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.

From the Department of Biochemistry and Institute for Research in Immunology and Cancer Université de Montréal Montréal Quebec H3T 1J4 Canada

Institut de Génomique Fonctionnelle Université de Montpellier 1 and 2 34090 Montpellier France

Institute of Molecular Genetics Academy of Science of the Czech Republic 14220 Prague 4 Czech Republic and

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Luttrell L. M., and Lefkowitz R. J. (2002) The role of {beta}-arrestins in the termination and transduction of G-protein-coupled receptor signals. J. Cell Sci. 115, 455–465 PubMed

White J. H., Wise a, Main M. J., Green A., Fraser N. J., Disney G. H., Barnes A. A., Emson P., Foord S. M., and Marshall F. H. (1998) Heterodimerization is required for the formation of a functional GABA(B) receptor. Nature 396, 679–682 PubMed

Kanaide M., and Uezono Y. (2007) Desensitization of GABAB receptor signaling by formation of protein complexes of GABAB2 subunit with GRK4 or GRK5. J. Cell Physiol. 210, 237–245 PubMed

Perroy J., Adam L., Qanbar R., Chénier S., and Bouvier M. (2003) Phosphorylation-independent desensitization of GABA(B) receptor by GRK4. EMBO J. 22, 3816–3824 PubMed PMC

Pontier S. M., Lahaie N., Ginham R., St-Gelais F., Bonin H., Bell D. J., Flynn H., Trudeau L.-E., McIlhinney J., White J. H., and Bouvier M. (2006) Coordinated action of NSF and PKC regulates GABAB receptor signaling efficacy. EMBO J. 25, 2698–2709 PubMed PMC

Kuramoto N., Wilkins M. E., Fairfax B. P., Revilla-Sanchez R., Terunuma M., Tamaki K., Iemata M., Warren N., Couve A., Calver A., Horvath Z., Freeman K., Carling D., Huang L., Gonzales C., Cooper E., Smart T. G., Pangalos M. N., and Moss S. J. (2007) Phospho-dependent functional modulation of GABA(B) receptors by the metabolic sensor AMP-dependent protein kinase. Neuron 53, 233–247 PubMed PMC

Couve A., Thomas P., Calver A. R., Hirst W. D., Pangalos M. N., Walsh F. S., Smart T. G., and Moss S. J. (2002) Cyclic AMP-dependent protein kinase phosphorylation facilitates GABA(B) receptor-effector coupling. Nat. Neurosci. 5, 415–424 PubMed

Schwenk J., Metz M., Zolles G., Turecek R., Fritzius T., Bildl W., Tarusawa E., Kulik A., Unger A., Ivankova K., Seddik R., Tiao J. Y., Rajalu M., Trojanova J., Rohde V., Gassmann M., Schulte U., Fakler B., and Bettler B. (2010) Native GABA(B) receptors are heteromultimers with a family of auxiliary subunits. Nature 465, 231–235 PubMed

Hanyaloglu A. C., and von Zastrow M. (2008) Regulation of GPCRs by endocytic membrane trafficking and its potential implications. Annu. Rev. Pharmacol. Toxicol. 48, 537–568 PubMed

Grampp T., Sauter K., Markovic B., and Benke D. (2007) γ-Aminobutyric acid type B receptors are constitutively internalized via the clathrin-dependent pathway and targeted to lysosomes for degradation. J. Biol. Chem. 282, 24157–24165 PubMed

Grampp T., Notz V., Broll I., Fischer N., and Benke D. (2008) Constitutive, agonist-accelerated, recycling and lysosomal degradation of GABA(B) receptors in cortical neurons. Mol. Cell Neurosci. 39, 628–637 PubMed

Vargas K. J., Terunuma M., Tello J. A., Pangalos M. N., Moss S. J., and Couve A. (2008) The availability of surface GABA B receptors is independent of γ-aminobutyric acid but controlled by glutamate in central neurons. J. Biol. Chem. 283, 24641–24648 PubMed PMC

