CharacterizationoftheRoleoftheRabGTPase-activating ... · previously shown that a Rab...

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Characterization of the Role of the Rab GTPase-activating Protein AS160 in Insulin-regulated GLUT4 Trafficking * S Received for publication, April 11, 2005, and in revised form, August 31, 2005 Published, JBC Papers in Press, September 8, 2005, DOI 10.1074/jbc.M503897200 Mark Larance ‡§1 , Georg Ramm ‡1 , Jacqueline Sto ¨ ckli ‡1,2 , Ellen M. van Dam , Stephanie Winata , Valerie Wasinger § , Fiona Simpson , Michael Graham , Jagath R. Junutula**, Michael Guilhaus § , and David E. James ‡3 From the Diabetes and Obesity Program, Garvan Institute of Medical Research, Sydney 2010, Australia, § Bioanalytical Mass Spectrometry Facility, University of New South Wales, Sydney 2052, Australia, Institute for Molecular Biology, University of Queensland, Brisbane 4072, Australia, Benitec, Inc., Mountain View, California 94043, and **Genentech Inc., South San Francisco, California 94080 Insulin stimulates the translocation of the glucose transporter GLUT4 from intracellular vesicles to the plasma membrane. In the present study we have conducted a comprehensive proteomic anal- ysis of affinity-purified GLUT4 vesicles from 3T3-L1 adipocytes to discover potential regulators of GLUT4 trafficking. In addition to previously identified components of GLUT4 storage vesicles including the insulin-regulated aminopeptidase insulin-regulated aminopeptidase and the vesicle soluble N-ethylmaleimide factor attachment protein (v-SNARE) VAMP2, we have identified three new Rab proteins, Rab10, Rab11, and Rab14, on GLUT4 vesicles. We have also found that the putative Rab GTPase-activating pro- tein AS160 (Akt substrate of 160 kDa) is associated with GLUT4 vesicles in the basal state and dissociates in response to insulin. This association is likely to be mediated by the cytosolic tail of insulin- regulated aminopeptidase, which interacted both in vitro and in vivo with AS160. Consistent with an inhibitory role of AS160 in the basal state, reduced expression of AS160 in adipocytes using short hairpin RNA increased plasma membrane levels of GLUT4 in an insulin-independent manner. These findings support an important role for AS160 in the insulin regulated trafficking of GLUT4. Glucose transport into mammalian muscle and fat cells is an impor- tant step in insulin action and is critical for the maintenance of glucose homeostasis within the body (1). In mammalian muscle and fat cells, insulin stimulation activates a phosphorylation cascade, which in turn causes intracellular vesicles that contain the glucose transporter GLUT4, 4 to translocate to the plasma membrane (PM) and fuse (2, 3). In the basal state GLUT4 is distributed between the endosomal system, the trans-Golgi network (TGN), and a GLUT4 storage vesicle (GSV) com- partment that is highly insulin-responsive (4 – 6). The protein kinase Akt is activated in response to insulin and plays a critical role in GLUT4 translocation (1, 7). However, the link between the insulin signaling pathway and GLUT4 translocation is not fully understood. The insulin-dependent movement of GLUT4 vesicles to the PM is an Akt-independent process, and this is followed by an Akt- dependent step likely involving the docking and fusion of vesicles with the PM (7–9). The mechanism by which Akt controls the docking and fusion of GLUT4 vesicles with the PM is not known. However, it was previously shown that a Rab GTPase-activating protein (RabGAP) known as AS160 is phosphorylated by Akt in response to insulin (10). How AS160 functions in GLUT4 trafficking and its cognate Rab pro- teins are not known. The role of a variety of Rab proteins in GLUT4 trafficking including Rab3d, Rab4, Rab5, and Rab11 has been examined (11–16). However, although these Rab proteins may participate in some aspects of GLUT4 trafficking, no compelling evidence for specific involvement in the insulin-regulated trafficking of GLUT4 has been found. In this study we describe four key findings that add to our under- standing of GLUT4 trafficking. Using mass spectrometry we have iden- tified three Rab proteins on GLUT4 vesicles that could potentially be substrates of AS160 and, thus, play an important role in insulin-regu- lated glucose transport. In addition, we have shown that AS160 is pres- ent on GLUT4 vesicles in the basal state and dissociates with insulin. We have gone on to show that the association of AS160 with GLUT4 vesi- cles is mediated at least in part via a direct interaction with the cytosolic tail of IRAP, and this interaction appears to be insulin-dependent. Fur- thermore, a decrease in AS160 expression by shRNA leads to increased levels of GLUT4 at the PM in the basal state. Together, these results provide novel insights into the regulatory mechanism of insulin-stimu- lated GLUT4 translocation. EXPERIMENTAL PROCEDURES Materials—3T3-L1 murine fibroblasts, C2C12 fibroblasts, and Chi- nese hamster ovary (CHO) cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA). Dulbecco’s modified Eagle’s medium and newborn calf serum were obtained from Invitro- gen, Myoclone-Plus fetal calf serum from Trace Scientific (Melbourne, * This work was supported in part by grants from the National Health and Medical Research Council of Australia and Diabetes Australia Research Trust (to D. E. J.). Mass spectrometric analysis for this work was carried out at the Bioanalytical Mass Spec- trometry Facility, University of New South Wales, and was supported in part by grants from the Australian Government Systemic Infrastructure Initiative and Major National Research Facilities Program (University of New South Wales node of the Australian Proteome Analysis Facility) and by the University of New South Wales Capital Grants Scheme. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. S The on-line version of this article (available at http://www.jbc.org) contains Supple- mental Table 2. 1 These authors contributed equally to this work. 2 Supported by the Swiss National Foundation and the Novartis Stiftung. 3 A National Health and Medical Research Council senior principal research fellow. To whom correspondence should be addressed: Diabetes and Obesity Program, Garvan Institute of Medical Research, 384 Victoria St., Darlinghurst, NSW 2010 Australia. Tel.: 61-2-92958210; Fax: 61-2-92958201; E-mail: [email protected]. 4 The abbreviations used are: GLUT4, glucose transporter 4; PM, plasma membrane; TGN, trans-Golgi network; GSV, GLUT4 storage vesicles; RabGAP, Rab GTPase-activating protein; AS160, Akt substrate of 160 kDa; shRNA, short hairpin RNA; CHO, Chinese hamster ovary; TfR, transferin receptor; IRAP, insulin-regulated aminopeptidase; VAMP, vesicle-associated membrane protein; LDM, low density microsomes; PBS, phosphate-buffered saline; Puro, puromyocin; Hygro, hygromyocin; HA, hemagglu- tinin; SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein recep- tors; CI-MPR, cation-independent mannose 6-phosphate receptor; CD-MPR, cation- dependent mannose 6-phosphate receptor; ATP7A, copper-transporting ATPase 1; SCAMP, secretory carrier-associated membrane protein; GST, glutathione S-transfer- ase; MES, 4-morpholineethanesulfonic acid; GSV, GLUT4 storage vesicle. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 280, NO. 45, pp. 37803–37813, November 11, 2005 © 2005 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. NOVEMBER 11, 2005 • VOLUME 280 • NUMBER 45 JOURNAL OF BIOLOGICAL CHEMISTRY 37803 by guest on April 11, 2019 http://www.jbc.org/ Downloaded from

