title: Post-endocytotic Deubiquitination and Degradation of the Metabotropic γ-Aminobutyric Acid Receptor by the Ubiquitin-specific Protease 14.
abstract: Mechanisms controlling the metabotropic γ-aminobutyric acid receptor (GABAB) cell surface stability are still poorly understood. In contrast with many other G protein-coupled receptors (GPCR), it is not subject to agonist-promoted internalization, but is constitutively internalized and rapidly down-regulated. In search of novel interacting proteins regulating receptor fate, we report that the ubiquitin-specific protease 14 (USP14) interacts with the GABAB(1b)subunit's second intracellular loop. Probing the receptor for ubiquitination using bioluminescence resonance energy transfer (BRET), we detected a constitutive and phorbol 12-myristate 13-acetate (PMA)-induced ubiquitination of the receptor at the cell surface. PMA also increased internalization and accelerated receptor degradation. Overexpression of USP14 decreased ubiquitination while treatment with a small molecule inhibitor of the deubiquitinase (IU1) increased receptor ubiquitination. Treatment with the internalization inhibitor Dynasore blunted both USP14 and IU1 effects on the receptor ubiquitination state, suggesting a post-endocytic site of action. Overexpression of USP14 also led to an accelerated degradation of GABABin a catalytically independent fashion. We thus propose a model whereby cell surface ubiquitination precedes endocytosis, after which USP14 acts as an ubiquitin-binding protein that targets the ubiquitinated receptor to lysosomal degradation and promotes its deubiquitination.
authors.affiliation: From the Department of Biochemistry and Institute for Research in Immunology and Cancer, Université de Montréal, Montréal, Quebec H3T 1J4, Canada.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Lahaie
authors.affiliation: Institute of Molecular Genetics, Academy of Science of the Czech Republic, 14220 Prague 4, Czech Republic, and.
authors.forename: Michaela
authors.initials: M
authors.lastname: Kralikova
authors.affiliation: Institut de Génomique Fonctionnelle, Université de Montpellier 1 and 2, 34090 Montpellier, France.
authors.forename: Laurent
authors.initials: L
authors.lastname: Prézeau
authors.affiliation: Institute of Molecular Genetics, Academy of Science of the Czech Republic, 14220 Prague 4, Czech Republic, and blahos@img.cas.cz.
authors.forename: Jaroslav
authors.initials: J
authors.lastname: Blahos
authors.affiliation: From the Department of Biochemistry and Institute for Research in Immunology and Cancer, Université de Montréal, Montréal, Quebec H3T 1J4, Canada, michel.bouvier@umontreal.ca.
authors.forename: Michel
authors.initials: M
authors.lastname: Bouvier
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D004705:Endocytosis / Q000187:drug effects; D017930:Genes, Reporter; D057809:HEK293 Cells; D006801:Humans; D006835:Hydrazones / Q000494:pharmacology; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism; D008969:Molecular Sequence Data; D011485:Protein Binding; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D059748:Proteolysis; D018080:Receptors, GABA-B / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D025801:Ubiquitin / Q000235:genetics / Q000378:metabolism*; D043222:Ubiquitin Thiolesterase / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination
chemical_list: D001426:Bacterial Proteins; D006835:Hydrazones; D008164:Luminescent Proteins; C511794:N'-(3,4-dihydroxybenzylidene)-3-hydroxy-2-naphthahydrazide; D018080:Receptors, GABA-B; C509299:USP14 protein, human; D025801:Ubiquitin; C054562:yellow fluorescent protein, Bacteria; D008156:Luciferases; D011493:Protein Kinase C; D043222:Ubiquitin Thiolesterase; D013755:Tetradecanoylphorbol Acetate
keywords: G protein-coupled receptor (GPCR); GABA receptor; bioluminescence resonance energy transfer (BRET); biosensor; deubiquitylation (deubiquitination); protein degradation; receptor endocytosis; ubiquitylation (ubiquitination)
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