Small Ubiquitin-like Modifier Modification of Arrestin-3Regulates Receptor Trafficking*□S

Received for publication, June 6, 2010, and in revised form, November 24, 2010 Published, JBC Papers in Press, November 30, 2010, DOI 10.1074/jbc.M110.152116

Debra Wyatt‡1, Rohit Malik§1, Alissa C. Vesecky‡, and Adriano Marchese‡§2

From the ‡Department of Pharmacology and the §Program in Molecular Biology, Stritch School of Medicine, Loyola UniversityChicago, Maywood, Illinois 60153

Nonvisual arrestins are regulated by direct post-transla-tional modifications, such as phosphorylation, ubiquitination,and nitrosylation. However, whether arrestins are regulated byother post-translational modifications remains unknown. Herewe show that nonvisual arrestins are modified by small ubiq-uitin-like modifier 1 (SUMO-1) upon activation of �2-adrener-gic receptor (�2AR). Lysine residues 295 and 400 in arrestin-3fall within canonical SUMO consensus sites, and mutagenicanalysis reveals that Lys-400 represents the main SUMOyla-tion site. Depletion of the SUMO E2 modifying enzyme Ubc9blocks arrestin-3 SUMOylation and attenuates �2AR internal-ization, suggesting that arrestin SUMOylation mediates G pro-tein-coupled receptor endocytosis. Consistent with this, ex-pression of a SUMO-deficient arrestin mutant failed topromote �2AR internalization as compared with wild-typearrestin-3. Our data reveal an unprecedented role forSUMOylation in mediating GPCR endocytosis and providenovel mechanistic insight into arrestin function and regulation.

Nonvisual arrestins play an important role in the regulationof G protein-coupled receptor (GPCR)3 desensitization, traf-ficking, and signaling. Arrestins bind to agonist-activated andGPCR kinase phosphorylated receptors to uncouple the asso-ciated heterotrimeric G protein from the receptor through aprocess likely to involve steric hindrance, culminating in thetermination of further signaling (1). Arrestins also promoteGPCR internalization by virtue of their ability to bind to com-ponents of the trafficking machinery, such as clathrin andAP2, thus enabling the recruitment of receptors to clathrin-coated pits where they are internalized (2, 3). In addition, ar-restins interact with and activate several downstream signal-ing molecules to initiate G protein-independent signalingevents (4–7).

Arrestins are regulated by post-translational modifications,including ubiquitination, phosphorylation, and nitrosylation(8–10). Ubiquitination of arrestin has been linked to its abilityto promote internalization of �2-adrenergic receptor (�2AR)(10). It has also been linked to G protein-independent andarrestin-dependent GPCR signaling whereby the arrestinubiquitination status correlates with its ability to stably asso-ciate with GPCRs upon internalization onto endosomes andto activate downstream signaling cascades (11). Dephosphor-ylation and nitrosylation facilitate the ability of arrestins topromote GPCR trafficking, possibly by facilitating interac-tions with the internalization machinery (8, 9). Whether ar-restins are regulated by other post-translational modificationsremains unknown.To our knowledge, other than a few studies, the role of

SUMO, another common post-translational modifier, inGPCR signaling remains relatively unexplored (12–14). Pro-teins that are modified by SUMO typically encode a SUMOconsensus motif, defined as �KX(D/E), where � representsan aliphatic amino acid followed by an acceptor lysine resi-due, and X represents any amino acid adjacent to an acidicresidue (Asp/Glu), although there are exceptions (15). SUMOis a member of the ubiquitin-like molecules family of proteins(16, 17). It is structurally related to ubiquitin, although at theprimary amino acid level they share very little amino acididentity (18). SUMO is attached to proteins via an enzymaticcascade, reminiscent of ubiquitination reactions but uses ded-icated SUMO E1, E2 and E3 enzymes. Unlike the ubiquitinsystem, which includes �40 E2s and �700 E3s, the SUMOsystem has only a single E2 (Ubc9) and approximately a dozenE3s (15, 19, 20). SUMO is attached to proteins via its C-termi-nal glycine residue, which forms an isopeptide bond with theepsilon amine group of the acceptor lysine residue on the tar-get protein. SUMOmodification of proteins has been linkedto several processes such as DNA repair, chromatin remodel-ing, and signal transduction (21–23). SUMO has also beenlinked to modulation of ion channel activity, possibly by regu-lating ion channel surface levels, but mechanistic insight islacking (24, 25).Here we show for the first time that nonvisual arrestins are

subject to stimulus-dependent SUMOylation. We identifylysine residue 400 of arrestin-3 as the main SUMOylationsite, and interestingly, SUMOylation of this site is requiredfor arrestin-3 to promote GPCR endocytosis. Our studyreveals for the first time a role for SUMO in GPCRtrafficking.

* This work was supported, in whole or in part, by National Institutes ofHealth Grants GM75159 and DA026040 (to A. M.). This work was also sup-ported by Predoctoral Fellowship 0910098G from the American HeartAssociation (to R. M.).

□S The on-line version of this article (available at http://www.jbc.org) con-tains supplemental Fig. S1.

1 Both authors contributed equally to this study.2 To whom correspondence should be addressed: Dept. of Pharmacology,

Loyola University Chicago, Stritch School of Medicine, 2160 S. 1st Ave.,Bldg. 101, Rm. 2721, Maywood, IL 60153. Tel.: 708-216-3456; Fax: 708-216-6596; E-mail: amarchese@lumc.edu.

