Identification of an N-formyl 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 displaying low binding affinity to fMet-Leu-Phe (fMLF). To characterize fMLF binding domain, we adopted a "gain-of-function" approach by selective replacement of non-conserved residues in the FPR2 portion of the chimeric receptors with those from the FPR. This led to the identification of 3 clusters of residues required for high-affinity fMLF binding. Introduction of 2 positively charged amino acids, Arg84 and Lys85, dramatically improved binding affinity of one chimeric receptor (Kd from 105 nM to 1.6 nM). Similarly, restoration of either Gly89/His90 or Phe102/Thr103 improved the binding affinity of another chimeric receptor from a Kd of 275 nM to a 2.3 Kd and 3.3 nM, respectively. Increased ligand binding affinity was accompanied by a gain in calcium mobilization capability, suggesting functional coupling to G proteins. 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. Copyright 1997 Academic Press.