Multiple activation steps of the N-formyl peptide receptor

The human N-formyl peptide receptor (FPR) is representative of a growing fa mily of G protein-coupled receptors (GPCR) that respond to chemokines and c hemoattractants. Despite the importance of this receptor class to immune fu nction, relatively little is known about the molecular mechanisms involved in their activation. To reveal steps required for the activation of GPCR re ceptors, we utilized mutants of the FPR which have previously been shown to be incapable of binding and activating G proteins. For this study, the FPR mutants were expressed in human myeloid U937 cells and characterized for f unctions in addition to G protein coupling, such as receptor phosphorylatio n and ligand-induced receptor internalization. The results demonstrated tha t one of the mutants, R123G, though being unable to activate G protein, was capable of undergoing ligand-induced phosphorylation as well as internaliz ation. Receptor internalization was monitored by following the fate of the ligand as well as by directly monitoring the fate of the receptor. The resu lts with the R123G mutant were in contrast to those obtained for mutants D7 1A and R309G/E310A/R311G which, though being expressed at the cell surface and binding ligand, were incapable of being phosphorylated or internalized upon agonist stimulation. These results suggest that following ligand bindi ng at least two "steps" are required for full activation of the wild-type F PR. That these observations may be of more general importance in GPCR-media ted signaling is suggested by the highly conserved nature of the mutants st udied: D71, R123, and the site represented by amino acids 309-311 are very highly conserved throughout the entire superfamily of G protein-coupled rec eptors, Models of receptor activation based on the observed results are dis cussed.