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-activated protein kinase activation and chemotaxis.
The formyl peptide receptor (FPR) is a G-protein-coupled receptor (GPCR) that 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 NPXXY motif in the seventh transmembrane domain (N297A and Y301A). These mutated 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 the D71A and N297A mutations resulted in defective endocytosis. The N297A substitution also prevented desensitization, as determined by intracellular calcium mobilization by sequential stimulation with ligand. In chemotaxis assays, the N297A mutation resulted in cell migration towards gradients of up to 100 nM N-formyl-methionyl-leucyl-phenylalanine (fMLF), whereas cells expressing the wild-type FPR and the Y301A mutant were no longer chemotactically responsive at 10-100 nM fMLF. Maximal activation of p42/44 MAPK occurred 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 microM fMLF. Since the MAPK kinase inhibitor PD98059 blocked fMLF-induced chemotaxis, our results suggest that the dose-dependent increase in p42/44 MAPK activation may correlate with the increased chemotactic migration of N297A transfectants at 10 nM-100 nM fMLF.
Gripentrog
J M
JM
Department of Microbiology, Montana State University, 109 Lewis Hall, Bozeman, MT 59717-3520, USA.
Jesaitis
A J
AJ
Miettinen
H M
HM
D019943:Amino Acid Substitution; D000818:Animals; D016466:CHO Cells; D002633:Chemotaxis; D006224:Cricetinae; D004705:Endocytosis; D004789:Enzyme Activation; D006801:Humans; D008024:Ligands; D008856:Microscopy, Fluorescence; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D010766:Phosphorylation; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D018000:Receptors, Peptide / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*
D008024:Ligands; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic; D018000:Receptors, Peptide; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases