title: Role of Cysteine Residues in the Carboxyl-Terminus of the Follicle-Stimulating Hormone Receptor in Intracellular Traffic and Postendocytic Processing.
abstract: Posttranslational modifications occurring during the biosynthesis of G protein-coupled receptors include glycosylation and palmitoylation at conserved cysteine residues located in the carboxyl-terminus of the receptor. In a number of these receptors, these modifications play an important role in receptor function and particularly, in intracellular trafficking. In the present study, the three cysteine residues present in the carboxyl-terminus of the human FSHR were replaced with glycine by site-directed mutagenesis. Wild-type and mutant (Cys627/629/655Gly) FSHRs were then transiently expressed in HEK-293 cells and analyzed for cell-surface plasma membrane expression, agonist-stimulated signaling and internalization, and postendocytic processing in the absence and presence of lysosome and/or proteasome inhibitors. Compared with the wild-type FSHR, the triple mutant FSHR exhibited ~70% reduction in plasma membrane expression as well as a profound attenuation in agonist-stimulated cAMP production and ERK1/2 phosphorylation. Incubation of HEK-293 cells expressing the wild-type FSHR with 2-bromopalmitate (palmitoylation inhibitor) for 6 h, decreased plasma membrane expression of the receptor by ~30%. The internalization kinetics and β-arrestin 1 and 2 recruitment were similar between the wild-type and triple mutant FSHR as disclosed by assays performed in non-equilibrium binding conditions and by confocal microscopy. Cells expressing the mutant FSHR recycled the internalized FSHR back to the plasma membrane less efficiently than those expressing the wild-type FSHR, an effect that was counteracted by proteasome but not by lysosome inhibition. These results indicate that replacement of the cysteine residues present in the carboxyl-terminus of the FSHR, impairs receptor trafficking from the endoplasmic reticulum/Golgi apparatus to the plasma membrane and its recycling from endosomes back to the cell surface following agonist-induced internalization. Since in the FSHR these cysteine residues are S-palmitoylated, the data presented emphasize on this posttranslational modification as an important factor for both upward and downward trafficking of this receptor.
authors.affiliation: Research Unit in Reproductive Medicine, Unidad Medica de Alta Especialidad Hospital de Ginecobstetricia "Luis Castelazo Ayala", Instituto Mexicano del Seguro Social Mexico City, Mexico.
authors.forename: Brenda
authors.initials: B
authors.lastname: Melo-Nava
authors.affiliation: Research Unit in Reproductive Medicine, Unidad Medica de Alta Especialidad Hospital de Ginecobstetricia "Luis Castelazo Ayala", Instituto Mexicano del Seguro Social Mexico City, Mexico.
authors.forename: Patricia
authors.initials: P
authors.lastname: Casas-González
authors.affiliation: Research Unit in Reproductive Medicine, Unidad Medica de Alta Especialidad Hospital de Ginecobstetricia "Luis Castelazo Ayala", Instituto Mexicano del Seguro Social Mexico City, Mexico.
authors.forename: Marco A
authors.initials: MA
authors.lastname: Pérez-Solís
authors.affiliation: Research Support Network, Universidad Nacional Autónoma de México and Instituto Nacional de Ciencias Médicas y Nutrición "Salvador Zubirán" Mexico City, Mexico.
authors.forename: Jean
authors.initials: J
authors.lastname: Castillo-Badillo
authors.affiliation: Research Support Network, Universidad Nacional Autónoma de México and Instituto Nacional de Ciencias Médicas y Nutrición "Salvador Zubirán" Mexico City, Mexico.
authors.forename: José L
authors.initials: JL
authors.lastname: Maravillas-Montero
authors.affiliation: Department of Earth Resources, Universidad Autónoma Metropolitana Lerma, Mexico.
authors.forename: Eduardo
authors.initials: E
authors.lastname: Jardón-Valadez
authors.affiliation: Research Support Network, Universidad Nacional Autónoma de México and Instituto Nacional de Ciencias Médicas y Nutrición "Salvador Zubirán" Mexico City, Mexico.
authors.forename: Teresa
authors.initials: T
authors.lastname: Zariñán
authors.affiliation: Research Unit in Reproductive Medicine, Unidad Medica de Alta Especialidad Hospital de Ginecobstetricia "Luis Castelazo Ayala", Instituto Mexicano del Seguro Social Mexico City, Mexico.
authors.forename: Arturo
authors.initials: A
authors.lastname: Aguilar-Rojas
authors.affiliation: BIOS Group, UMR85, Unité Physiologie de la Reproduction et des Comportements, Centre National de la Recherche Scientifique, Institut National de la Recherche Agronomique, UMR7247, Université François Rabelais Tours, France.
authors.forename: Nathalie
authors.initials: N
authors.lastname: Gallay
authors.affiliation: BIOS Group, UMR85, Unité Physiologie de la Reproduction et des Comportements, Centre National de la Recherche Scientifique, Institut National de la Recherche Agronomique, UMR7247, Université François Rabelais Tours, France.
authors.forename: Eric
authors.initials: E
authors.lastname: Reiter
authors.affiliation: Research Support Network, Universidad Nacional Autónoma de México and Instituto Nacional de Ciencias Médicas y Nutrición "Salvador Zubirán" Mexico City, Mexico.
authors.forename: Alfredo
authors.initials: A
authors.lastname: Ulloa-Aguirre
keywords: follicle-stimulating hormone; follicle-stimulating hormone receptor; follitropin; internalization; palmitoylation; recycling