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Characterization of the Role of the Rab GTPase-activatingProtein AS160 in Insulin-regulated GLUT4 Trafficking*□S

Received for publication, April 11, 2005, and in revised form, August 31, 2005 Published, JBC Papers in Press, September 8, 2005, DOI 10.1074/jbc.M503897200

Mark Larance‡§1, Georg Ramm‡1, Jacqueline Stockli‡1,2, Ellen M. van Dam‡, Stephanie Winata‡, Valerie Wasinger§,Fiona Simpson¶, Michael Graham�, Jagath R. Junutula**, Michael Guilhaus§, and David E. James‡3

From the ‡Diabetes and Obesity Program, Garvan Institute of Medical Research, Sydney 2010, Australia, §Bioanalytical MassSpectrometry Facility, University of New South Wales, Sydney 2052, Australia, ¶Institute for Molecular Biology, University ofQueensland, Brisbane 4072, Australia, �Benitec, Inc., Mountain View, California 94043, and **Genentech Inc.,South San Francisco, California 94080

Insulin stimulates the translocation of the glucose transporterGLUT4 from intracellular vesicles to the plasma membrane. In thepresent study we have conducted a comprehensive proteomic anal-ysis of affinity-purified GLUT4 vesicles from 3T3-L1 adipocytes todiscover potential regulators of GLUT4 trafficking. In addition topreviously identified components of GLUT4 storage vesiclesincluding the insulin-regulated aminopeptidase insulin-regulatedaminopeptidase and the vesicle soluble N-ethylmaleimide factorattachment protein (v-SNARE) VAMP2, we have identified threenew Rab proteins, Rab10, Rab11, and Rab14, on GLUT4 vesicles.We have also found that the putative Rab GTPase-activating pro-tein AS160 (Akt substrate of 160 kDa) is associated with GLUT4vesicles in the basal state and dissociates in response to insulin. Thisassociation is likely to be mediated by the cytosolic tail of insulin-regulated aminopeptidase, which interacted both in vitro and invivowith AS160. Consistent with an inhibitory role of AS160 in thebasal state, reduced expression of AS160 in adipocytes using shorthairpin RNA increased plasma membrane levels of GLUT4 in aninsulin-independent manner. These findings support an importantrole for AS160 in the insulin regulated trafficking of GLUT4.

