Differential roles of the NPXXY motif in formyl peptide receptor signaling.
The NPXXY motif (X represents any amino acid) in the seventh transmembrane domain of the chemotactic formyl peptide receptor (FPR) is highly conserved among G protein-coupled receptors. Recent work suggested that this motif contributes to G protein-coupled receptor internalization and signal transduction; however, its role in FPR signaling remains unclear. In this study we replaced Asn(297) and Tyr(301) in the NPXXY motif of the human FPR with Ala (N297A) and Ala/Phe (Y301A/Y301F), respectively, and determined the effects of the substitutions on FPR functions in transfected rat basophilic leukemia cells. Whereas all the mutant receptors were expressed on the cell surface, the N297A receptor exhibited reduced binding affinity and was unable to mediate activation of phospholipase C-beta and the p42/44 mitogen-activated protein kinase (MAP kinase). The Y301F receptor displayed significantly decreased ligand-stimulated internalization and MAP kinase activation, suggesting that the hydrogen bonding at Tyr(301) is critical for these functions. The Y301F receptor showed a chemotactic response similar to that of wild-type FPR, indicating that cell chemotaxis does not require receptor internalization and hydrogen bonding at the Tyr(301) position. In contrast, the Y301A receptor displayed a left-shifted, but overall reduced, chemotaxis response that peaked at 0.1-1 nM. Finally, using a specific MAP kinase kinase inhibitor, we found that activation of MAP kinase is required for efficient FPR internalization, but is not essential for chemotaxis. These findings demonstrate that residues within the NPXXY motif differentially regulate the functions of FPR.
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Department of Pharmacology, University of Illinois College of Medicine, Chicago, IL 60612, USA.
Browning
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D020816:Amino Acid Motifs / Q000235:genetics / Q000276:immunology; D000818:Animals; D002070:Butadienes / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002634:Chemotaxis, Leukocyte / Q000187:drug effects / Q000276:immunology; D017124:Conserved Sequence / Q000235:genetics / Q000276:immunology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000378:metabolism*; D009570:Nitriles / Q000494:pharmacology; D010446:Peptide Fragments / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics / Q000276:immunology; D051381:Rats; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D018000:Receptors, Peptide / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D015398:Signal Transduction / Q000235:genetics / Q000276:immunology*; D014407:Tumor Cells, Cultured
D002070:Butadienes; D004791:Enzyme Inhibitors; D009570:Nitriles; D010446:Peptide Fragments; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic; D018000:Receptors, Peptide; C113580:U 0126; D009240:N-Formylmethionine Leucyl-Phenylalanine; D020928:Mitogen-Activated Protein Kinases; D002118:Calcium