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• W3089063994 abstract "The glucagon receptor (GCGR) activated by the peptide hormone glucagon is a seven-transmembrane G protein–coupled receptor (GPCR) that regulates blood glucose levels. Ubiquitination influences trafficking and signaling of many GPCRs, but its characterization for the GCGR is lacking. Using endocytic colocalization and ubiquitination assays, we have identified a correlation between the ubiquitination profile and recycling of the GCGR. Our experiments revealed that GCGRs are constitutively ubiquitinated at the cell surface. Glucagon stimulation not only promoted GCGR endocytic trafficking through Rab5a early endosomes and Rab4a recycling endosomes, but also induced rapid deubiquitination of GCGRs. Inhibiting GCGR internalization or disrupting endocytic trafficking prevented agonist-induced deubiquitination of the GCGR. Furthermore, a Rab4a dominant negative (DN) that blocks trafficking at recycling endosomes enabled GCGR deubiquitination, whereas a Rab5a DN that blocks trafficking at early endosomes eliminated agonist-induced GCGR deubiquitination. By down-regulating candidate deubiquitinases that are either linked with GPCR trafficking or localized on endosomes, we identified signal-transducing adaptor molecule–binding protein (STAMBP) and ubiquitin-specific protease 33 (USP33) as cognate deubiquitinases for the GCGR. Our data suggest that USP33 constitutively deubiquitinates the GCGR, whereas both STAMBP and USP33 deubiquitinate agonist-activated GCGRs at early endosomes. A mutant GCGR with all five intracellular lysines altered to arginines remains deubiquitinated and shows augmented trafficking to Rab4a recycling endosomes compared with the WT, thus affirming the role of deubiquitination in GCGR recycling. We conclude that the GCGRs are rapidly deubiquitinated after agonist-activation to facilitate Rab4a-dependent recycling and that USP33 and STAMBP activities are critical for the endocytic recycling of the GCGR. The glucagon receptor (GCGR) activated by the peptide hormone glucagon is a seven-transmembrane G protein–coupled receptor (GPCR) that regulates blood glucose levels. Ubiquitination influences trafficking and signaling of many GPCRs, but its characterization for the GCGR is lacking. Using endocytic colocalization and ubiquitination assays, we have identified a correlation between the ubiquitination profile and recycling of the GCGR. Our experiments revealed that GCGRs are constitutively ubiquitinated at the cell surface. Glucagon stimulation not only promoted GCGR endocytic trafficking through Rab5a early endosomes and Rab4a recycling endosomes, but also induced rapid deubiquitination of GCGRs. Inhibiting GCGR internalization or disrupting endocytic trafficking prevented agonist-induced deubiquitination of the GCGR. Furthermore, a Rab4a dominant negative (DN) that blocks trafficking at recycling endosomes enabled GCGR deubiquitination, whereas a Rab5a DN that blocks trafficking at early endosomes eliminated agonist-induced GCGR deubiquitination. By down-regulating candidate deubiquitinases that are either linked with GPCR trafficking or localized on endosomes, we identified signal-transducing adaptor molecule–binding protein (STAMBP) and ubiquitin-specific protease 33 (USP33) as cognate deubiquitinases for the GCGR. Our data suggest that USP33 constitutively deubiquitinates the GCGR, whereas both STAMBP and USP33 deubiquitinate agonist-activated GCGRs at early endosomes. A mutant GCGR with all five intracellular lysines altered to arginines remains deubiquitinated and shows augmented trafficking to Rab4a recycling endosomes compared