck Research Laboratories, Rahway, NJ). Bovine serum albumin (BSA), tyrphostin 51, paraformaldehyde, coumermycin, and novobiocin were from Sigma. Staurosporine and GF109203X were from BIOMOL Research Laboratories. Dimethyl sulfoxide from Fisher, and lipid-free BSA was from Calbiochem. AG 1-X8 resin was from Bio-Rad, and DEAE-dextran was purchased from Amersham Biosciences. PAF was from the Cayman Chemical Co. (Ann Arbor, MI), [3H]hexadecyl-PAF and myo-[3H]inositol were from Amersham Biosciences. WEB2086 and FuGENE 6 were from Roche Applied Science. [3H]WEB2086 was purchased from PerkinElmer Life Sciences. Antibodies (Abs) used were rabbit polyclonal anti-c-Myc (A-14) (Santa Cruz Biotechnology, Santa Cruz, CA), monoclonal anti-c-Myc (9E10) (ATCC, Manassas, VA), and monoclonal anti-hemagglutinin (HA) (12CA5) (BAbCO, Richmond, CA). Horseradish peroxidase-conjugated goat anti-mouse and donkey anti-rabbit Abs were from Amersham Biosciences. Fluorescein isothiocyanate-conjugated goat anti-mouse and rhodamine-conjugated goat anti-mouse Abs were obtained from Bio/Can Scientific (Mississauga, ON, Canada).Construction of Epitope-tagged PAFR-GyrB—Human PAF receptor was epitope tagged in the amino-terminal extracellular domain with either a c-Myc or a HA epitope, as described previously (12Le 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) and subcloned into the pcDNA3 vector. To construct PAFR-GyrB, NheI, NotI, and ApaI sites were introduced in the pcDNA3-c-Myc-hPAFR by digestion using the oligonucleotides 5′-CTGGCAATTCCCTCAAAAATGCTAGCTAGGCGGCCGCGGGC-3′ corresponding to the last 23 nucleic acids of the hPAFR. The stop codon was then replaced at the NheI-NotI sites with the coding region of GyrB isolated from pcDNA3-Raf-GyrB at XbaI and NotI sites. Constructs were verified by restriction enzyme digestion.Cell Culture and Transfections—COS-7 and CHO cells were maintained at 37 °C in a 5% CO2 atmosphere in Dulbecco's modified Eagle's medium (high glucose) and Dulbecco's modified Eagle's medium F-12 (Ham's medium, high glucose), respectively, supplemented with 5% fetal bovine serum. Cells were grown in 100-mm dishes to 70–80% confluence and transiently transfected the following day with 7 ml of a mixture of 100 μm chloroquine and 0.25 mg/ml DEAE-dextran containing 4 μg of plasmid DNA (pcDNA3-c-Myc-hPAFR, pcDNA3-HA-hPAFR, or pcDNA3-c-Myc-hPAFR-GyrB) in Dulbecco's modified Eagle's medium with 5% fetal bovine serum. After 2 h at 37 °C, the solution was removed, and the cells were treated for 1 min at room temperature with 10% dimethyl sulfoxide in phosphate-buffered saline (PBS), rinsed twice with PBS, and returned to the 37 °C incubator in growth medium supplemented with 5% fetal bovine serum. For confocal microscopy experiments, cells were seeded into 6-well plates (1–1.5 × 105 cells/ml) and transiently transfected with 1 μg of plasmid DNA/well by using the FuGENE 6 transfection reagent (Roche Applied Science) according to the manufacturer's instructions. Stable CHO transfectants expressing the pcDNA3-c-Myc-hPAFR were generated as described previously (33Parent 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). CHO stably transfected with the pcDNA3-c-Myc-hPAFR-GyrB were established as above except that positive cells were selected with a FACS-Vantage cell sorter (BD Biosciences) after labeling with anti-c- Myc monoclonal antibody and fluorescein isothiocyanate-conjugated goat anti-mouse Ab.Immunoprecipitation and Western Blotting—COS-7 were rinsed 48 h after transfection with PBS. Cells were then removed from 100-mm plates and collected by centrifugation in PBS. The cell pellet from each plate was disrupted in 500 μl of radioimmunoprecipitation assay buffer (150 mm NaCl, 50 mm Tris-HCl, pH 