3 The abbreviations used are: GPCR, G protein-coupled receptor; SUMO,small ubiquitin-like modifier; �2AR, �2-adrenergic receptor; IP, immuno-precipitated/immunoprecipitation; IB, immunoblot/immunoblotting;HEK, human embryonic kidney.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 5, pp. 3884 –3893, February 4, 2011© 2011 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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EXPERIMENTAL PROCEDURES

Cell Culture, Transfection, and Reagents—Human embry-onic kidney (HEK) 293 cells, obtained fromMicrobix(Toronto, Canada), and COS-1 and HeLa cells, obtained fromAmerican Tissue Type Culture Collection (Manassas, VA),were maintained in DMEM (Media Tech, Manassas, VA) sup-plemented with 10% FBS (Hyclone (Logan, UT)). The cellswere transfected using TransIT-LT1 transfection reagent ac-cording to the manufacturer’s recommendations (Mirus,Madison, WI). Lipofectamine 2000 transfection reagent wasfrom Invitrogen and was used to transfect siRNA alone orco-transfected with DNA. The His antibody was from Qiagen(Valencia, CA). The FLAGM2 horseradish peroxidase andalkaline phosphatase-conjugated antibodies were from Sigma.Monoclonal and polyclonal HA antibodies were from Co-vance (Emeryville, CA). The GST antibody was from Rock-land (Gilbertsville, PA). The SUMO-1 and Ubc9 polyclonalantibodies were from Boston Biochem (Cambridge, MA) andEnzo Life Sciences (Plymouth Meeting, PA). The arrestin (21-B1) monoclonal antibody was from Santa Cruz Biotechnology(Santa Cruz, CA). The arrestin polyclonal antibody was kindlyprovided by Dr. Jeffrey Benovic (Thomas Jefferson University,Philadelphia, PA). The GAPD and Ubc9 (siGenome Smartpool M-004910) siRNA were from Dharmacon RNAi Tech-nologies (Lafayette, CO). Alexa Fluor-conjugated secondaryantibodies and transferrin were from Invitrogen.Constructs, Cloning, and Mutagenesis—HA-tagged bovine

arrestin-2 and arrestin-3 were as described previously (26).DNA encoding SUMO-1 was from Dr. Frauke Melchior (Uni-versity of Heidelberg, Heidelberg, Germany) and was used tomake FLAG-tagged SUMO-1. Site-directed mutagenesis bysequential PCR steps was employed to generate the single andmultiple point mutants described in this study. The sequencesof all of the constructs were verified by dideoxy sequencing.SUMOylation Assay—To detect SUMOylation of arrestin-2

and arrestin-3, HEK293 cells were transiently transfected withHA-tagged arrestin-2 and arrestin-3 and empty vector(pcDNA) plus FLAG-tagged SUMO-1 or empty vector(pCMV) for 48 h. The cells were lysed in immunoprecipita-tion buffer 1 (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM

EDTA, 0.5% (w/v) sodium deoxycholate, 1% Nonidet P-40,0.1% SDS, 20 mM N-ethylmaleimide, 10 �g/ml leupeptin, 10�g/ml pepstatin A, and 10 �g/ml aprotinin), sonicated, andclarified by centrifugation. The lysates were incubated with ananti-HA polyclonal antibody to immunoprecipitate taggedarrestins followed by 7% SDS-PAGE and immunoblottingwith the FLAGM2 HRP-conjugated antibody to detect theincorporated FLAG-tagged SUMO-1. To detect endogenousarrestin SUMOylation, HEK293 cells grown to confluency in10-cm dishes were lysed in 1 ml of immunoprecipitationbuffer, and clarified lysates were divided into equal aliquotsand incubated with IgG (Santa Cruz) control and an arrestinmonoclonal antibody followed by immunoblotting with apolyclonal SUMO-1 antibody. To detect arrestin-3 SUMOyla-tion in COS-1 cells, cells grown in six-well plates were co-transfected with 1 �g of HA-arrestin-3 (WT; K400R,K295R;2K/R; and 4K/R) plus FLAG-Ubc9 (1 �g), His-SUMO-1 (1

�g) and pcDNA alone, or with FLAG-Ubc9 and His-SUMO-1together. For siRNA transfections, HeLa cells grown to �80%confluency in six-well plates were co-transfected with 1 �g ofHA-arrestin-3, His-SUMO-1, or empty vector plus 200 pmolof GAPDH or Ubc9 siRNA using Lipofectamine 2000. Thecells were grown for 24 h and lysed directly in 2� samplebuffer (0.0375 M Tris-HCl, pH 6.5, 8% SDS, 10% glycerol, 5%�-mercaptoethanol, 0.003% bromphenol blue), and equalamounts were analyzed by immunoblotting. For agonist-de-pendent SUMOylation, COS-1 cells grown in six-well plateswere co-transfected with DNA encoding �2-adrenergic recep-tor (1 �g), HA-arrestins (Arr2; Arr3; K400R; K2/R; 1 �g), andHis-SUMO-1 (1 �g). Twenty-four hours later, the cells werewashed once in warm DMEM and treated with DMEM sup-plemented with 20 mM HEPES, pH 7.0, containing either ve-hicle (0.01 mM ascorbic acid) or isoproterenol (10 �M) for 5min. The cells were washed once on ice with cold PBS andcollected in immunoprecipitation buffer 2 (50 mM Tris-HCl,pH 7.4, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1%SDS, 20 mM N-ethylmaleimide, and 10 �g/ml each of leupep-tin, aprotonin, and pepstatin A), followed by sonication on iceand centrifugation at 21,000 � g, for 20 min at 4 °C. Clarifiedsamples were incubated with an anti-HA polyclonal antibodyand protein A-agarose to immunoprecipitate HA-tagged ar-restins, followed by SDS-PAGE and immunoblotting using ananti-His monoclonal antibody to detect incorporated His-tagged SUMO-1. Lysates were also subject to immunoblottingto detect expression of the various constructs.Co-immunoprecipitation Assay—HEK293 cells were tran-