with the WT, thus affirming the role of deubiquitination in GCGR recycling. We conclude that the GCGRs are rapidly deubiquitinated after agonist-activation to facilitate Rab4a-dependent recycling and that USP33 and STAMBP activities are critical for the endocytic recycling of the GCGR. Glucagon is a peptide hormone secreted by the alpha cells of the islets of Langerhans in response to hypoglycemia. Glucagon exerts its effect by binding to the seven-transmembrane glucagon receptor (GCGR) and activating adenylyl cyclase via stimulatory heterotrimeric G protein (Gs) coupling (1Lefkowitz R.J. Seven transmembrane receptors: a brief personal retrospective.Biochim. Biophys. Acta. 2007; 1768 (17173855): 748-75510.1016/j.bbamem.2006.11.001Crossref PubMed Scopus (47) Google Scholar, 2Authier F. Desbuquois B. Glucagon receptors.Cell Mol. Life Sci. 2008; 65 (18292967): 1880-189910.1007/s00018-008-7479-6Crossref PubMed Scopus (70) Google Scholar). GCGR-induced phosphorylation cascades triggered by cAMP-dependent protein kinase A results in gluconeogenesis and glycogenolysis. The GCGR is an emerging target in anti-diabetic therapy, particularly in the development of GCGR/GLP-1R co-agonists, which are predicted to have additive effects on body weight reduction and glycemia (3Axelsen L.N. Keung W. Pedersen H.D. Meier E. Riber D. Kjølbye A.L. Petersen J.S. Proctor S.D. Holstein-Rathlou N.H. Lopaschuk G.D. Glucagon and a glucagon-GLP-1 dual-agonist increases cardiac performance with different metabolic effects in insulin-resistant hearts.Br. J. Pharmacol. 2012; 165 (22014161): 2736-274810.1111/j.1476-5381.2011.01714.xCrossref PubMed Scopus (25) Google Scholar, 4Ambery P. Parker V.E. Stumvoll M. Posch M.G. Heise T. Plum-Moerschel L. Tsai L.F. Robertson D. Jain M. Petrone M. Rondinone C. Hirshberg B. Jermutus L. MEDI0382, a GLP-1 and glucagon receptor dual agonist, in obese or overweight patients with type 2 diabetes: a randomised, controlled, double-blind, ascending dose and phase 2a study.Lancet. 2018; 391 (29945727): 2607-261810.1016/S0140-6736(18)30726-8Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). Dysregulated GCGR trafficking and signaling are implicated in the development of pancreatic α-cell hyperplasia and pancreatic neuroendocrine tumors (5Zhou C. Dhall D. Nissen N.N. Chen C.R. Yu R. Homozygous P86S mutation of the human glucagon receptor is associated with hyperglucagonemia, α cell hyperplasia, and islet cell tumor.Pancreas. 2009; 38 (19657311): 941-94610.1097/MPA.0b013e3181b2bb03Crossref PubMed Scopus (93) Google Scholar, 6Roberts R.E. Zhao M. Whitelaw B.C. Ramage J. Diaz-Cano S. Le Roux C.W. Quaglia A. Huang G.C. Aylwin S.J. GLP-1 and glucagon secretion from a pancreatic neuroendocrine tumor causing diabetes and hyperinsulinemic hypoglycemia.J. Clin. Endocrinol. Metab. 2012; 97 (22774207): 3039-304510.1210/jc.2011-2005Crossref PubMed Scopus (13) Google Scholar, 7Li M. Dean E.D. Zhao L. Nicholson W.E. Powers A.C. Chen W. Glucagon receptor inactivation leads to α-cell hyperplasia in zebrafish.J. Endocrinol. 2015; 227 (26446275): 93-10310.1530/JOE-15-0284Crossref PubMed Scopus (23) Google Scholar). Glucagon also induces phosphorylation of the GCGR by the GPCR kinases (GRKs) 2, 3, and 5 and protein kinase Cα, promoting recruitment of the endocytic adaptor proteins β-arrestin 1 and β-arrestin 2, leading to clathrin-dependent internalization (8Krilov L. Nguyen A. Miyazaki T. Unson C.G. Williams R. Lee N.H. Ceryak S. Bouscarel B. Dual mode of glucagon receptor internalization: role of PKCα, GRKs and β-arrestins.Exp. Cell Res. 2011; 317 (22001118): 2981-299410.1016/j.yexcr.2011.10.001Crossref PubMed Scopus (20) Google