title: Regulation of stably transfected platelet activating factor receptor in RBL-2H3 cells. Role of multiple G proteins and receptor phosphorylation.
abstract: Platelet activating factor (PAF) interacts with cell surface receptors to mediate inflammatory responses. To determine the mechanisms of PAF receptor regulation, we constructed epitope-tagged human PAF receptor cDNA (ET-PAFR) and generated stable transfectants in a rat basophilic cell line (RBL-2H3 cells). The expressed receptors displayed ligand binding and functional properties similar to the native receptors in neutrophils. PAF-stimulated intracellular Ca2+ mobilization was not inhibited by pertussis toxin (PTx), whereas phosphoinositide hydrolysis and secretion were blocked by approximately 40%. The PTx-resistant secretion mediated by PAF was, however, inhibited by guanosine 5'-O-(2-thio-diphosphate) in permeabilized RBL-2H3 cells, indicating a role for PTx-insensitive G protein. In contrast to the PAF receptor, responses mediated by formylpeptide and C5a chemoattractants were inhibited by PTx. PAF stimulated a dose- and time-dependent phosphorylation of its receptor. ET-PAFR was also phosphorylated by phorbol 12-myristate 13-acetate (PMA) and dibutyryl cyclic AMP. Staurosporine caused complete inhibition of ET-PAFR phosphorylation by PMA but only partial inhibition by PAF. Receptor phosphorylation by PAF and PMA correlated with desensitization as measured by a decrease in both PAF-stimulated GTPase activity in membranes and Ca2+ mobilization in intact cells. Phosphorylation of ET-PAFR by dibutyryl cyclic AMP was not, however, associated with desensitization. These data demonstrate that a single PAF receptor population interacts with multiple G proteins to mediate its biological responses. Moreover, ET-PAFR, unlike the formylpeptide or C5a receptors, is phosphorylated by at least three kinases (most likely protein kinases A and C and a receptor kinase). The functional consequences of cellular activation by various chemoattractants may depend upon the G protein to which their receptor is coupled.
authors.affiliation: Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710.
authors.forename: H
authors.initials: H
authors.lastname: Ali
authors.forename: R M
authors.initials: RM
authors.lastname: Richardson
authors.forename: E D
authors.initials: ED
authors.lastname: Tomhave
authors.forename: R A
authors.initials: RA
authors.lastname: DuBose
authors.forename: B
authors.initials: B
authors.lastname: Haribabu
authors.forename: R
authors.initials: R
authors.lastname: Snyderman
mesh_terms: D000470:Alkaloids / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D003994:Bucladesine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D019204:GTP-Binding Proteins / Q000502:physiology*; D006153:Guanosine Diphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D008969:Molecular Sequence Data; D037342:Pertussis Toxin; D010766:Phosphorylation; D010972:Platelet Activating Factor / Q000494:pharmacology; D010980:Platelet Membrane Glycoproteins / Q000235:genetics / Q000502:physiology*; D051381:Rats; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; D019311:Staurosporine; D013873:Thionucleotides / Q000494:pharmacology; D014162:Transfection; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
chemical_list: D000470:Alkaloids; D010972:Platelet Activating Factor; D010980:Platelet Membrane Glycoproteins; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; D013873:Thionucleotides; D010566:Virulence Factors, Bordetella; C039351:platelet activating factor receptor; D006153:Guanosine Diphosphate; D003994:Bucladesine; C023427:guanosine 5'-O-(2-thiodiphosphate); D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D019311:Staurosporine; D002118:Calcium