title: A phosphorylation cluster of five serine and threonine residues in the C-terminus of the follicle-stimulating hormone receptor is important for desensitization but not for beta-arrestin-mediated ERK activation.
abstract: Classically, the FSH receptor (FSH-R) mediates its effects through coupling to guanine nucleotide-binding protein alpha S subunit (Galpha(s)) and activation of the cAMP/protein kinase A (PKA) signaling pathway. beta-Arrestins are rapidly recruited to the FSH-activated receptor and play key roles in its desensitization and internalization. Here, we show that the FSH-R expressed in HEK 293 cells activated ERK by two temporally distinct pathways dependent, respectively, on Galpha(s)/PKA and beta-arrestins. Galpha(s)/PKA-dependent ERK activation was rapid, transient, and blocked by H89 (a PKA inhibitor), but it was insensitive to small interfering RNA-mediated depletion of beta-arrestins. beta-Arrestin-dependent ERK activation was slower but more sustained and was insensitive to H89. We identified five Ser/Thr residues in the C terminus of the receptor (638-644) as a major phosphorylation site. Mutation of these residues into Ala (5A FSH-R) significantly reduced the stability of FSH-induced beta-arrestin 1 and 2 interaction when compared with the wild-type receptor. As expected, the 5A FSH-R-mediated cAMP accumulation was enhanced, and its internalization was reduced. In striking contrast, the ability of the 5A FSH-R to activate ERK via the beta-arrestin-dependent pathway was increased. G protein-coupled receptor kinase 5 (GRK5) and GRK6 were required for beta-arrestin-dependent ERK activation by both the wild-type and 5A FSH-R. By contrast, GRK2 depletion enhanced ERK activation by the wild-type FSH-R but not by the 5A FSH-R. In conclusion, we demonstrate the existence of a beta-arrestin-dependent, GRK-regulated mechanism for ERK activation by the FSH-R. A phosphorylation cluster in the C terminus of the FSH-R, identified as a site of beta-arrestin recruitment, positively regulated both desensitization and internalization but negatively regulated beta-arrestin-dependent ERK activation.
authors.affiliation: Institut National de la Recherche Agronomique, Centre National de Recherche Scientifique/Université de Tours/Haras Nationaux/Institut Fédératif de Recherche 135, 37380 Nouzilly, France.
authors.forename: Elodie
authors.initials: E
authors.lastname: Kara
authors.forename: Pascale
authors.initials: P
authors.lastname: Crépieux
authors.forename: Christophe
authors.initials: C
authors.lastname: Gauthier
authors.forename: Nadine
authors.initials: N
authors.lastname: Martinat
authors.forename: Vincent
authors.initials: V
authors.lastname: Piketty
authors.forename: Florian
authors.initials: F
authors.lastname: Guillou
authors.forename: Eric
authors.initials: E
authors.lastname: Reiter
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019390:Arrestins / Q000378:metabolism*; D002478:Cells, Cultured; D004705:Endocytosis; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D054769:G-Protein-Coupled Receptor Kinase 2; D006801:Humans; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011962:Receptors, FSH / Q000737:chemistry / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000378:metabolism; D015398:Signal Transduction; D013912:Threonine / Q000378:metabolism; D051552:beta-Adrenergic Receptor Kinases / Q000378:metabolism; D000071559:beta-Arrestin 1; D000071557:beta-Arrestins
chemical_list: C000605253:ARRB1 protein, human; D019390:Arrestins; D050505:Mutant Proteins; D011962:Receptors, FSH; D000071559:beta-Arrestin 1; D000071557:beta-Arrestins; D013912:Threonine; D012694:Serine; C496567:GRK2 protein, human; D051552:beta-Adrenergic Receptor Kinases; D054769:G-Protein-Coupled Receptor Kinase 2; D048049:Extracellular Signal-Regulated MAP Kinases