Scholar). GCGR interacts with a single transmembrane protein called receptor activity–modifying protein 2, which reduces the cell-surface expression and dampens cAMP activation by glucagon (9Klein K.R. Matson B.C. Caron K.M. The expanding repertoire of receptor activity modifying protein (RAMP) function.Crit. Rev. Biochem. Mol. Biol. 2016; 51 (26740457): 65-7110.3109/10409238.2015.1128875Crossref PubMed Scopus (22) Google Scholar, 10Cegla J. Jones B.J. Gardiner J.V. Hodson D.J. Marjot T. McGlone E.R. Tan T.M. Bloom S.R. RAMP2 influences glucagon receptor pharmacology via trafficking and signaling.Endocrinology. 2017; 158 (28586439): 2680-269310.1210/en.2016-1755Crossref PubMed Scopus (13) Google Scholar, 11Sexton P.M. Morfis M. Tilakaratne N. Hay D.L. Udawela M. Christopoulos G. Christopoulos A. Complexing receptor pharmacology: modulation of family B G protein-coupled receptor function by RAMPs.Ann. N. Y. Acad. Sci. 2006; 1070 (16888151): 90-10410.1196/annals.1317.076Crossref PubMed Scopus (74) Google Scholar, 12Weston C. Lu J. Li N. Barkan K. Richards G.O. Roberts D.J. Skerry T.M. Poyner D. Pardamwar M. Reynolds C.A. Dowell S.J. Willars G.B. Ladds G. Modulation of glucagon receptor pharmacology by receptor activity-modifying protein-2 (RAMP2).J. Biol. Chem. 2015; 290 (26198634): 23009-2302210.1074/jbc.M114.624601Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Despite these characterizations, the molecular mechanisms that regulate GCGR trafficking and plasma membrane recycling are not completely understood. Ubiquitination of mammalian GPCRs, demonstrated initially for the β-adrenergic receptor and the CXCR4 chemokine receptor, has been identified as an important post-translational modification that directs the endocytic sorting and lysosomal degradation of internalized GPCRs (13Jean-Charles P.Y. Zhang L. Wu J.H. Han S.O. Brian L. Freedman N.J. Shenoy S.K. Ubiquitin-specific protease 20 regulates the reciprocal functions of β-arrestin2 in Toll-like receptor 4-promoted nuclear factor κB (NFκB) activation.J. Biol. Chem. 2016; 291 (26839314): 7450-746410.1074/jbc.M115.687129Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 14Marchese A. Benovic J.L. Agonist-promoted ubiquitination of the G protein-coupled receptor CXCR4 mediates lysosomal sorting.J. Biol. Chem. 2001; 276 (11641392): 45509-4551210.1074/jbc.C100527200Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar, 15Shenoy S.K. McDonald P.H. Kohout T.A. Lefkowitz R.J. Regulation of receptor fate by ubiquitination of activated β2-adrenergic receptor and β-arrestin.Science. 2001; 294 (11588219): 1307-131310.1126/science.1063866Crossref PubMed Scopus (684) Google Scholar). Expanding work in this area has revealed that ubiquitination of a GPCR can also provoke signal transduction: protease-activated receptor 1 (PAR1) traffics independently of PAR1 ubiquitination but requires PAR1 ubiquitination to trigger signaling cascades through p38 mitogen-activated protein kinases (16Dores M.R. Trejo J. Endo-lysosomal sorting of G-protein-coupled receptors by ubiquitin: diverse pathways for G-protein-coupled receptor destruction and beyond.Traffic. 