HUMAN FORMYL PEPTIDE RECEPTOR-LIGAND BINDING DOMAIN(S) - STUDIES USING AN IMPROVED MUTAGENESIS EXPRESSION VECTOR REVEAL A NOVEL MECHANISM FOR THE REGULATION OF RECEPTOR OCCUPANCY/

Recently, we reported the domain requirements for the binding of formy l peptide to its specific receptor Based on experiments using receptor chimeras, we also postulated an importance for the amino terminal dom ain of the receptor in ligand binding (Perez, H. D., Holmes, R., Vilan der, L., Adams, R., Manzana, W., Jolley, D., and Andrews, W. H. (1993) J. Biol. Chem. 268, 2292-2295). We have begun to perform a detailed a nalysis of the regions within the formyl peptide receptor involved in ligand binding. To address the importance of the receptor amino-termin al domain, we substituted (or inserted) hydrophilic sequences within t he amino terminal domain, expressed the receptors, and determined thei r ability to bind ligand. A stretch of nine amino acids next to the in itial methionine was identified as crucial for receptor occupancy. A p eptide containing such a sequence specifically competed binding of the ligand to the receptor. Alanine screen mutagenesis of the second extr acellular domain also identified amino acids involved in ligand bindin g as well as a disulfide bond (Cys(98) to Cys(176)) crucial for mainta ining the binding pocket. These studies provide evidence for a novel m echanism involved in regulation of receptor occupancy. Binding of the ligand induces conformational changes in the receptor that result in t he aposition of the amino-terminal domain over the ligand, providing a lid to the binding pocket.