Multiple domains of the N-formyl peptide receptor are required for high-affinity ligand binding. Construction and analysis of chimeric N-formyl peptide receptors.

Binding of the chemotactic tripeptide fMet-Leu-Phe (fMLP) to its receptor on phagocytes activates these cells through a G protein-coupled pathway. To delineate the structural requirement of the N-formyl peptide receptor (FPR) for ligand binding and signaling, we constructed chimeric receptors between FPR and a recently identified granulocyte receptor, FPR2 (Ye, R. D., Cavanagh, S. L., Quehenberger, O., Prossnitz, E. R., and Cochrane, C. G. (1992) Biochem. Biophys. Res. Commun. 184, 582-589). FPR2 shares 69% sequence homology with the FPR; yet it binds fMLP with a low affinity (Kd = 430 nM), as compared with the high affinity (Kd = 1 nM) displayed by the FPR. This property of the FPR2 was utilized for mapping the FPR ligand binding domains. Seven chimeric FPR/FPR2 receptors were generated by sequential replacement of the FPR segments with the corresponding regions from FPR2. Three reciprocal FPR2/FPR chimeric receptors were also constructed by selective substitution of the FPR segments into FPR2. These chimeric receptors were stably expressed in transfected fibroblasts and analyzed for their ligand binding and transmembrane signaling properties. Replacement of the FPR domains, including the first and the third extracellular loops, resulted in 275- and 85-fold decrease in ligand binding affinity, respectively. Introduction of both domains into the FPR2 significantly increased ligand binding affinity (Kd = 18 nM), whereas substitution of the domains containing the first or third extracellular loop alone improved ligand binding to a lesser degree (Kd = 90 and 372 nM, respectively). In contrast, substitution of either the amino or the carboxyl-terminal regions with those of the FPR2 had little effect on ligand binding affinity. An analysis of the sequences of the two receptors revealed several key residues in the first and the third extracellular loops of the FPR and their adjacent transmembrane domains that may be essential for binding of fMLP. We propose that multiple domains of the FPR are required for high-affinity ligand binding, with a major determinant located in the first extracellular loop and its adjacent transmembrane domains.