Terunuma M., Vargas K. J., Wilkins M. E., Ramirez O. A., Jaureguiberry-Bravo M., Pangalos M. N., Smart T. G., Moss S. J., and Couve A. (2010) Prolonged activation of NMDA receptors promotes dephosphorylation and alters postendocytic sorting of GABAB receptors. Proc. Natl. Acad. Sci. 107, 13918–13923 PubMed PMC

Guetg N., Aziz S. A., Holbro N., Turecek R., Rose T., Seddik R., Gassmann M., Moes S., Jenoe P., Oertner T. G., Casanova E., and Bettler B. (2010) NMDA receptor-dependent GABAB receptor internalization via CaMKII phosphorylation of serine 867 in GABAB1. Proc. Natl. Acad. Sci. U.S.A. 107, 13924–13929 PubMed PMC

Maier P. J., Marin I., Grampp T., Sommer A., and Benke D. (2010) Sustained glutamate receptor activation down-regulates GABA(B) receptors by shifting the balance from recycling to lysosomal degradation. J. Biol. Chem. 285, 35606–35614 PubMed PMC

Shenoy S. K., McDonald P. H., Kohout T. A., and Lefkowitz R. J. (2001) Regulation of receptor fate by ubiquitination of activated β2-adrenergic receptor and β-arrestin. Science 294, 1307–1313 PubMed

Sarker S., Xiao K., and Shenoy S. K. (2011) A tale of two sites: How ubiquitination of a G protein-coupled receptor is coupled to its lysosomal trafficking from distinct receptor domains. Commun. Integr. Biol. 4, 528–531 PubMed PMC

Wolfe B. L., Marchese A., and Trejo J. (2007) Ubiquitination differentially regulates clathrin-dependent internalization of protease-activated receptor-1. J. Cell Biol. 177, 905–916 PubMed PMC

Shenoy S. K., Xiao K., Venkataramanan V., Snyder P. M., Freedman N. J., and Weissman A. M. (2008) Nedd4 mediates agonist-dependent ubiquitination, lysosomal targeting, and degradation of the β2-adrenergic receptor. J. Biol. Chem. 283, 22166–22176 PubMed PMC

Nabhan J. F., Pan H., and Lu Q. (2010) Arrestin domain-containing protein 3 recruits the NEDD4 E3 ligase to mediate ubiquitination of the β2-adrenergic receptor. EMBO Rep. 11, 605–611 PubMed PMC

Berthouze M., Venkataramanan V., Li Y., and Shenoy S. K. (2009) The deubiquitinases USP33 and USP20 coordinate β2 adrenergic receptor recycling and resensitization. EMBO J. 28, 1684–1696 PubMed PMC

Marchese A., Raiborg C., Santini F., Keen J. H., Stenmark H., and Benovic J. L. (2003) The E3 ubiquitin ligase AIP4 mediates ubiquitination and sorting of the G protein-coupled receptor CXCR4. Dev. Cell 5, 709–722 PubMed

Mines M. A., Goodwin J. S., Limbird L. E., Cui F.-F., and Fan G.-H. (2009) Deubiquitination of CXCR4 by USP14 is critical for both CXCL12-induced CXCR4 degradation and chemotaxis but not ERK ativation. J. Biol. Chem. 284, 5742–5752 PubMed PMC

Berlin I., Higginbotham K. M., Dise R. S., Sierra M. I., and Nash P. D. (2010) The deubiquitinating enzyme USP8 promotes trafficking and degradation of the chemokine receptor 4 at the sorting endosome. J. Biol. Chem. 285, 37895–37908 PubMed PMC

Zemoura K., Schenkel M., Acuña M. A., Yévenes G. E., Zeilhofer H. U., and Benke D. (2013) Endoplasmic Reticulum-associated Degradation Controls Cell Surface Expression of γ-Aminobutyric Acid, Type B Receptors. J. Biol. Chem. 288, 34897–34905 PubMed PMC

