MUTATION OF A PUTATIVE AMPHIPATHIC ALPHA-HELIX IN THE 3RD INTRACELLULAR DOMAIN OF THE PLATELET-ACTIVATING-FACTOR RECEPTOR DISRUPTS RECEPTORG-PROTEIN COUPLING AND SIGNALING

Platelet-activating factor (PAF) is a potent phospholipid mediator tha t interacts with G protein-coupled PAF receptors to elicit diverse phy siological and pathophysiological actions. We recently demonstrated th at the third intracellular domain of the rat PAF receptor (rPAFR) is a critical determinant in its coupling to phosphoinositide phospholipas e C-activating G proteins. Here, we report identification of a putativ e 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 thir d intracellular domain and adjacent transmembrane regions of the rPAFR identified a single amphipathic helix located in the amino-terminal r egion of the third intracellular domain of the receptor. Baby hamster kidney cells were transiently transfected with cDNAs encoding the rPAF R or rPAFR mutants in which nonconserved substitutions were made separ ately in the hydrophobic or polar face of this amphipathic helix. The number and affinity of binding sites for specific PAF receptor antagon ist WEB2086 were identical in membranes prepared from rPAFR and amphip athic helix mutant PAFR transfectants. However, only membranes derived from rPAFR transfectants possessed high affinity PAF binding sites th at were sensitive to the G protein-uncoupling effects of guanosine-5'- O-(3-thio)triphosphate. These results show that substitutions into eit her face of the amphipathic helical domain abolished the ability of th e rPAFR to undergo coupling to G proteins to form a high affinity agon ist/receptor/G protein ternary complex. To examine the effects of thes e mutations on rPAFR signaling, PAF-stimulated inositol phosphate accu mulation was determined in cells transfected with cDNAs encoding the w ild-type or amphipathic helix mutant PAFRs. Although PAF stimulated 10 -fold in creases in inositol phosphate accumulation in rPAFR transfect ants, it had no effects on inositol phosphate accumulation in amphipat hic helix mutant PAFR transfectants. These results suggest that an amp hipathic helix located in the amino-terminal region of the third intra cellular domain of the rPAFR is required for its coupling to and activ ation of G proteins. This study provides the first insight into the st ructure of the receptor interface for G protein coupling of a PAFR and suggests a conserved role of amphipathic helices in G protein couplin g of receptors ranging from those for biogenic amines to the phospholi pid mediator PAF.