7.5, 5 mm EDTA, 1% IGEPAL, 0.5% deoxycholic acid, 0.1% SDS) containing protease inhibitors (1 μg/ml soybean trypsin inhibitors, 1 μm leupeptin, 20 μg/ml aprotinin, 40 μg/ml prefabloc, and 40 μg/ml 1-chloro-3-tosylamido-7-amino-2-heptanone) and incubated 30 min on ice. Lysates were then precleared with 25 μg of protein A-Sepharose for 30 min at 4 °C and incubated with anti-HA or anti-c-Myc Ab overnight at 4 °C. The protein A-Sepharose was shaken gently in lysis buffer containing 1% BSA for 30 min at room temperature before use. Epitope-tagged hPAFRs were precipitated by incubation with 100 μg of protein A-Sepharose for 2 h at 4 °C. After washing three times in lysis buffer, complexes were dissolved in 1 × Laemmli sample buffer (34Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206024) Google Scholar). Immunoprecipitated proteins were then separated by 10% Tris-glycine precast gels (Invitrogen) and transferred electrophoretically to nitrocellulose membranes. Phorbol 12-myristate 13-acetate (PMA)-stimulated cells (80 nm for 0, 30, 60, and 90 min) were lysed in radioimmunoprecipitation assay buffer, and immunoprecipitation was performed as described above. For coumarin experiments, CHO cells stably expressing c-Myc epitope-tagged hPAFR-GyrB and nontransfected cells were resuspended in ice-cold buffer containing 10 mm Tris-HCl, pH 8.0, and 1 mm EDTA. The cells were then disrupted by sonication and pelleted by centrifuging at 12,500 rpm for 30 min at 4 °C. The supernatants were collected as total cell lysates, and protein concentration was determined by the Bio-Rad procedure with BSA as standard. Samples containing 20 μg of protein were resolved using 6% Tris-glycine precast gels under nonreducing conditions. For Western blotting analysis, nitrocellulose membranes were blocked in PBS and 0.1% Tween containing 10% dried milk for 1 h and incubated with anti-HA or anti-Myc in PBS and 1% dried milk 1 h at room temperature. After washing with PBS-Tween and incubation with secondary Abs, a chemiluminescence detection system was used for protein detection (PerkinElmer Life Sciences).Flow Cytometry Studies—Receptor internalization was determined as the level of receptor loss from the cell surface. Stably transfected CHO cells were seeded on 6-well plates (4 × 105 cells/well) 1 day prior to the assay. Cells were then washed with PBS and incubated in Dulbecco's modified Eagle's medium F-12 with the indicated concentration of coumarins for a specific time period. Cells were rinsed once with PBS and treated with 1 μm PAF for the appropriate time in medium containing 0.2% BSA. Cells were then washed once with PBS containing 2% BSA and harvested in ice-cold PBS. Cells were first labeled with or without anti-Myc antibody on ice for 1 h. After washing twice with ice-cold PBS, cells were incubated for an additional 45 min with fluorescein isothiocyanate-conjugated goat anti-mouse Ab on ice and washed as described above. Antibody-labeled cells were analyzed for fluorescence intensity on a FACScan flow cytometer (BD Biosciences) with dead cells excluded by gating on forward and side scatter.Ligand Internalization—The ligand internalization kinetics had been evaluated in CHO cells stably transfected with the c-Myc-hPAFR-GyrB in 12-well plates (2 × 105 cells/well). After 24 h, cells were pretreated or not with coumermycin (15 μm for 10, 45, 80, 120, 180, and 210 min). Cells were 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 0.2% lipid-free BSA for 45 min. After the incubation period, cells were washed twice with 1 ml of the same buffer but containing 2% BSA. Cells were then lysed in 0.1 n NaOH, and internalized radioactivity was measured by liquid scintillation.Confocal Microscopy—COS-7 cells