Partial phosphorylation of the N-formyl peptide receptor inhibits G protein association independent of arrestin binding.
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It is now well accepted that G protein-coupled receptors activated by agonist binding become targets for phosphorylation, leading to desensitization of the receptor. Using a series of phosphorylation deficient mutants of the N-formyl peptide receptor (FPR), we have explored the role of phosphorylation on the ability of the receptor to interact with G proteins and arrestins. Using a fluorometric assay in conjunction with solubilized receptors, we demonstrate that phosphorylation of the wild type FPR lowers its affinity for G protein, whereas mutant receptors lacking four potential phosphorylation sites retain their ability to couple to G protein. Phosphorylated mutant receptors lacking only two potential phosphorylation sites are again unable to couple to G protein. Furthermore, whereas stimulated wild type FPR in whole cells colocalizes with arrestin-2, and the solubilized, phosphorylated FPR binds arrestin-2, the stimulated receptors lacking four potential phosphorylation sites display no interaction with arrestin-2. However, the mutant receptors lacking only two potential phosphorylation sites are restored in their ability to bind and colocalize with arrestin-2. Thus, there is a submaximal threshold of FPR phosphorylation that simultaneously results in an inhibition of G protein binding and an induction of arrestin binding. These results are the first to demonstrate that less than maximal levels of receptor phosphorylation can block G protein binding, independent of arrestin binding. We therefore propose that phosphorylation alone may be sufficient to desensitize the FPR in vivo, raising the possibility that for certain G protein-coupled receptors, desensitization may not be the primary function of arrestin.