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.
Highlights
Platelet activatingfactor (PAF)interacts with cellsur- released frommast cells, platelets, neutrophilsm, onocytes, and face receptors to mediate inflammatory responses
To macrophages [1].PAF is a potent chemoattractant for neutrodetermine the mechanisms of PAF receptor regulation, phils and elicits various otherresponses such as coronary vaswe constructed epitope-tagged human PAF receptor oconstriction and increased vasculaprermeability (reviewed in cDNA (ET-PAFR)and generated stable transfectants in a Refs. 2 and 3).The effects of PAF on neutrophils are mediated, rat basophilic cell line (RBL-2H3 cells)
PAF-stimulated intracellular Ca2+ mobilization was not mobilization and exocytosis in a pertussis toxin (PTx)-sensitive inhibited by pertussis toxin (PTx), whereas phospho- manner, different receptor subtypes are thoughtto be involved inositide hydrolysis and secretion wereblocked by in PAF-stimulated responses [7,8,9]
Summary
Dept. of Medicine, Duke University MedicalCtr., Box 3680, Durham, NC 27710. Tel.: 919-. The5'-oligonucleotide, in a 5' to 3' order, Labeling of ET-PAFR with [125Z]Iodine-RBL-2H3 cells (5 x lo6)excontained 3 miscellaneous bases, 6 bases encoding a HindIII site, 3 pressing ET-PAFR, preincubated or not with 10nM0 PAF for 5 min, were bases from the 5'-untranslated sequence preceding the ATG codon of resuspended in200 pl of phosphate-buffered saline inan iodogen-coated the human PAF receptor,3basesencodingamethionine,27bases glass tube (100 &tube). Cells were removed, washed, the cDNA sequence encoding amino acids 2-9of PAF receptor. The cells were washed monolayer cultures in Earle's modified Eagle's medium supplemented with phosphate-buffered saline and lysed with 0.75 omf lysis bufferof with 15% fetal bovine serum, 2mM glutamine, penicillin(100unitdml), the following composition: 150 mM NaCl, 50 mM Tris, pH 8.0, 1.0%.
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