siently transfected with HA-tagged wild-type arrestin-3, ar-restin-3–4K/R, and empty vector (pcDNA3) plus His-SUMO-1 or pcDNA3 using TransIT-LT1 transfectionreagent. Twenty-four hours later, the cells were lysed in im-munoprecipitation buffer 3 (20 mM Na2PO4, pH 6.5, 150 mM

NaCl, 1% (v/v) Triton X-100, 10 �g/ml leupeptin, 10 �g/mlaprotinin, 10 �g/ml pepstatin A) and incubated at 4 °C for 30min. The cells were sonicated and centrifuged, and clarifiedlysates were incubated with an anti-HA polyclonal antibodyto immunoprecipitate HA-tagged arrestin followed by immu-noblotting to detect bound �2-adaptin.Receptor Internalization Assay—To measure receptor inter-

nalization, we employed an enzyme-linked immunosorbantassay, as described previously (26). COS-1 cells grown in six-well plates to �70–80% confluency were transfected withFLAG-�2AR (1 �g) or FLAG-AT1aR plus 6–12 ng of HA-tagged wild-type arrestin-3 and the 4K/R or 2K/R arrestin-3SUMO-deficient mutants, respectively. For internalizationmeasured in HEK293 cells, the cells grown in six-well plateswere co-transfected with 250 ng of FLAG-�2AR DNA plus 50pmol of GAPDH or Ubc9 siRNA using Lipofectamine 2000,according to the manufacturer’s recommendations (Invitro-gen). The following day, the cells were passaged onto 24-wellplates coated with poly-L-lysine (0.1 mg/ml) and grown for anadditional 24 h. On the day of the experiment, the cells werewashed once in warm DMEM and were treated with vehicleand 10 �M isoproterenol for 15 min in DMEM containing 20mM HEPES and 0.01 mM ascorbic acid, after which receptorinternalization was measured using the M2-alkaline phos-

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phatase-conjugated FLAG antibody (Sigma), using a protocolwe have described previously (27).Confocal Fluorescence Microscopy—HeLa cells transiently

transfected with FLAG-�2AR and HA-arrestin-3 wild-type orHA-arrestin-3–4K/R were passaged onto poly-L-lysine-coatedcoverslips and grown for 24 h. The cells were serum-starvedby incubating with warm DMEM containing 20 mM HEPES,pH 7.5, for 3–4 h at 37 °C followed by treatment with 10 �M

isoproterenol or vehicle (0.01 mM ascorbic acid) for 5 min.The cells were fixed with 3.7% paraformaldehyde and perme-abilized with 0.05% (w/v) saponin for 10 min, similar to a pro-tocol that we have described previously (26). Briefly, after per-meabilization and fixation, the cells were incubated with 1%BSA in 0.01% saponin-PBS for 30 min at 37 °C, followed byimmunostaining with FLAG polyclonal antibody used at 1:100dilution. For HA-arrestin-3–4K/R, the cells were also immu-nostained with anti-HA monoclonal antibody. The cells were

washed five times with 0.01% saponin-PBS, followed by incu-bating with Alexa Fluor 594- or 488-conjugated secondaryantibodies for 30 min at 37 °C. Finally, the cells were washedwith PBS and fixed again in 3.7% formaldehyde-PBS andmounted onto glass slides using mounting medium contain-ing DAPI (Vector Laboratories, Burlingame, CA). The sam-ples were analyzed using a Zeiss LSM 510 laser scanning con-focal microscope equipped with a Plan-Apo 63�/1.4 oil lensobjective. The images were acquired using a 1.4 megapixelcooled extended spectra range RGB digital camera set at512 � 512 resolution. Acquired images were analyzed usingImage J software (version 1.41o).To determine a role for Ubc9 in internalization of FLAG-

�2AR and transferrin receptor in HeLa cells, the cells grownin 10-cm dishes were co-transfected with DNA encodingFLAG-�2AR (3 �g) and Ubc9 or GAPDH siRNA (600 pmol)using Lipofectamine 2000. The next day, the cells were pas-

FIGURE 1. Arrestins are modified by SUMO. A, schematic representation of arrestin-2 and arrestin-3 depicting the putative SUMO consensus sites (under-lined) and the surrounding amino acid residues. The SUMO acceptor lysine residues are in bold type. B, SUMOylation analysis of nonvisual arrestins. HEK293cells were transiently transfected with HA-tagged arrestin-2, HA-arrestin-3, empty vector (pcDNA) plus FLAG-tagged SUMO-1, or empty vector (pCMV) for48 h. Transfections with empty vector are indicated with a minus sign. HA-tagged arrestins were immunoprecipitated (IP) and followed by immunoblotting(IB) to detect the incorporated FLAG-SUMO-1. The top blot was stripped and reprobed with an anti-HA monoclonal antibody to determine the level oftagged arrestin in the IP. Cross-reactivity to IgG is indicated. The lysates were analyzed by IB to detect the expression of the various constructs. Representa-tive blots from one of three independent experiments are shown. The positions of the molecular mass markers in kDa are shown. C, SUMOylation of endog-enous arrestins. Endogenous arrestins were IP from HEK293 cells, followed by IB for endogenous SUMO-1. SUMOylated arrestin is indicated with arrows. Theasterisk represents possible cross-reactive bands present in samples incubated with arrestin and IgG antibodies. Representative blots from one of three in-dependent experiments are shown. The positions of the molecular mass markers in kDa are shown.