Perroy J., Pontier S., Charest P., Aubry M., and Bouvier M. (2004) Real-time monitoring of ubiquitination in living cells by BRET. Nat. Methods 1, 203–208 PubMed

Villemure J.-F., Adam L., Bevan N. J., Gearing K., Chénier S., and Bouvier M. (2005) Subcellular distribution of GABA(B) receptor homo- and hetero-dimers. Biochem. J. 388, 47–55 PubMed PMC

Issafras H., Angers S., Bulenger S., Blanpain C., Parmentier M., Labbé-Jullié C., Bouvier M., and Marullo S. (2002) Constitutive agonist-independent CCR5 oligomerization and antibody-mediated clustering occurring at physiological levels of receptors. J. Biol. Chem. 277, 34666–34673 PubMed

Breton B., Lagacé M., and Bouvier M. (2010) Combining resonance energy transfer methods reveals a complex between the α2A-adrenergic receptor, Gαi1β1γ2, and GRK2. FASEB J. 24, 4733–4743 PubMed

Nagai A., Kadowaki H., Maruyama T., Takeda K., Nishitoh H., and Ichijo H. (2009) USP14 inhibits ER-associated degradation via interaction with IRE1α. Biochem. Biophys. Res. Commun. 379, 995–1000 PubMed

Lee B.-H., Lee M. J., Park S., Oh D.-C., Elsasser S., Chen P.-C., Gartner C., Dimova N., Hanna J., Gygi S. P., Wilson S. M., King R. W., and Finley D. (2010) Enhancement of proteasome activity by a small-molecule inhibitor of USP14. Nature 467, 179–184 PubMed PMC

Mercier J.-F., Salahpour A., Angers S., Breit A., and Bouvier M. (2002) Quantitative assessment of β1- and β2-adrenergic receptor homo- and heterodimerization by bioluminescence resonance energy transfer. J. Biol. Chem. 277, 44925–44931 PubMed

Margeta-Mitrovic M., Jan Y. N., and Jan L. Y. (2001) Function of GB1 and GB2 subunits in G protein coupling of GABA(B) receptors. Proc. Natl. Acad. Sci. U.S.A. 98, 14649–14654 PubMed PMC

Na C. H., Jones D. R., Yang Y., Wang X., Xu Y., and Peng J. (2012) Synaptic protein ubiquitination in rat brain revealed by antibody-based ubiquitome analysis. J. Proteome Res. 11, 4722–4732 PubMed PMC

Mukherjee R. S., Mcbride E. W., Beinborn M., Dunlap K., and Kopin A. S. (2006) Point mutations in either subunit of the GABAB receptor confer constitutive activity to the heterodimer. Mol. Pharmacol. 70, 1406–1413 PubMed

Dutar P., and Nicoll R. A. (1988) Pre- and postsynaptic GABAB receptors in the hippocampus have different pharmacological properties. Neuron 1, 585–591 PubMed

Jacob C., Cottrell G. S., Gehringer D., Schmidlin F., Grady E. F., and Bunnett N. W. (2005) c-Cbl mediates ubiquitination, degradation, and down-regulation of human protease-activated receptor 2. J. Biol. Chem. 280, 16076–16087 PubMed

Marchese A., and Benovic J. L. (2001) Agonist-promoted ubiquitination of the G protein-coupled receptor CXCR4 mediates lysosomal sorting. J. Biol. Chem. 276, 45509–45512 PubMed

Fairfax B. P., Pitcher J. A., Scott M. G. H., Calver A. R., Pangalos M. N., Moss S. J., and Couve A. (2004) Phosphorylation and chronic agonist treatment atypically modulate GABAB receptor cell surface stability. J. Biol. Chem. 279, 12565–12573 PubMed

Clague M. J., Barsukov I., Coulson J. M., Liu H., Rigden D. J., and Urbé S. (2013) Deubiquitylases from genes to organism. Physiol. Rev. 93, 1289–1315 PubMed

