Agonist-independent desensitization and internalization of the human platelet-activating factor receptor by coumermycin-gyrase B-induced 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 delta-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.
Perron
Amelie
A
Immunology Division, Department of Pediatrics, Faculty of Medicine, University of Sherbrooke, Sherbrooke, Quebec J1H 5N4, Canada.
Chen
Zhang-Guo
ZG
Gingras
Denis
D
Dupre
Denis J
DJ
Stankova
Jana
J
Rola-Pleszczynski
Marek
M
D049933:Aminocoumarins; D000818:Animals; D001667:Binding, Competitive; D015153:Blotting, Western; D016466:CHO Cells; D019556:COS Cells; D003374:Coumarins / Q000494:pharmacology*; D006224:Cricetinae; D027081:DNA Gyrase / Q000737:chemistry*; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D000939:Epitopes; D005434:Flow Cytometry; D006801:Humans; D015151:Immunoblotting; D007211:Indoles / Q000494:pharmacology; D007249:Inflammation; D008301:Maleimides / Q000494:pharmacology; D018613:Microscopy, Confocal; D010980:Platelet Membrane Glycoproteins / Q000737:chemistry* / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D011487:Protein Conformation; D011493:Protein Kinase C / Q000378:metabolism; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000737:chemistry* / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction; D013997:Time Factors; D014162:Transfection
D049933:Aminocoumarins; D003374:Coumarins; D004791:Enzyme Inhibitors; D000939:Epitopes; D007211:Indoles; D008301:Maleimides; D010980:Platelet Membrane Glycoproteins; D016271:Proto-Oncogene Proteins c-myc; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; D011993:Recombinant Fusion Proteins; C039351:platelet activating factor receptor; D011493:Protein Kinase C; D027081:DNA Gyrase; C070515:bisindolylmaleimide I; C004628:coumermycin