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saged onto poly-L-lysine-coated coverslips and grown for anadditional 24 h. The cells were serum-starved, followed bytreatment in the absence and presence of 10 �M isoproterenolfor 30 min. During the final 5 min of the incubation, the cellswere treated with 25 �g/�l transferrin conjugated to AlexaFluor 594 (Invitrogen). The cells were processed as describedabove. FLAG-�2AR was labeled with a FLAG polyclonal anti-body (1:100 dilution) and an Alexa Fluor 483-conjugated anti-rabbit IgG secondary antibody (1:200 dilution). The cells ex-pressing FLAG-�2AR were counted from 10 random fieldscontaining 5–10 cells/field, and the amount of receptor inter-nalized was calculated as the percentage of FLAG-�2AR-ex-pressing cells showing only punctate staining. The cells thatshowed staining on the surface of cells were indicative of cellsin which receptor internalization had not occurred or wasincomplete. Transferrin receptor internalization was deter-mined in the same cells used to calculate FLAG-�2AR inter-nalization. The fluorescence intensity of Alex Fluor 594-con-

jugated transferrin was calculated using the histogram featureof Zeiss LSM 510 image analysis software (4.2 SP1).

RESULTS

Nonvisual Arrestins Are Modified by SUMO—As part ofour efforts to understand how ubiquitin-like molecules gov-ern GPCR regulation, we noticed the presence of two SUMOconsensus sequences within nonvisual arrestin-2 and arres-tin-3 (Fig. 1A), suggesting that they are modified by SUMO.To determine whether arrestins are indeed SUMOylated,HEK293 cells were transiently transfected with DNA encod-ing HA-tagged arrestin-2, arrestin-3, or empty vector(pcDNA3) with FLAG-tagged SUMO-1 or empty vector(pCMV-10). HA-tagged arrestins were immunoprecipitatedwith an anti-HA antibody followed by SDS-PAGE and immu-noblotting to detect incorporation of FLAG tagged-SUMO-1.As shown in Fig. 1B, several migrating species were detectedin cells transfected with HA-arrestin-2 or HA-arrestin-3 and

FIGURE 2. Identification of the SUMO acceptor sites on arrestin-3. A, SUMOylation analysis of arrestin-3 by co-expressing SUMO-1 and Ubc9 in COS-1cells. COS-1 cells grown in six-well plates were transfected either with empty vector (pcDNA) or HA-arrestin-3, plus either empty vector (pcDNA), FLAG-Ubc9alone, His-SUMO-1 alone, or together with FLAG-Ubc9. Twenty-fours hours later, the cells were lysed directly in 2� sample buffer, and equal amounts wereanalyzed by IB. Unmodified and SUMOylated arrestins are indicated. Shown are representative IBs from one of three independent experiments. B, SUMOyla-tion analysis of arrestin-3 lysine mutants. Lysine residues 295 and 400, both of which reside within SUMOylation consensus sites, were changed individuallyto arginine residues. Lysine residues 398 and 400 were changed simultaneously to create the 2K/R mutant, and lysine residues 293 and 295 were changedsimultaneously to arginine residues in the 2K/R background to create the 4K/R mutant. The SUMOylation status of these mutants was assessed as describedin A. Shown are representative IBs from one of three independent experiments.

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FLAG-SUMO-1 but not in cells transfected with either arres-tin or SUMO-1 alone, suggesting that these migrating speciesrepresent SUMOylated arrestins. The presence of multiplespecies suggests that arrestins are modified by SUMO-1 onmultiple lysine residues, consistent with the presence of twoSUMO consensus sites in both arrestins (Fig. 1A). We alsoobserved that endogenous arrestins are modified by endoge-nous SUMO-1 in HEK293 cells (Fig. 1C). The migration ofendogenous SUMOylated arrestin is faster than that of exoge-nous arrestin, likely because of the absence of the HA andFLAG epitope tags on endogenous arrestin and SUMO,respectively.Arrestin-3 Is Modified by SUMO on Lysine Residue 400—The

SUMO consensus site represents a binding site for theSUMO-specific E2 enzyme Ubc9 (28) and to facilitate the de-tection of arrestin SUMOylation, we co-expressed HA-taggedarrestin-3 with exogenous FLAG-tagged Ubc9 alone or to-gether with His-tagged SUMO-1 in COS-1 cells and analyzedwhole cell lysates for the presence of SUMOylated arrestin byimmunoblotting. As shown in Fig. 2A, two main species wereobserved that migrated more slowly than unmodified arrestinin cells transfected with His-SUMO-1 alone and were moreabundant in cells that were co-transfected with His-SUMO-1and FLAG-Ubc9, suggesting that these species represented