2019; 20 (30353650): 101-10910.1111/tra.12619Crossref PubMed Scopus (16) Google Scholar, 17Grimsey N.J. Lin Y. Narala R. Rada C.C. Mejia-Pena H. Trejo J. G protein-coupled receptors activate p38 MAPK via a non-canonical TAB1-TAB2- and TAB1-TAB3-dependent pathway in endothelial cells.J. Biol. Chem. 2019; 294 (30760523): 5867-587810.1074/jbc.RA119.007495Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). Although not extensive, published studies also point to a critical regulatory role for deubiquitinases that reverse protein ubiquitination in GPCR trafficking and signaling pathways (18Berthouze M. Venkataramanan V. Li Y. Shenoy S.K. The deubiquitinases USP33 and USP20 coordinate β2 adrenergic receptor recycling and resensitization.EMBO J. 2009; 28 (19424180): 1684-169610.1038/emboj.2009.128Crossref PubMed Scopus (116) Google Scholar, 19Yu S.M. Jean-Charles P.Y. Abraham D.M. Kaur S. Gareri C. Mao L. Rockman H.A. Shenoy S.K. The deubiquitinase ubiquitin-specific protease 20 is a positive modulator of myocardial β1-adrenergic receptor expression and signaling.J. Biol. Chem. 2019; 294 (30538132): 2500-251810.1074/jbc.RA118.004926Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar, 20Jean-Charles P.Y. Snyder J.C. Shenoy S.K. Ubiquitination and deubiquitination of G protein-coupled receptors.Prog. Mol. Biol. Transl. Sci. 2016; 141 (27378754): 1-5510.1016/bs.pmbts.2016.05.001Crossref PubMed Scopus (20) Google Scholar). Whereas the effect of receptor ubiquitination has been reported for more than forty different members of the superfamily of GPCRs (20Jean-Charles P.Y. Snyder J.C. Shenoy S.K. Ubiquitination and deubiquitination of G protein-coupled receptors.Prog. Mol. Biol. Transl. Sci. 2016; 141 (27378754): 1-5510.1016/bs.pmbts.2016.05.001Crossref PubMed Scopus (20) Google Scholar), whether the GCGR is regulated by ubiquitination has remained unknown. Additionally, there are no detailed studies addressing ubiquitination of related class B subfamily of receptors, namely, glucagon-like peptide-1 (GLP-1) and GLP-2 receptors. On the other hand, glucose-dependent insulinotropic polypeptide (GIP) receptor has been reported to be ubiquitinated and down-regulated in isolated rat pancreatic islets upon prolonged exposure to high glucose concentration in culture medium (21Zhou J. Livak M.F. Bernier M. Muller D.C. Carlson O.D. Elahi D. Maudsley S. Egan J.M. Ubiquitination is involved in glucose-mediated downregulation of GIP receptors in islets.Am. J. Physiol. Endocrinol. Metab. 2007; 293: E538-E54710.1152/ajpendo.00070.2007Crossref PubMed Scopus (53) Google Scholar). In this study, we have ascertained the effects of the endogenous agonist glucagon on GCGR ubiquitination and correlated them with the endocytic trafficking of the receptor. We have also generated a mutant GCGR that is defective in ubiquitination and compared its trafficking with the WT GCGR. Additionally, by systematically screening different deubiquitinases involved in trafficking pathways, we have linked the GCGR with its cognate deubiquitinating enzymes. About 40 different GPCRs have been shown to be ubiquitinated, with a vast majority of these GPCRs undergoing ubiquitin-directed intracellular trafficking (20Jean-Charles P.Y. Snyder J.C. Shenoy S.K. Ubiquitination and deubiquitination of G protein-coupled receptors.Prog. Mol. Biol. Transl. Sci. 2016; 141 (27378754): 1-5510.1016/bs.pmbts.2016.05.001Crossref PubMed Scopus (20) Google Scholar, 22Dores M.R. Trejo J. Ubiquitination of G protein-coupled receptors: functional implications and drug discovery.Mol. Pharmacol. 2012; 82 (22700696): 563-57010.1124/mol.112.079418Crossref PubMed Scopus (36) Google Scholar). However, GCGR ubiquitination had not been characterized thus far. To define whether the GCGR is ubiquitinated and whether this is modulated by agonist stimulation, we treated human embryonic kidney (HEK-293) cells that have been stably transfected with GCGR with 200 nm glucagon for different times and determined the ubiquitination profile using FLAG immunoprecipitation by methods that we and others have reported before (14Marchese A. Benovic J.L. Agonist-promoted ubiquitination of the G protein-coupled receptor CXCR4 