Differential phosphorylation paradigms dictate desensitization and internalization of the N-formyl peptide receptor

Following activation by ligand, most G protein-coupled receptors undergo ra pid phosphorylation, This is accompanied by a drastic decrease in the effic acy of continued or repeated stimulation, due to receptor uncoupling from G protein and receptor internalization. Such processing steps have been show n to be absolutely dependent on receptor phosphorylation in the case of the N-formyl peptide receptor (FPR), In this study, we report results that ind icate that the mechanisms responsible for desensitization and internalizati on are distinct. Using site-directed mutagenesis of the serine and threonin e residues of the FPR carboxyl terminus, we have characterized regions that differentially regulate these two processes. Whereas substitution of all 1 1 Ser/Thr residues in the carboxyl terminus prevents both desensitization a nd internalization, substitution of four Ser/Thr residues between 328-332 b locks desensitization but has no effect on internalization, Similarly, subs titution of four Ser/Thr residues between positions 334 and 339 results in a deficit in desensitization but again no decrease in internalization, sugg esting that phosphorylation at either site evokes receptor internalization, whereas maximal desensitization requires phosphorylation at both sites. Th ese results also indicate that receptor internalization is not involved in the process of desensitization. Further analysis of the residues between 32 8-332 revealed that restoration either of Ser(328) and Thr(329) or of Thr(3 31) and Ser(332) was sufficient to restore desensitization, suggesting that phosphorylation within either of these two sites, in addition to sites bet ween residues 334 and 339, is sufficient to produce desensitization. Taken together, these results indicate that the mechanisms involved in FPR proces sing ( uncoupling from G; proteins and internalization) are regulated diffe rentially by phosphorylation at distinct sites within the carboxyl terminus of the FPR, The relevance of this paradigm to other G protein-coupled rece ptors is discussed.