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 recep tor on phagocytes activates these cells through a G protein-coupled pa thway. To delineate the structural requirement of the N-formyl peptide receptor (FPR) for ligand binding and signaling, we constructed chime ric receptors between FPR and a recently identified granulocyte recept or, 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 fM LP with a low affinity (K(d) = 430 nM), as compared with the high affi nity (K(d) = 1 nM) displayed by the FPR. This property of the FPR2 was utilized for mapping the FPR ligand binding domains. Seven chimeric F PR/FPR2 receptors were generated by sequential replacement of the FPR segments with the corresponding regions from FPR2. Three reciprocal FP R2/FPR chimeric receptors were also constructed by selective substitut ion of the FPR segments into FPR2. These chimeric receptors were stabl y expressed in transfected fibroblasts and analyzed for their ligand b inding and transmembrane signaling properties. Replacement of the FPR domains, including the first and the third extracellular loops, result ed in 275- and 85-fold decrease in ligand binding affinity, respective ly. Introduction of both domains into the FPR2 significantly increased ligand binding affinity (K(d) = 18 nM), whereas substitution of the d omains containing the first or third extracellular loop alone improved ligand binding to a lesser degree (K(d) = 90 and 372 nM, respectively ). In contrast, substitution of either the amino or the carboxyl-termi nal 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 essentia l for binding of fMLP. We propose that multiple domains of the FPR are required for high-affinity ligand binding, with a major determinant l ocated in the first extracellular loop and its adjacent transmembrane domains.