SUMOylated arrestin-3. These species were not present incells transfected with vector, HA-arrestin-3, or FLAG-Ubc9alone, further suggesting that they represent SUMOylatedarrestin-3. We observed similar results from three indepen-dent experiments. It is important to note that SUMOylatedarrestin represents a small percentage of the total cellularcomplement of arrestin. This is not unusual, because fornearly all proteins that have been shown to be SUMOylated,the SUMOylated form represents a small percentage of thetotal cellular complement of that protein (15, 29, 30).We next set out to identify the lysine residues that are

modified by SUMO. As noted in Fig. 1A, arrestin-3 has twoconsensus sites for SUMOylation and thus two possible lysineacceptor sites: Lys-295 and Lys-400. To determine whetherthese residues are SUMO acceptor sites, they were changed toconserved arginine residues, and the SUMOylation status ofthese arrestin mutants (K295R and K400R) was assessed byimmunoblot analysis, as described above. As shown in Fig. 2B,the SUMOylation status of the K295R mutant was indistin-guishable from wild-type arrestin-3; however, in sharp con-trast, the K400R mutant was not modified by SUMO-1, sug-gesting that Lys-400 is the primary SUMO-1 site. However,upon longer exposure the presence of additional species, al-beit less abundant, were evident, suggesting that additional

FIGURE 3. Role of receptor activation on arrestin SUMOylation. A, examination of nonvisual arrestin SUMOylation by GPCR activation. Stimulus-depen-dent SUMOylation of arrestin-2 and arrestin-3 was examined in COS-1 cells co-transfected with FLAG-tagged �2AR, His-tagged SUMO-1, and HA-taggedarrestin-2, arrestin-3, or empty vector (pcDNA). The cells were treated with vehicle (0.01 mM ascorbic acid) and 10 �M isoproterenol (ISO) for 5 min, followedby IP of tagged arrestin and IB to detect incorporated His-SUMO-1. Shown are IBs from one of three independent experiments. B, role of lysine residues inarrestin-3 SUMOylation by GPCR activation. Stimulus-dependent SUMOylation was examined in COS-1 cells transfected with FLAG-tagged �2AR and His-tagged SUMO-1 plus HA-tagged arrestin-3, wild-type, K400R, 2K/R, or empty vector (pcDNA). Treatment and analysis of arrestin SUMOylation was as de-scribed in A. Shown are IBs from one of three independent experiments. C, arrestin SUMOylation is represented graphically. The data represent the averageresults from the densitometric analysis of immunoblots from three independent experiments. The fold increase upon isoproterenol (ISO) treatment as com-pared with vehicle (VEH) treatment was normalized to HA-arrestin-3 levels present in the immunoprecipitates. The error bars represent the standard error ofthe mean. The data were analyzed by an unpaired Student’s t test (*, p � 0.05).

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lysine residues are modified by SUMO-1. To rule out the pos-sibility that neighboring lysine residues to Lys-400 and Lys-295 are SUMOylated, we created two additional arrestin-3lysine residue mutants. In one mutant, referred to as the 2K/Rmutant, Lys-400 and Lys-398 were changed simultaneously toarginine residues, whereas in another mutant, referred to as4K/R, Lys-295 and Lys-293 were changed simultaneously toarginine residues in the 2K/R background. As shown in Fig.2B, SUMOylation of the 2K/R mutant was observed onlyupon a long exposure of the immunoblot and was indistin-guishable from that of the K400R mutant, suggesting thatother lysine residues, possibly Lys-295 and/or Lys-293, weremodified by SUMO-1 in the 2K/R mutant. Accordingly, when

these residues were changed to arginine residues in the 2K/Rbackground, SUMOylation of this mutant (i.e. 4K/R) was notobserved. Taken together, these data suggest that Lys-400 isthe predominant SUMOylation site, whereas Lys-295 andother lysine residues (Lys-293 and Lys-398) may also be modi-fied by SUMO-1, although to a lesser degree.Receptor Activation Promotes Arrestin SUMOylation—

Upon binding to ligand activated and phosphorylated recep-tors, arrestins are thought to undergo several conformationalchanges, exposing the C-tail that ultimately facilitates down-stream interactions (31, 32). Because Lys-400 resides withinthe C-tail of arrestin-3, it is possible that arrestin SUMOyla-tion is regulated by receptor activation. To examine this, we

FIGURE 4. The role of Ubc9 on arrestin SUMOylation and receptor trafficking. A, HeLa cells transfected with control (CON, GAPDH) and Ubc9 siRNA plusHis-tagged SUMO or empty vector (pcDNA) were solubilized in 2� sample buffer and subjected to IB to detect HA-tagged arrestin-3. SUMOylated arrestin isindicated. Shown are IBs from one of three independent experiments. B, the role of Ubc9 on GPCR internalization was examined in HeLa cells co-transfectedwith DNA encoding FLAG-�2AR and control (Ctrl, GAPDH) or Ubc9 siRNA. The cells were treated with 10 �M isoproterenol for 30 min, and during the final 5min of the incubation, the cells were treated with 25 �g/�l Alexa Fluor 594-conjugated transferrin (Tfn). The cells were processed as described under “Ex-perimental Procedures.” Shown are representative confocal fluorescence microscopy images of cells analyzed from three independent experiments. Bar, 20�m. C, shown is a graph representing FLAG-�2AR internalization calculated in control (GAPDH) and Ubc9 siRNA-transfected cells shown in B. The percent-age of receptor internalization was determined by counting cells showing punctate staining and no membrane staining in 10 randomly selected fields con-taining 5–10 cells/field. The data represent the averages from three independent experiments, and the error bars represent the standard error of the mean.The data were analyzed by a Student’s t test (*, p � 0.0001). D, internalization of the transferrin receptor was determined in the same cells used for the anal-ysis performed in C. Internalization of the transferrin receptor was calculated by measuring the fluorescence intensity of transferrin Alexa Fluor 594 usingLSM 510 image analysis software, as described under “Experimental Procedures.” The percentage of fluorescence intensity in Ubc9 transfected cells wasnormalized to control (GAPDH) transfected cells. The error bars represent the standard error of the mean. E, levels of Ubc9 and actin were detected by im-munoblotting whole cell lysates from control and Ubc9 siRNA-transfected cells. F, FLAG-�2AR internalization was examined in HEK293 cells co-transfectedwith DNA encoding FLAG-�2AR and control (GAPDH) or Ubc9 siRNA, as described under “Experimental Procedures.” The cells were treated with vehicle(0.01 mM ascorbic acid) and 10 �M isoproterenol for 15 min, and receptor internalization was measured by cell surface ELISA. The data represent the aver-ages from three or four independent experiments, and the error bars represent the standard error of the mean. The data were analyzed by a Student’s t test(*, p � 0.05). Shown in the inset are representative IBs to detect Ubc9 and actin levels.