mediates lysosomal sorting.J. Biol. Chem. 2001; 276 (11641392): 45509-4551210.1074/jbc.C100527200Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar, 19Yu S.M. Jean-Charles P.Y. Abraham D.M. Kaur S. Gareri C. Mao L. Rockman H.A. Shenoy S.K. The deubiquitinase ubiquitin-specific protease 20 is a positive modulator of myocardial β1-adrenergic receptor expression and signaling.J. Biol. Chem. 2019; 294 (30538132): 2500-251810.1074/jbc.RA118.004926Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar, 23Han S.O. Kommaddi R.P. Shenoy S.K. Distinct roles for β-arrestin2 and arrestin-domain-containing proteins in β2 adrenergic receptor trafficking.EMBO Rep. 2013; 14 (23208550): 164-17110.1038/embor.2012.187Crossref PubMed Scopus (71) Google Scholar, 24Xiao K. Shenoy S.K. β2-adrenergic receptor lysosomal trafficking is regulated by ubiquitination of lysyl residues in two distinct receptor domains.J. Biol. Chem. 2011; 286 (21330366): 12785-1279510.1074/jbc.M110.203091Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Interestingly, we not only observed an agonist-dependent mobility shift of the immunoprecipitated GCGR (see GCGR immunoblot in Fig. 1A) engendered by receptor phosphorylation (25Komolov K.E. Du Y. Duc N.M. Betz R.M. Rodrigues J. Leib R.D. Patra D. Skiniotis G. Adams C.M. Dror R.O. Chung K.Y. Kobilka B.K. Benovic J.L. Structural and functional analysis of a β2-adrenergic receptor complex with GRK5.Cell. 2017; 169 (28431242): 407-421.e1610.1016/j.cell.2017.03.047Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 26Stadel J.M. Nambi P. Shorr R.G. Sawyer D.F. Caron M.G. Lefkowitz R.J. Catecholamine-induced desensitization of turkey erythrocyte adenylate cyclase is associated with phosphorylation of the β-adrenergic receptor.Proc. Natl. Acad. Sci. U. S. A. 1983; 80 (6304694): 3173-317710.1073/pnas.80.11.3173Crossref PubMed Scopus (152) Google Scholar), but also detected an agonist-induced decrease in ubiquitination (i.e. deubiquitination) of the GCGR within 5 min, which was unchanged until 60 min of glucagon stimulation, when the GCGR ubiquitination returned to basal levels (Fig. 1, A and B). Within this duration of agonist exposure, we did not observe a significant change in total receptor protein, suggesting that the decrease in ubiquitination is not from a corresponding decrease in the GCGR expression. We also failed to detect GCGR ubiquitin signals with an antibody that is selective to monoubiquitin (P4D1), but antibodies that are selective to polyubiquitin (clone FK1 and rabbit polyclonal ubiquitin antibodies) yielded reproducible signals, which were detected only in receptor pulldowns and not in control immunoprecipitates; therefore, we conclude that the GCGR is polyubiquitinated (Fig. 1A). To discern a correlation between glucagon-induced deubiquitination and internalization kinetics of the GCGR, we assessed agonist-induced changes in cell-surface expression of GCGR. We used a GCGR construct with an N-terminal MYC tag that could be detected at the plasma membrane in nonpermeabilized cells (Fig. 1C). We determined the amount of cell-surface GCGR in quiescent and agonist-stimulated cells using an ELISA (Fig. 1D). We mainly focused on 15 and 60 min of agonist stimulation, which showed distinct patterns of deubiquitination and reubiquitination (Fig. 1, A and B). At 15 min of glucagon stimulation, we detected a significant decrease in cell-surface GCGR expression compared with unstimulated conditions, and furthermore, after 60 min of glucagon stimulation, we observed a significant level of recovery of cell-surface receptors, although it did not attain levels in unstimulated cells (Fig. 1D). These data strongly