The rabbit neutrophil N-formyl peptide receptor. cDNA cloning, expression, and structure/function implications.

The rabbit neutrophil N-formyl peptide receptor (FPR) has been well studied for its ligand binding properties. Recent gene cloning experiments have established the existence of a subfamily of G protein-coupled receptors that share extensive sequence homology with the FPR, yet lack the capability of high affinity binding to FMLP. These findings prompted us to identify the structural requirement for formyl peptide ligand binding by delineation of the primary structure of the rabbit FPR. A rabbit neutrophil cDNA library was screened with a cloned human FPR cDNA probe and the insert of one positive isolate (B6) was sequenced. The 1268-bp cDNA insert encodes a peptide of 352 amino acids. Stably transfected L cell fibroblasts expressing the rabbit cDNA displayed specific binding of the ligand fMet-Leu-[3H]Phe with two affinities (Kd = 0.31 and 7.5 nM). Addition of the nonhydrolyzable guanosine triphosphate analogue, GTP gamma S, converted > or = 85% of the high affinity sites to the low affinity sites. FMLP induced mobilization of intracellular calcium in the transfected cells (EC50 = 0.5 nM), a response sensitive to pertussis toxin. FMLP stimulation desensitized the receptor such that subsequent stimulation with the same ligand produced a significantly reduced signal. These results indicate that the cloned rabbit receptor represents a high affinity FPR, and that FPR-mediated early signal transduction events can be fully reconstituted in transfected mammalian cells. The rabbit FPR sequence is 78% identical to that of the human FPR, and 68% identical to FPR2, a homologue of FPR with a low binding affinity (Kd > or = 400 nM) for FMLP. Analysis of the aligned sequences of these three proteins revealed that: 1) the amino termini and the second extracellular loops have the lowest sequence homology; 2) sequence in the intracellular domains that couple to G protein are highly conserved; and 3) the first and the third extracellular loops and their adjacent transmembrane domains of the FPR may contain residues essential for the high affinity binding of FMLP.