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used �2AR, a prototypical GPCR used to study many aspectsof arrestin function on GPCR signaling and trafficking (33).As shown in Fig. 3A, treatment of COS-1 cells expressingFLAG-�2AR and His-SUMO-1 with the nonselective �-adre-nergic agonist isoproterenol (10 �M) for 5 min enhancedSUMOylation of both arrestin-2 and arrestin-3, as comparedwith vehicle-treated cells. In contrast, the K400R and 2K/Rarrestin mutants were not modified by SUMO-1, as comparedwith wild-type arrestin-3 (Fig. 3B). Densitometric analysis ofdata from three independent experiments reveals that recep-tor activation significantly enhances arrestin-3 SUMOylationon Lys-400 (Fig. 3C).Arrestin SUMOylation Regulates GPCR Internalization—

Because nonvisual arrestins mediate GPCR endocytosis, it ispossible that SUMOylation modulates the role of arrestin inGPCR trafficking. To confirm a role for SUMOylation inGPCR endocytosis, we examined arrestin SUMOylation andinternalization of �2AR in cells depleted of the SUMO E2 en-zyme Ubc9. We first determined whether depletion of Ubc9modulated arrestin-3 SUMOylation. As shown in Fig. 4A,siRNA-mediated depletion of Ubc9 attenuated arrestinSUMOylation as compared with control siRNA-transfectedcells. We next examined the role of Ubc9 on agonist-inducedinternalization of �2AR. HeLa cells co-transfected withFLAG-�2AR and GAPDH or Ubc9 siRNA were treated with10 �M isoproterenol for 30 min, and receptor internalizationwas assessed by confocal immunofluorescene microscopy, asdescribed under “Experimental Procedures.” As shown in Fig.4B, upon isoproterenol treatment in cells transfected withGAPDH siRNA, FLAG-�2AR staining was mostly punctate,indicating that the receptor had internalized into endocyticvesicles. In contrast, upon agonist treatment of cells trans-fected with Ubc9 siRNA, most FLAG-�2AR staining was re-stricted to the cell surface, indicating that the receptor hadfailed to internalize (Fig. 4B, bottom panels). Quantification ofFLAG-�2AR internalization in multiple cells from 10 ran-domly selected fields from three independent experimentsrevealed that agonist-induced internalization of FLAG-�2ARwas significantly inhibited in cells transfected with Ubc9siRNA as compared with GAPDH siRNA (Fig. 4C). To ruleout the possibility that Ubc9 siRNA has a global effect on en-docytosis, we also examined internalization of the transferrinreceptor in the same cells expressing FLAG-�2AR. As shownin Fig. 4 (B and D), internalization of the transferrin receptorwas not impaired in cells transfected with Ubc9 siRNA ascompared with GAPDH siRNA-transfected cells. The extentof Ubc9 depletion in these experiments was greater than 90%as revealed by immunoblotting of whole cell lysates (Fig. 4E).To rule out the possibility that Ubc9 may modulate GPCRendocytosis in a cell type-selective manner, we also examinedthe role of Ubc9 on �2AR internalization in HEK293 cells.Similar to HeLa cells, agonist-induced internalization of �2ARwas significantly (Student’s t test, p � 0.05) attenuated inHEK293 cells transfected with Ubc9 siRNA as compared withGAPDH siRNA-transfected cells (Fig. 4F). Taken together,these data suggest that SUMOylation is important for arrestinfunction in promoting GPCR internalization. To further linkarrestin SUMOylation to �2AR internalization, we examined

internalization of �2AR in COS-1 cells co-expressing wild-type HA-arrestin-3 and the SUMOylation-deficient 4K/R mu-tant. As shown in Fig. 5A, when expressed to equal levels, theSUMOylation-deficient 4K/R mutant failed to enhance �2ARinternalization as compared with wild-type arrestin-3, furthersuggesting that arrestin SUMOylation mediates �2AR endo-cytosis. To examine the possibility that arrestin SUMOylationmay modulate the internalization of other GPCRs, we alsoexamined the role of arrestin SUMOylation on internalizationof the angiotension 1A receptor (AT1aR). As shown in Fig.5B, the SUMOylation-deficient 2K/R mutant failed to en-hance AT1aR internalization as compared with wild-type ar-restin-3, suggesting that arrestin SUMOylation regulates in-ternalization of AT1aR and that it may play a broad role inmediating GPCR endocytosis.To rule out the possibility that the SUMOylation-deficient

arrestin-3 mutants may be impaired in their ability to be re-cruited to the receptor upon agonist treatment, we examinedtheir distribution in cells upon receptor activation by confocalimmunofluoresence microscopy. As shown in Fig. 6A, in vehi-cle-treated cells, wild-type HA-arrestin-3 was diffusely dis-tributed throughout the cytoplasm; however, upon treatmentwith isoproterenol for 5 min, HA-arrestin-3 rapidly translo-cated to the plasma membrane where it co-localized with�2AR. Similarly, the SUMOylation-deficient 4K/R mutantrapidly translocated to the plasma membrane upon agonisttreatment where it co-localized with �2AR (Fig. 6B). In addi-

