IDENTIFICATION OF AN N-FOUMYL PEPTIDE RECEPTOR-LIGAND BINDING DOMAIN BY A GAIN-OF-FUNCTION APPROACH

Replacement of N-formyl peptide receptor (FPR) domains with those from a homologous receptor, FPR2, resulted in chimeric receptors displayin g low binding affinity to fMet-Leu-Phe (fMLF). To characterize fMLF bi nding domain, we adopted a ''gain-of-function'' approach by selective replacement of non-conserved residues in the FPR2 portion of the chime ric receptors with those from the FPR. This led to the identification of 3 clusters of residues required for high-affinity fMLF binding. Int roduction of 2 positively charged amino acids, Arg(84) and Lys(85), dr amatically improved binding a affinity of one chimeric receptor (K-d f rom 105 nM to 1.6 nM). Similarly, restoration of either Gly(89)/His(90 ) or Phe(102)/Thr(103) improved the binding-affinity of another chimer ic receptor from a K-d Of 275 nM to a 2.3 K-d and 3.3 nM, respectively . Increased ligand binding affinity was accompanied by a gain in calci um mobilization capability, suggesting functional coupling to G protei ns. These results demonstrate the presence of structural determinants in the first extracellular loop and its adjacent transmembrane domains that are essential for high affinity fMLF binding. (C) 1997 Academic Press.