Regulation of stably transfected platelet activating factor receptor in RBL-2H3 cells. Role of multiple G proteins and receptor phosphorylation.

Published

Journal Article

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.

Full Text

Duke Authors

Cited Authors

  • Ali, H; Richardson, RM; Tomhave, ED; DuBose, RA; Haribabu, B; Snyderman, R

Published Date

  • October 7, 1994

Published In

Volume / Issue

  • 269 / 40

Start / End Page

  • 24557 - 24563

PubMed ID

  • 7929127

Pubmed Central ID

  • 7929127

International Standard Serial Number (ISSN)

  • 0021-9258

Language

  • eng

Conference Location

  • United States