FIGURE 5. Role of arrestin-3 SUMOylation on GPCR internalization.FLAG-�2AR (A) and FLAG-AT1aR (B) internalization was assessed in COS-1cells co-transfected with HA-arrestin-3 WT, 4K/R, 2K/R, or empty vector(pcDNA) upon treatment with vehicle (0.01 mM ascorbic acid) and 10 �M

isoproterenol for 30 min by ELISA. The data represent the averages fromthree independent experiments, and the error bars represent the standarderror of the mean. The data were analyzed by a one-way analysis of variancefollowed by Bonferroni’s post-hoc test. Wild-type arrestin-3, but not theSUMO-deficient mutants, was significantly different from pcDNA (p � 0.05).Shown in the insets are representative IBs to detect expression of HA-tagged arrestins.

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tion, we also examined the role of SUMOylation in the distri-bution of arrestin-3 in response to angiotensin II treatment.In cells co-expressing HA-AT1aR and wild-type arrestin-3-yellow fluorescent protein, angiotensin II treatment for 30min promoted co-internalization of both the receptor andarrestin-3, as assessed by confocal fluorescence microscopy(supplemental Fig. S1A). Similarly, the SUMO-deficientK400R mutant co-internalized with HA-AT1aR (supplemen-tal Fig. S1B). Quantification of the percentage of co-localiza-tion between receptor and arrestin puncta revealed no differ-ence between wild-type and the SUMO-deficient arrestin(supplemental Fig. S1C), which is consistent with the idea thatSUMOylation is not involved in arrestin binding to the recep-tor. Taken together, these data suggest that SUMOylation isnot involved in arrestin recruitment to GPCRs but rather islikely involved in mediating interactions with factors of theinternalization machinery to promote GPCR endocytosis.To explore this possibility, we examined the role of arrestin

SUMOylation on binding to �2-adaptin by co-immunopre-cipitation. The �2-adaptin subunit of AP2 binds to nonvisualarrestins, and this interaction is important for promotingGPCR endocytosis. HA-tagged wild-type arrestin-3 and the

4K/R mutant transiently transfected in HEK293 cells weresubject to immunoprecipitation followed by immunoblottingto detect bound endogenous �2-adaptin. As shown in Fig. 7,both wild-type arrestin-3 and the 4K/R mutant bound to �2-adaptin to equal levels. However, when His-tagged SUMO-1was co-expressed, binding of �2-adaptin to wild-type arres-tin-3 was markedly enhanced, as compared with the 4K/Rmutant, suggesting that arrestin SUMOylation promotesbinding to �2-adaptin.

DISCUSSION

Our data reveal for the first time that arrestins are subjectto stimulus-dependent SUMOylation. Lysine residue 400 inarrestin-3 is the main SUMOylation site, and the arrestinSUMOylation status correlates with its ability to mediateGPCR endocytosis, possibly by promoting binding to AP2.Whether SUMOylation also modulates the role of arrestin inGPCR desensitization and/or signaling remains to be deter-mined. Our data reveal an unprecedented function for proteinSUMOylation in GPCR endocytosis and extends our knowl-edge about the molecular mechanisms by which arrestinsgovern GPCR trafficking.

FIGURE 6. Confocal microscopy analysis of arrestin distribution upon agonist activation. HeLa cells co-transfected with FLAG-�2AR and HA-arrestin-3-WT (A) or HA-Arrestin-3– 4K/R (B) were serum-starved before being treated with vehicle (0.01 mM ascorbic acid) and 10 �M isoproterenol (ISO) for 5 min.The cells were then fixed, permeabilized, and immunostained with anti-FLAG and anti-HA antibodies. Yellow in the merged images represent co-localiza-tion between FLAG-�2AR and HA-arrestin-3. The differential interference contrast (DIC) image is shown. Shown are representative images from cells ana-lyzed from three independent experiments in which at least 10 cells were analyzed per experiment. Bar, 20 �m.