Hu M., Li P., Song L., Jeffrey P. D., Chenova T. A., Wilkinson K. D., Cohen R. E., and Shi Y. (2005) Structure and mechanisms of the proteasome-associated deubiquitinating enzyme USP14. EMBO J. 24, 3747–3756 PubMed PMC

Leithe E., and Rivedal E. (2004) Ubiquitination and down-regulation of gap junction protein connexin-43 in response to 12-O-tetradecanoylphorbol 13-acetate treatment. J. Biol. Chem. 279, 50089–50096 PubMed

Li S., Zhang Q., and You G. (2013) Three ubiquitination sites of organic anion transporter-1 synergistically mediate protein kinase C-dependent endocytosis of the transporter. Mol. Pharmacol. 84, 139–146 PubMed PMC

Haglund K., and Dikic I. (2012) The role of ubiquitylation in receptor endocytosis and endosomal sorting. J. Cell Sci. 125, 265–275 PubMed

Zemoura K., and Benke D. (2014) Proteasomal degradation of γ-aminobutyric acidB receptors is mediated by the interaction of the GABAB2 C terminus with the proteasomal ATPase Rtp6 and regulated by neuronal activity. J. Biol. Chem. 289, 7738–7746 PubMed PMC

Peth A., Besche H. C., and Goldberg A. L. (2009) Ubiquitinated proteins activate the proteasome by binding to Usp14/Ubp6, which causes 20S gate opening. Mol. Cell 36, 794–804 PubMed PMC

Peth A., Kukushkin N., Bossé M., and Goldberg A. L. (2013) Ubiquitinated proteins activate the proteasomal ATPases by binding to Usp14 or Uch37 homologs. J. Biol. Chem. 288, 7781–7790 PubMed PMC

Walters B. J., Hallengren J. J., Theile C. S., Ploegh H. L., Wilson S. M., and Dobrunz L. E. (2014) A catalytic independent function of the deubiquitinating enzyme USP14 regulates hippocampal synaptic short-term plasticity and vesicle number. J. Physiol. 592, 571–586 PubMed PMC

Kuramoto N., Ito M., Saito Y., Niihara H., Tanaka N., Yamada K.-I., Yamamura Y., Iwasaki K., Onishi Y., and Ogita K. (2013) Dephosphorylation of endogenous GABA(B) receptor R2 subunit and AMPK α subunits which were measured by in vitro method using transfer membrane. Neurochem. Int. 62, 137–144 PubMed

Yao Y., Kelly M. T., Sajikumar S., Serrano P., Tian D., Bergold P. J., Frey J. U., and Sacktor T. C. (2008) PKMζ maintains late-LTP by NSF/GluR2-dependent trafficking of postsynaptic AMPAR receptors. J. Neurosci. 28, 7820–7827 PubMed PMC

Wilson S. M., Bhattacharyya B., Rachel R. A., Coppola V., Tessarollo L., Householder D. B., Fletcher C. F., Miller R. J., Copeland N. G., and Jenkins N. A. (2002) Synaptic defects in ataxia mice result from a mutation in Usp14, encoding a ubiquitin-specific protease. Nat. Genet. 32, 420–425 PubMed

Lappe-Siefke C., Loebrich S., Hevers W., Waidmann O. B., Schweizer M., Fehr S., Fritschy J.-M., Dikic I., Eilers J., Wilson S. M., and Kneussel M. (2009) The ataxia (axJ) mutation causes abnormal GABAA receptor turnover in mice. PLoS Genet. 5, e1000631. PubMed PMC

Bhattacharyya B. J., Wilson S. M., Jung H., and Miller R. J. (2012) Altered neurotransmitter release machinery in mice deficient for the deubiquitinating enzyme Usp14. Am. J. Physiol. Cell Physiol. 302, C698–708 PubMed PMC

Sakaba T., and Neher E. (2003) Direct modulation of synaptic vesicle priming by GABA(B) receptor activation at a glutamatergic synapse. Nature 424, 775–778 PubMed