suggest a correlation between GCGR deubiquitination and internalization at 15 min and GCGR reubiquitination and cell-surface recovery at 60 min. The GCGR is expressed at higher levels in the liver than in other organs like kidney or pancreatic islets; hence, we also tested GCGR ubiquitination in the commonly used hepatocyte model cell line, HepG2. Glucagon-induced deubiquitination of the GCGR observed in HEK-293 cells is recapitulated in the HepG2 cells (Fig. 1, E and F) as we detected basal polyubiquitination of the GCGR, which decreased by ∼65% after 15 min of glucagon exposure. Thus, the GCGR presents a unique pattern of agonist-induced deubiquitination that is not observed with other GPCRs that have been tested for ubiquitination (16Dores M.R. Trejo J. Endo-lysosomal sorting of G-protein-coupled receptors by ubiquitin: diverse pathways for G-protein-coupled receptor destruction and beyond.Traffic. 2019; 20 (30353650): 101-10910.1111/tra.12619Crossref PubMed Scopus (16) Google Scholar, 20Jean-Charles P.Y. Snyder J.C. Shenoy S.K. Ubiquitination and deubiquitination of G protein-coupled receptors.Prog. Mol. Biol. Transl. Sci. 2016; 141 (27378754): 1-5510.1016/bs.pmbts.2016.05.001Crossref PubMed Scopus (20) Google Scholar). Based on the correlation between glucagon-induced deubiquitination and internalization (Fig. 1), we hypothesized that the plasma membrane–localized receptors represent the ubiquitinated species and that internalized receptors are deubiquitinated units. If this were true, then inhibition of internalization should prevent glucagon-induced deubiquitination of the GCGR. The GCGR has been reported to utilize multiple pathways of internalization invoking both clathrin and caveolin-dependent mechanisms (8Krilov L. Nguyen A. Miyazaki T. Unson C.G. Williams R. Lee N.H. Ceryak S. Bouscarel B. Dual mode of glucagon receptor internalization: role of PKCα, GRKs and β-arrestins.Exp. Cell Res. 2011; 317 (22001118): 2981-299410.1016/j.yexcr.2011.10.001Crossref PubMed Scopus (20) Google Scholar, 27Krilov L. Nguyen A. Miyazaki T. Unson C.G. Bouscarel B. Glucagon receptor recycling: role of carboxyl terminus, β-arrestins, and cytoskeleton.Am. J. Physiol. Cell Physiol. 2008; 295 (18787074): C1230-C123710.1152/ajpcell.00240.2008Crossref PubMed Scopus (15) Google Scholar). When we pretreated GCGR-stable cells with 0.4 m sucrose, which blocks clathrin-dependent and non-clathrin-dependent endocytosis (28Carpentier J.L. Sawano F. Geiger D. Gorden P. Perrelet A. Orci L. Potassium depletion and hypertonic medium reduce “non-coated” and clathrin-coated pit formation, as well as endocytosis through these two gates.J. Cell. Physiol. 1989; 138 (2466853): 519-52610.1002/jcp.1041380311Crossref PubMed Scopus (72) Google Scholar), the agonist-induced deubiquitination of the GCGR was completely blocked (Fig. 2, A and B), suggesting that only internalized GCGRs are deubiquitinated. To assess the contribution of dynamin and clathrin-dependent endocytosis, we employed Dyngo-4a, a widely used chemical inhibitor of dynamin GTPase (29Robertson M.J. Deane F.M. Robinson P.J. McCluskey A. Synthesis of Dynole 34-2, Dynole 2-24 and Dyngo 4a for investigating dynamin GTPase.Nat. Protoc. 2014; 9 (24651498): 851-87010.1038/nprot.2014.046Crossref PubMed Scopus (34) Google Scholar, 30Tsvetanova N.G. Trester-Zedlitz M. Newton B.W. Riordan D.P. Sundaram A.B. Johnson J.R. Krogan N.J. von Zastrow M. G protein-coupled receptor endocytosis confers uniformity in responses to chemically distinct ligands.Mol. Pharmacol. 