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.

• Klíčová slova
• G protein-coupled receptor (GPCR), GABA receptor, bioluminescence resonance energy transfer (BRET), biosensor, deubiquitylation (deubiquitination), protein degradation, receptor endocytosis, ubiquitylation (ubiquitination),
• MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• buněčná membrána účinky léků   metabolismus   MeSH
• endocytóza účinky léků   MeSH
• HEK293 buňky MeSH
• hydrazony farmakologie   MeSH
• lidé MeSH
• luciferasy genetika   metabolismus   MeSH
• luminescentní proteiny genetika   metabolismus   MeSH
• lyzozomy metabolismus   MeSH
• molekulární sekvence - údaje MeSH
• posttranslační úpravy proteinů * MeSH
• proteinkinasa C genetika   metabolismus   MeSH
• proteolýza MeSH
• receptory GABA-B genetika   metabolismus   MeSH
• reportérové geny MeSH
• sekvence aminokyselin MeSH
• signální transdukce MeSH
• tetradekanoylforbolacetát farmakologie   MeSH
• thiolesterasa ubikvitinu genetika   metabolismus   MeSH
• ubikvitin genetika   metabolismus   MeSH
• ubikvitinace MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny MeSH
• hydrazony MeSH
• luciferasy MeSH
• luminescentní proteiny MeSH
• N'-(3,4-dihydroxybenzylidene)-3-hydroxy-2-naphthahydrazide MeSH Prohlížeč
• proteinkinasa C MeSH
• receptory GABA-B MeSH
• tetradekanoylforbolacetát MeSH
• thiolesterasa ubikvitinu MeSH
• ubikvitin MeSH
• USP14 protein, human MeSH Prohlížeč
• yellow fluorescent protein, Bacteria MeSH Prohlížeč