title: Identification and functional characterization of the phosphorylation sites of the neuropeptide FF2 receptor.
abstract: The neuropeptide FF2 (NPFF2) receptor belongs to the rhodopsin family of G protein-coupled receptors and mediates the effects of several related RFamide neuropeptides. One of the main pharmacological interests of this system resides in its ability to regulate endogenous opioid systems, making it a potential target to reduce the negative effects of chronic opioid use. Phosphorylation of intracellular residues is the most extensively studied post-translational modification regulating G protein-coupled receptor activity. However, until now, no information concerning NPFF2 receptor phosphorylation is available. In this study, we combined mass spectrometric analysis and site-directed mutagenesis to analyze for the first time the phosphorylation pattern of the NPFF2 receptor and the role of the various phosphorylation sites in receptor signaling, desensitization, and trafficking in a SH-SY5Y model cell line. We identified the major, likely GRK-dependent, phosphorylation cluster responsible for acute desensitization, (412)TNST(415) at the end of the C terminus of the receptor, and additional sites involved in desensitization ((372)TS(373)) and internalization (Ser(395)). We thus demonstrate the key role played by phosphorylation in the regulation of NPFF2 receptor activity and trafficking. Our data also provide additional evidence supporting the concept that desensitization and internalization are partially independent processes relying on distinct phosphorylation patterns.
authors.affiliation: From the Institut de Pharmacologie et Biologie Structurale, UMR5089 CNRS, Université de Toulouse, 31077 Toulouse, France.
authors.forename: Lauriane
authors.initials: L
authors.lastname: Bray
authors.affiliation: From the Institut de Pharmacologie et Biologie Structurale, UMR5089 CNRS, Université de Toulouse, 31077 Toulouse, France.
authors.forename: Carine
authors.initials: C
authors.lastname: Froment
authors.affiliation: From the Institut de Pharmacologie et Biologie Structurale, UMR5089 CNRS, Université de Toulouse, 31077 Toulouse, France.
authors.forename: Pierre
authors.initials: P
authors.lastname: Pardo
authors.affiliation: From the Institut de Pharmacologie et Biologie Structurale, UMR5089 CNRS, Université de Toulouse, 31077 Toulouse, France.
authors.forename: Cédric
authors.initials: C
authors.lastname: Candotto
authors.affiliation: From the Institut de Pharmacologie et Biologie Structurale, UMR5089 CNRS, Université de Toulouse, 31077 Toulouse, France.
authors.forename: Odile
authors.initials: O
authors.lastname: Burlet-Schiltz
authors.affiliation: From the Institut de Pharmacologie et Biologie Structurale, UMR5089 CNRS, Université de Toulouse, 31077 Toulouse, France.
authors.forename: Jean-Marie
authors.initials: JM
authors.lastname: Zajac
authors.affiliation: From the Institut de Pharmacologie et Biologie Structurale, UMR5089 CNRS, Université de Toulouse, 31077 Toulouse, France catherine.mollereau-manaute@ipbs.fr.
authors.forename: Catherine
authors.initials: C
authors.lastname: Mollereau
authors.affiliation: From the Institut de Pharmacologie et Biologie Structurale, UMR5089 CNRS, Université de Toulouse, 31077 Toulouse, France Lionel.mouledous@ipbs.fr.
authors.forename: Lionel
authors.initials: L
authors.lastname: Moulédous
mesh_terms: D000595:Amino Acid Sequence; D045744:Cell Line, Tumor; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism*; D010449:Peptide Mapping; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D021381:Protein Transport; D018013:Receptors, Neuropeptide / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D015398:Signal Transduction
chemical_list: D009842:Oligopeptides; D018013:Receptors, Neuropeptide; C083929:neuropeptide FF receptor; C053651:phenylalanyl-leucyl-phenylalanyl-glutaminyl-prolyl-glutaminyl-arginyl-phenylalaninamide
keywords: Desensitization; G Protein-coupled Receptor (GPCR); Internalization; Mass Spectrometry; Mass Spectrometry (MS); Neuropeptide FF; Phosphorylation; Post-translational Modification (PTM); Signaling