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


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


  • eng

Conference Location

  • United States