were grown on 25-mm coverslips and transiently transfected with pcDNA3-c-Myc-hPAFR-GyrB and green fluorescent protein (GFP)-conjugated arrestin 2 or arrestin 3 (kind gifts from Dr. J. Benovic, Philadelphia) using FuGENE 6 and processed as described previously (35Lukashova 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). Cells were incubated at 37 °C in the presence or absence of coumermycin or novobiocin (15 μm, 180 min) and then treated or not with 1 μm PAF for 80 min. Cells were fixed with 4% paraformaldehyde (15 min at room temperature) and permeabilized in 0.1% saponin. Cells were then incubated with anti-Myc Ab followed by rhodamine-conjugated goat anti-mouse Ab. Cells were examined, as described (35Lukashova 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), with a scanning confocal microscope (NORAN Instruments, Inc., Middleton, WI) equipped with a krypton/argon laser and coupled to an inverted microscope with a 40 × oil immersion objective (Nikon). Optical sections were collected at 1-μm intervals with a 10-μm pinhole aperture. Digitized images were obtained with 256 × line averaging and enhanced with Intervision software (NORAN Instruments, Inc.) on a Silicon Graphics O2-work station.Inositol Phosphate (IP) Determination—Stably transfected CHO cells expressing c-Myc-hPAFR-GyrB were plated on 6-well dishes (4 × 105 cells/well) and incubated at 37 °C in complete medium 12 h before the assay. Cells were then washed once in PBS and labeled for 18–24 h with myo-[3H]inositol at 3 μCi/ml in Dulbecco's modified Eagle's medium (high glucose, without inositol). After labeling, cells were washed once in PBS, preincubated at 37 °C in prewarmed modified medium containing 20 mm LiCl for 10 min. Medium was then removed, and cells were pretreated, or not, with 15 μm coumermycin or 15 μm novobiocin for the indicated periods of time in modified medium containing 20 mm LiCl. Cells were washed with PBS and incubated in prewarmed medium containing 0.2% BSA and 20 mm LiCl for 10 min with 1 μm PAF. The reactions were terminated with the addition of perchloric acid followed by a 30-min incubation on ice. IPs were extracted as described previously (30Le Gouill C. Parent J.L. Caron C.A. Gaudreau R. Volkov L. Rola-Pleszczynski M. Stankova J. J. Biol. Chem. 1999; 274: 12548-12554Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar) and separated on Dowex AG 1-X8 columns. Total labeled IPs were then counted by liquid scintillation.Radioligand Binding Assay—Competition binding curves were done on CHO cells expressing the c-Myc wild-type or GyrB receptor species, and binding reactions with 10 nm [3H]WEB2086 were done as described before (12Le 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). Internalization experiments were done on stably transfected CHO cells seeded onto 100-mm dishes 24 h prior to assay. Cells were washed once with PBS and pretreated or not with the indicated inhibitors (3 μm staurosporine, 20 min; 2 μm GF109203X, 30 min; and 10 μm tyrphostin 51, 25 min) or 80 nm PKC activator PMA, 30 min at 37 °C in Dulbecco's modified Eagle's medium F-12. Cells were then washed with PBS and incubated at 37 °C with 15 μm coumarins for the indicated periods of time. Binding reactions were done on ice as described above.RESULTSDimerization of hPAFR—To study whether hPAFR can form dimers, albeit under conditions of overexpression, we used COS-7 cells transiently expressing c-Myc epitope-tagged hPAFR. Immunoblotting of c-Myc epitope-tagged receptor with rabbit anti-c-Myc antibody consistently revealed the presence of molecular species corresponding to the expected positions of the monomeric receptor (38–45 kDa). Forms corresponding to twice that of the monomer (80–85 kDa) were also