Vol. 53, Issue 3, 451-458, March 1998

Mutation of a Putative Amphipathic -Helix in the Third Intracellular Domain of the Platelet-Activating Factor Receptor Disrupts Receptor/G Protein Coupling and Signaling

Steve A. Carlson, Tapan K. Chatterjee, Kenneth P. Murphy, and Rory A. Fisher

Departments of Biochemistry (K.P.M.) and Pharmacology (S.A.C., T.K.C., R.A.F.), University of Iowa College of Medicine, Iowa City, Iowa 52242

Platelet-activating factor (PAF) is a potent phospholipid mediator that interacts with G protein-coupled PAF receptors to elicit diverse physiological and pathophysiological actions. We recently demonstrated that the third intracellular domain of the rat PAF receptor (rPAFR) is a critical determinant in its coupling to phosphoinositide phospholipase C-activating G proteins. Here, we report identification of a putative amphipathic helix in the third intracellular domain of the rPAFR and the effects of mutational disruption of its amphipathic character on G protein coupling of and signaling by the rPAFR. Modeling of the third intracellular domain and adjacent transmembrane regions of the rPAFR identified a single amphipathic helix located in the amino-terminal region of the third intracellular domain of the receptor. Baby hamster kidney cells were transiently transfected with cDNAs encoding the rPAFR or rPAFR mutants in which nonconserved substitutions were made separately in the hydrophobic or polar face of this amphipathic helix. The number and affinity of binding sites for specific PAF receptor antagonist WEB2086 were identical in membranes prepared from rPAFR and amphipathic helix mutant PAFR transfectants. However, only membranes derived from rPAFR transfectants possessed high affinity PAF binding sites that were sensitive to the G protein-uncoupling effects of guanosine-5-O-(3-thio)triphosphate. These results show that substitutions into either face of the amphipathic helical domain abolished the ability of the rPAFR to undergo coupling to G proteins to form a high affinity agonist/receptor/G protein ternary complex. To examine the effects of these mutations on rPAFR signaling, PAF-stimulated inositol phosphate accumulation was determined in cells transfected with cDNAs encoding the wild-type or amphipathic helix mutant PAFRs. Although PAF stimulated 10-fold increases in inositol phosphate accumulation in rPAFR transfectants, it had no effects on inositol phosphate accumulation in amphipathic helix mutant PAFR transfectants. These results suggest that an amphipathic helix located in the amino-terminal region of the third intracellular domain of the rPAFR is required for its coupling to and activation of G proteins. This study provides the first insight into the structure of the receptor interface for G protein coupling of a PAFR and suggests a conserved role of amphipathic helices in G protein coupling of receptors ranging from those for biogenic amines to the phospholipid mediator PAF.