MUTATION OF AN ASPARTATE AT POSITION-63 IN THE HUMAN PLATELET-ACTIVATING-FACTOR RECEPTOR AUGMENTS BINDING-AFFINITY BUT ABOLISHES G-PROTEIN-COUPLING AND INOSITOL PHOSPHATE PRODUCTION

Platelet-activating factor is a potent phospholipid mediator which bin ds to a specific, high affinity receptor of the G protein-coupled rece ptor (GPCR) family. In the present report, we demonstrate that the hig hly conserved aspartate 63 is critical in G protein coupling of the PA F receptor: substitution of an asparagine for the aspartate 63 (D63N) abolished inositol phosphate production following agonist stimulation; moreover, binding isotherms of the D63N mutant were monophasic and un affected by GTP gamma S. We also demonstrate that aspartate 63 is not involved in direct interaction with the agonist: the D63N mutant displ ayed a higher intrinsic affinity for PAF than the uncoupled WT recepto r. Sodium decreased specific H-3-PAF and antagonist H-3-WEB2086 bindin g to the PAF receptor, but the aspartate 63 residue was not involved i n this regulation, contrary to cognate aspartate residues in other GPC Rs. Our data suggest that aspartate 63 in the PAF receptor may be invo lved in the structural requirement for G protein coupling to the recep tor and may contribute to receptor affinity for tile ligand. (C) 1996 Academic Press, Inc.