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 c ontributes to G protein-coupled receptor internalization and signal transdu ction; 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 Al a (N297A) and Ala/Phe (Y301A/Y301F), respectively, and determined the effec ts of the substitutions on FPR functions in transfected rat basophilic leuk emia cells. Whereas all the mutant receptors were expressed on the cell sur face, the N297A receptor exhibited reduced binding affinity and was unable to mediate activation of phospholipase C-beta and the p42/44 mitogen-activa ted protein kinase (MAP kinase). The Y301F receptor displayed significantly decreased ligand-stimulated internalization and MAP kinase activation, sug gesting that the hydrogen bonding at Tyr(301) is critical for these functio ns. The Y301F receptor showed a chemotactic response similar to that of wil d-type FPR, indicating that cell chemotaxis does not require receptor inter nalization and hydrogen bonding at the Tyr(301) position. In contrast, the Y301A receptor displayed a left-shifted, but overall reduced, chemotaxis re sponse 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 efficien t FPR internalization, but is not essential for chemotaxis, These findings demonstrate that residues within the NPXXY motif differentially regulate th e functions of FPR.