A single amino acid substitution (N297A) in the conserved NPXXY sequence of the human N-formyl peptide receptor results in inhibition of desensitization and endocytosis, and a dose-dependent shift in p42/44 mitogen-activatedprotein kinase activation and chemotaxis

The formyl peptide receptor (FPR) is a G-protein-coupled receptor (GPCR) th at mediates chemotaxis and stimulates the mitogen-activated protein kinase (MAPK)/extracellular-signal-regulated kinase pathway. We have examined the functional effects of substitutions of a conserved aspartic acid residue in the second transmembrane domain (D71A) and of residues in the conserved NP XXY motif in the seventh transmembrane domain (N297A and Y301A). These muta ted receptors, expressed in Chinese hamster ovary (CHO) cells, bind ligand with affinities similar to wild-type FPR, but the D71A mutant is uncoupled from G-protein [Miettinen, Mills, Gripentrog, Dratz, Granger and Jesaitis ( 1997) J. Immunol 159, 4045-4054]. In the present study, we show that both t he D71A and N297A mutations resulted in defective endocytosis. The N297A su bstitution also prevented desensitization, as determined by intracellular c alcium mobilization by sequential stimulation with ligand. In chemotaxis as says, the N297A mutation resulted in cell migration towards gradients of up to 100 nM N-formyl-methionyl-leucyl-phenylalanine (fMLF), whereas cells ex pressing the wild-type FPR and the Y301A mutant were no longer chemotactica lly responsive at 10-100 nM fMLF. Maximal activation of p42/44 MAPK occurre d in CHO cells expressing wild-type FPR at 10 nM-100 nM fMLF, whereas cells expressing the N297A mutant showed a dose-dependent increase in the amount of phosphorylated p42/44 MAPK up to 1-10 muM fMLF. Since the MAPK kinase i nhibitor PD98059 blocked fMLF-induced chemotaxis, our results suggest that the dose-dependent increase in p42/44 MAPK activation may correlate with th e increased chemotactic migration of N297A transfectants at 10 nM-100 nM fM LF.