2017; 91 (27879340): 145-15610.1124/mol.116.106369Crossref PubMed Scopus (16) Google Scholar, 31Jensen D.D. Lieu T. Halls M.L. Veldhuis N.A. Imlach W.L. Mai Q.N. Poole D.P. Quach T. Aurelio L. Conner J. Herenbrink C.K. Barlow N. Simpson J.S. Scanlon M.J. Graham B. et al.Neurokinin 1 receptor signaling in endosomes mediates sustained nociception and is a viable therapeutic target for prolonged pain relief.Sci. Transl. Med. 2017; 9 (28566424)eaal3447 10.1126/scitranslmed.aal3447Crossref PubMed Scopus (76) Google Scholar). When cells were pretreated with 60 μm Dyngo-4a, we detected a modest increase in basal ubiquitination, and although we observed a glucagon-induced decrease in ubiquitination, it was not significant (Fig. 2, C and D). Thus, blocking clathrin-dependent internalization prevented deubiquitination of most but perhaps not all activated GCGRs. We next tested whether internalized GCGR is deubiquitinated when intracellular trafficking of the GCGR is disrupted. We used the carboxylic ionophore monensin, which has no effect on receptor internalization but blocks trafficking of internalized receptor complexes into recycling vesicles. These blocking effects of monensin on trafficking have been documented for several GPCRs as well as for other cell-surface receptors (32Brown M.S. Anderson R.G. Basu S.K. Goldstein J.L. Recycling of cell-surface receptors: observations from the LDL receptor system.Cold Spring Harb. Symp. Quant. 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When GCGR-stable cells were pretreated for 60 min with 50 μm monensin, basal ubiquitination of the GCGR was unaltered compared with cells that were not treated with monensin. However, glucagon stimulation did not induce GCGR deubiquitination in just monensin-treated cells (Fig. 2, E and F). These results suggest that agonist-activated GCGRs have to be mobilized into specific compartments after internalization to be deubiquitinated. Monensin can block GPCR recycling from different populations of endosomal vesicles, including early and perinuclear endosomes (36Li G. Shi Y. Huang H. Zhang Y. Wu K. Luo J. Sun Y. Lu J. Benovic J.L. Zhou N. Internalization of the human nicotinic acid receptor GPR109A is regulated by Gi, GRK2, and arrestin3.J. Biol. Chem. 2010; 285 (20460384): 22605-2261810.1074/jbc.M109.087213Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Therefore, we next determined the effects of monensin on the subcellular localization of GCGR after 15 min of agonist activation that obtains the maximal extent of deubiquitination (Fig. 1). We first tested colocalization of GCGR and GFP-Rab5a, a small GTPase that is a well-known marker for the early endosome population (37Novick P. Zerial M. The diversity of Rab proteins in vesicle transport.Curr. Opin. Cell Biol. 1997; 9 (9261061): 496-50410.1016/S0955-0674(97)80025-7Crossref PubMed Scopus (655) Google Scholar, 38Wandinger-Ness A. Zerial M. Rab proteins and the compartmentalization of the endosomal system.Cold Spring Harb. Perspect. Biol. 2014; 6 (25341920)a022616 10.1101/cshperspect.a022616Crossref PubMed Scopus (228) Google Scholar). As shown in the confocal images in Fig. 3A, in unstimulated GCGR stable cells, the staining for the GCGR was mostly detected at the plasma membrane, and GFP-Rab5a was mostly distributed in cytoplasmic vesicles. In cells stimulated with glucagon, we detected a significant increase in the colocalization of GCGR and GFP-Rab5a (Fig. 3, A and B). Monensin treatment alone led to a modest increase in GCGR localization in vesicles that were Rab5a-positive, resulting in a 20–30% increase in GCGR and Rab5a colocalization compared with unstimulated vehicle-treated cells. Furthermore, in cells exposed to both monensin and glucagon, we observed a significant increase in the colocalization of GCGR with