observed, suggesting the existence of an SDS-resistant dimeric species of hPAFR (Fig. 1A, lane 2). Higher molecular mass forms of hPAFR were also detected, possibly corresponding to putative higher order structures, such as receptor tetramers. No immunoreactive bands were detected in lysates prepared from non-transfected cells (Fig. 1A, lane 1). The identity of the dimer was confirmed by immunoprecipitation experiments using differentially tagged receptors. In cells coexpressing both HA- and c-Myc-tagged hPAFR, blotting of the anti-c-Myc immunoprecipitate with the anti-HA antibody revealed the presence of hPAFR, indicating that dimerization occurred between the two different epitope-tagged receptors (Fig. 1B, lane 1). The specificity of the antibodies is illustrated by absence of cross-reactivity in cells expressing only one tagged receptor species (Fig. 1B, lane 2).Characterization of the hPAFR-GyrB—To study the effect of artificially induced dimerization of hPAFR on cells, we constructed a chimeric cDNA encoding hPAFR and GyrB as a fusion protein (hPAFR-GyrB) and generated a stable c-Myc-hPAFR-GyrB-expressing CHO cell line. Coumermycin acts as a natural dimerizer of GyrB because it binds GyrB with a stoichiometry of 1:2, whereas a related coumarin antibiotic, novobiocin, binds GyrB as a 1:1 complex and thus serves as a nondimerizing control (36Ali J.A. Jackson A.P. Howells A.J. Maxwell A. Biochemistry. 1993; 32: 2717-2724Crossref PubMed Scopus (309) Google Scholar). We next determined the effect of fusing GyrB on hPAFR properties by comparing CHO cells stably expressing the c-Myc-wild-type hPAFR with CHO cells stably transfected with c-Myc-hPAFR-GyrB. Flow cytometry studies showed that wild-type hPAFR and hPAFR-GyrB had similar cell surface expression levels (Fig. 2, A and B), indicating that GyrB had no significant effect on cellular trafficking and distribution of the receptor. Binding characteristics were determined using the PAF receptor antagonist, [3H]WEB2086, by competition with WEB2086. Competition binding experiments showed that the affinity of WEB2086 for hPAFR-GyrB was the same as for the wild-type receptor (Fig. 2C). To investigate the signaling capacity of the chimeric receptor, we examined the ability of hPAFR-GyrB to mediate the stimulation of phosphatidylinositol hydrolysis. Agonist-independent basal IP production and agonist-induced increased IP production were very similar in both c-Myc-hPAFR and c-Myc-hPAFR-GyrB transfected cells (Fig. 2D). Thus, the addition of the GyrB moiety did not lead to detectable changes in hPAFR coupling.Fig. 2Expression, binding, and signaling of wild-type hPAFR and hPAFR-GyrB receptors in CHO cells. Flow cytometric analysis of c-Myc-wild-type (A) and -GyrB (B) receptors in stably transfected CHO cells. Dotted line, labeling with fluorescein isothiocyanate-goat anti-mouse antibody. Solid line, labeling with anti-c-Myc antibody. C, competition binding isotherms of [3H]WEB2086 by WEB2086 in stably transfected CHO cells. Cells were incubated with increasing concentrations of WEB2086, and binding responses were determined as described previously (12Le 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). WT, wild-type. D, basal and stimulated IP levels in CHO cells expressing wild-type or GyrB PAF receptors. The results are representative of three independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Coumermycin Treatment Induces the Dimerization of hPAFR-GyrB—To verify that coumermycin modulates dimerization of hPAFR-GyrB as predicted by the system, CHO cells stably expressing c-Myc-hAPFR-GyrB were treated for various periods of time with coumarins (novobiocin or coumermycin) at 37
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