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 binds to a specific, high affinity receptor of the G protein-coupled receptor (GPCR) family. In the present report, we demonstrate that the highly conserved aspartate 63 is critical in G protein coupling of the PAF 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 unaffected by GTPgammaS. We also demonstrate that aspartate 63 is not involved in direct interaction with the agonist: the D63N mutant displayed a higher intrinsic affinity for PAF than the uncoupled WT receptor. Sodium decreased specific (3)H-PAF and antagonist (3)H-WEB2086 binding to the PAF receptor, but the aspartate 63 residue was not involved in this regulation, contrary to cognate aspartate residues in other GPCRs. Our data suggest that aspartate 63 in the PAF receptor may be involved in the structural requirement for G protein coupling to the receptor and may contribute to receptor affinity for the ligand.