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We identified lysine residues 295 and 400 as the primarySUMOylation sites in arrestin-3 (Fig. 2A). Each of these resi-dues falls within a canonical SUMO consensus motif, and al-though it is possible that other lysine residues may be modi-fied by SUMO, Lys-400 and Lys-295 likely represent the mainsites. Arrestin SUMOylation is regulated by GPCR activationbecause SUMOylation of Lys-400 is enhanced by agonist acti-vation of the �2AR (Fig. 3). Upon agonist exposure and bind-ing to phosphorylated receptor, arrestin is thought to undergoa conformational change that exposes elements in the C-tail.Because the SUMO site resides within the C-tail, it is likelythat agonist activation and a subsequent conformationalchange in arrestin facilitates Ubc9 binding and subsequentSUMOmodification of Lys-400. SUMOmodification of thissite enables arrestin to regulate GPCR trafficking. This isconsistent with our data showing that 1) overexpression of aSUMO-deficient arrestin fails to promote �2AR and AT1aRinternalization (Fig. 5); and 2) siRNA-mediated depletion ofUbc9 inhibits arrestin SUMOylation and �2AR internalization(Fig. 4, A–C). To our knowledge, these data link SUMOyla-tion for the first time to endocytic trafficking of GPCRs. It ispossible that SUMOylation has a broad role in regulating en-docytosis. Dynamin, a key component of the internalizationmachinery, may be regulated by the SUMOmachinery (34).Therefore, it is possible that Ubc9 siRNA may also affectother factors of the trafficking machinery, in addition to ar-restin-3, to modulate GPCR endocytosis. However, depletionof Ubc9 siRNA did not have a global effect on endocytosis,because transferrin receptor internalization was not affectedby depletion of Ubc9 (Fig. 4, B and D). The fact that the arres-tin SUMOylation-deficient mutants are unable to support

GPCR endocytosis indicates that arrestin SUMOylation isrequired for this process (Fig. 5).Arrestins are also regulated by other post-translational

modifications including phosphorylation, nitrosylation, andubiquitination. Stimulus-dependent dephosphorylation ofarrestin-3 (also called �-arrestin-2) on serine (Ser-361) andthreonine (Thr-383) residues is required for internalization of�2AR, likely by facilitating arrestins association with clathrin(8). Ubiquitination of arrestin-3 is required to support inter-nalization of �2AR, although mechanistic insight is lacking(10). Nitrosylation of the C-terminal cysteine residue of arres-tin-3 has been linked to its ability to regulate GPCR internal-ization through a mechanism that may involve release of theburied C-tail, thus facilitating binding to clathrin and �2-adaptin, thereby promoting GPCR endocytosis (9). It is possi-ble that SUMOylation of Lys-400 may favor the release of theburied C-tail, thereby facilitating these interactions to pro-mote GPCR endocytosis. Our data in Fig. 7 are consistentwith this idea, because expression of His-SUMO-1 enhanced�2-adaptin binding to wild-type arrestin-3 but not to theSUMO-deficient 4K/R mutant. It is unlikely that this mutantdoes not interact with the receptor, because the 4K/R mutantwas not impaired in its ability to be recruited to �2AR at theplasma membrane (Fig. 6), suggesting that SUMOylation isnot involved in the ability of arrestins to bind to the receptor.It is possible that the attached SUMOmoiety may play a

more direct role in GPCR endocytosis. SUMO often interactswith SUMO interaction motifs. SUMO interaction motifs arefound in several proteins and are characterized by a shortstretch of hydrophobic amino acids near an acidic cluster ofresidues that interact noncovalently with the SUMOmoietywhile it is attached to its target protein (35). It is possible thatthe SUMOmoiety on arrestin interacts with a SUMO interac-tion motif-harboring protein to execute its function in GPCRendocytosis. Alternatively, SUMOylation may be linked toarrestin ubiquitination. For example, SUMOmodification hasbeen shown to prevent a protein from being ubiquitinated,because some ubiquitin acceptor sites may also serve asSUMO acceptor sites (36). As mentioned above, ubiquitina-tion of arrestin facilitates its role in promoting GPCR inter-nalization (10), as we observed with SUMOylation in thisstudy. Interestingly, the SUMO acceptor site Lys-400 in arres-tin-3 may not be a ubiquitination site (11). Therefore, it ispossible that ubiquitination and SUMOylation have distinctroles in mediating arrestin-dependent GPCR internalization.We observed that only a small percentage of the total cellu-

lar pool of arrestin-3 is SUMOylated. This is not uncommon,because with most proteins that are SUMOylated, only asmall fraction of the total cellular pool of a protein is in factSUMOylated at any given time (15, 30). Although the reasonremains poorly understood, it may be because only a smallfraction of a protein is in the appropriate location to be modi-fied by SUMO, and once it performs its action it may be rap-idly de-SUMOylated to return the protein to its basal state.This may be true for arrestin because SUMOylation may oc-cur before or once arrestin is recruited to clathrin-coated pits,where we propose it facilitates interactions with factors of theinternalization machinery. De-SUMOylation would antago-

FIGURE 7. Role of arrestin SUMOylation on binding to AP2. HEK293 cellswere transfected with HA-tagged wild-type arrestin-3 and the 4K/R mutantplus His-SUMO-1 or empty vector (pcDNA). The next day, HA-tagged ar-restins were subjected to IP followed by IB to detect bounds �2-adaptin, asdescribed under “Experimental Procedures.” Shown are representative IBsfrom one of three independent experiments.

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nize these interactions and subserve to terminate SUMOy-lated arrestin activity. We anticipate that the identification ofthe machinery, such as the SUMO E3 ligase and the de-SUMOylase, that regulates the arrestin SUMOylation statuswill provide significant insight into these events.In summary, we provide evidence that SUMOylation of

arrestin-3 on lysine 400 promotes internalization of �2AR.This study reveals for the first time a role for SUMOylation inpromoting GPCR endocytosis. Whether SUMOylation plays arole in other aspects of GPCR signaling remains to beexamined.

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Debra Wyatt, Rohit Malik, Alissa C. Vesecky and Adriano MarcheseTrafficking

Small Ubiquitin-like Modifier Modification of Arrestin-3 Regulates Receptor

doi: 10.1074/jbc.M110.152116 originally published online November 30, 20102011, 286:3884-3893.J. Biol. Chem. 

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