title: Partial phosphorylation of the N-formyl peptide receptor inhibits G protein association independent of arrestin binding.
abstract: 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.
authors.affiliation: Department of Cell Biology and Physiology, Cancer Research and Treatment Center, University of New Mexico Health Sciences Center, Albuquerque, New Mexico 87131, USA.
authors.forename: T A
authors.initials: TA
authors.lastname: Bennett
authors.forename: T D
authors.initials: TD
authors.lastname: Foutz
authors.forename: V V
authors.initials: VV
authors.lastname: Gurevich
authors.forename: L A
authors.initials: LA
authors.lastname: Sklar
authors.forename: E R
authors.initials: ER
authors.lastname: Prossnitz
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019393:Arrestin / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018000:Receptors, Peptide / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013050:Spectrometry, Fluorescence / Q000379:methods
chemical_list: D019393:Arrestin; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic; D018000:Receptors, Peptide; D019204:GTP-Binding Proteins