GFP-Rab5a (Fig. 3, A and B). Accordingly, monensin potentiates glucagon-induced localization of GCGR in Rab5a-positive early endosomes. We next repeated the confocal experiments to determine trafficking of the GCGR with or without monensin with another Rab marker protein, GFP-Rab4a, which localizes predominantly at recycling endosomes. As shown in Fig. 4 (A and B), glucagon stimulation leads to a robust increase in GCGR and GFP-Rab4a colocalization compared with unstimulated cells. On the other hand, in a majority of monensin-treated cells, glucagon stimulation caused GCGR to prevail in large vesicles that did not colocalize with GFP-Rab4a. Accordingly, monensin prevents the trafficking of internalized GCGRs into Rab4a-marked recycling vesicles. Taken together with the effects on GCGR ubiquitination (Fig. 2), these data suggest that in monensin-treated cells, ubiquitinated GCGRs are accumulated in Rab5a-positive early endosomes. To further dissect the contrasting effects produced by monensin on the colocalization of the internalized GCGR with Rab4a and Rab5a, we next employed the inactive (dominant negative) mutants in our ubiquitination assays. Whereas the WT Rab GTPases bind GTP and trigger GTP hydrolysis and endosomal vesicle fusion, Rab4a S22N and Rab5a S34N mutants have increased affinity for GDP and are unable to engender vesicle membrane fusion (39Stenmark H. Parton R.G. Steele-Mortimer O. Lütcke A. Gruenberg J. Zerial M. Inhibition of rab5 GTPase activity stimulates membrane fusion in endocytosis.EMBO J. 1994; 13 (8137813): 1287-129610.1002/j.1460-2075.1994.tb06381.xCrossref PubMed Scopus (740) Google Scholar, 40McCaffrey M.W. Bielli A. Cantalupo G. Mora S. Roberti V. Santillo M. Drummond F. Bucci C. Rab4 affects both recycling and degradative endosomal trafficking.FEBS Lett. 2001; 495 (11322941): 21-3010.1016/S0014-5793(01)02359-6Crossref PubMed Scopus (126) Google Scholar). These mutants act as dominant negatives by sequestering endogenous guanine nucleotide exchange factors because of the low affinity for GTP (41Lee M.T. Mishra A. Lambright D.G. Structural mechanisms for regulation of membrane traffic by Rab GTPases.Traffic. 2009; 10 (19522756): 1377-138910.1111/j.1600-0854.2009.00942.xCrossref PubMed Scopus (93) Google Scholar). Exogenous expression of Rab4a S22N blocks recycling of membrane receptors to the plasma membrane, and Rab5a S34N has been shown to either inhibit internalization and retain receptors at the plasma membrane or trap receptors in small endocytic vesicles that are destined to fuse with early endosomes (Fig. 5A). Co-expression of GFP vector preserved basal ubiquitination of GCGRs as well as agonist-induced deubiquitination of the GCGR (Fig. 5, B and C). In cells transfected with GFP-Rab4a S22N, basal GCGR ubiquitination was modestly reduced compared with GFP-transfected cells, and agonist stimulation led to further deubiquitination. In marked contrast, in cells transfected with GFP-Rab5a S34N, we detected a 25% increase in ubiquitination after agonist activation, compared with unstimulated samples. We also assessed the effect of GFP-Rab5a S34N on GCGR subcellular distribution by confocal microscopy. As shown in Fig. S1, in cells expressing GFP-Rab5a S34N, we detected GCGR internalization into smal" @default.
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• W3089063994 date "2020-12-01" @default.
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• W3089063994 title "Agonist-activated glucagon receptors are deubiquitinated at early endosomes by two distinct deubiquitinases to facilitate Rab4a-dependent recycling" @default.
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