Glucose transport into mammalian muscle and fat cells is an impor-tant step in insulin action and is critical for the maintenance of glucosehomeostasis within the body (1). In mammalian muscle and fat cells,insulin stimulation activates a phosphorylation cascade, which in turncauses intracellular vesicles that contain the glucose transporterGLUT4,4 to translocate to the plasmamembrane (PM) and fuse (2, 3). Inthe basal stateGLUT4 is distributed between the endosomal system, the

trans-Golgi network (TGN), and a GLUT4 storage vesicle (GSV) com-partment that is highly insulin-responsive (4–6).The protein kinase Akt is activated in response to insulin and plays a

critical role in GLUT4 translocation (1, 7). However, the link betweenthe insulin signaling pathway and GLUT4 translocation is not fullyunderstood. The insulin-dependent movement of GLUT4 vesicles tothe PM is an Akt-independent process, and this is followed by an Akt-dependent step likely involving the docking and fusion of vesicles withthe PM (7–9). The mechanism by which Akt controls the docking andfusion of GLUT4 vesicles with the PM is not known. However, it waspreviously shown that a Rab GTPase-activating protein (RabGAP)known as AS160 is phosphorylated by Akt in response to insulin (10).How AS160 functions in GLUT4 trafficking and its cognate Rab pro-teins are not known. The role of a variety of Rab proteins in GLUT4trafficking including Rab3d, Rab4, Rab5, and Rab11 has been examined(11–16). However, although these Rab proteinsmay participate in someaspects of GLUT4 trafficking, no compelling evidence for specificinvolvement in the insulin-regulated trafficking of GLUT4 has beenfound.In this study we describe four key findings that add to our under-

standing of GLUT4 trafficking. Using mass spectrometry we have iden-tified three Rab proteins on GLUT4 vesicles that could potentially besubstrates of AS160 and, thus, play an important role in insulin-regu-lated glucose transport. In addition, we have shown that AS160 is pres-ent onGLUT4 vesicles in the basal state and dissociateswith insulin.Wehave gone on to show that the association of AS160 with GLUT4 vesi-cles is mediated at least in part via a direct interaction with the cytosolictail of IRAP, and this interaction appears to be insulin-dependent. Fur-thermore, a decrease in AS160 expression by shRNA leads to increasedlevels of GLUT4 at the PM in the basal state. Together, these resultsprovide novel insights into the regulatory mechanism of insulin-stimu-lated GLUT4 translocation.

EXPERIMENTAL PROCEDURES

Materials—3T3-L1 murine fibroblasts, C2C12 fibroblasts, and Chi-nese hamster ovary (CHO) cells were purchased from the AmericanType Culture Collection (ATCC, Manassas, VA). Dulbecco’s modifiedEagle’s medium and newborn calf serum were obtained from Invitro-gen, Myoclone-Plus fetal calf serum from Trace Scientific (Melbourne,

* This work was supported in part by grants from the National Health and MedicalResearch Council of Australia and Diabetes Australia Research Trust (to D. E. J.). Massspectrometric analysis for this work was carried out at the Bioanalytical Mass Spec-trometry Facility, University of New South Wales, and was supported in part by grantsfrom the Australian Government Systemic Infrastructure Initiative and Major NationalResearch Facilities Program (University of New South Wales node of the AustralianProteome Analysis Facility) and by the University of New South Wales Capital GrantsScheme. The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked “advertisement” inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

□S The on-line version of this article (available at http://www.jbc.org) contains Supple-mental Table 2.

1 These authors contributed equally to this work.2 Supported by the Swiss National Foundation and the Novartis Stiftung.3 A National Health and Medical Research Council senior principal research fellow. To

whom correspondence should be addressed: Diabetes and Obesity Program, GarvanInstitute of Medical Research, 384 Victoria St., Darlinghurst, NSW 2010 Australia. Tel.:61-2-92958210; Fax: 61-2-92958201; E-mail: [email protected].

4 The abbreviations used are: GLUT4, glucose transporter 4; PM, plasma membrane; TGN,trans-Golgi network; GSV, GLUT4 storage vesicles; RabGAP, Rab GTPase-activatingprotein; AS160, Akt substrate of 160 kDa; shRNA, short hairpin RNA; CHO, Chinesehamster ovary; TfR, transferin receptor; IRAP, insulin-regulated aminopeptidase;

VAMP, vesicle-associated membrane protein; LDM, low density microsomes; PBS,phosphate-buffered saline; Puro, puromyocin; Hygro, hygromyocin; HA, hemagglu-tinin; SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein recep-tors; CI-MPR, cation-independent mannose 6-phosphate receptor; CD-MPR, cation-dependent mannose 6-phosphate receptor; ATP7A, copper-transporting ATPase 1;SCAMP, secretory carrier-associated membrane protein; GST, glutathione S-transfer-ase; MES, 4-morpholineethanesulfonic acid; GSV, GLUT4 storage vesicle.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 280, NO. 45, pp. 37803–37813, November 11, 2005© 2005 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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Australia), and antibiotics were from Invitrogen. Paraformaldehyde wasfromProSciTech (ThuringowaCentral, Australia). Insulinwas obtainedfrom Calbiochem, and bovine serum albumin from United States Bio-chemical Corp. (Cleveland, OH). Bicinchoninic acid reagent, GF-2000beads, Supersignal West Pico chemiluminescent substrate, and proteinG-agarose beads were from Pierce. Lipofectamine 2000 was fromInvitrogen. Centrifuge tubes (11 � 60mm) were from Beckman Instru-ments. Polyvinylidene difluoride membrane was from Millipore (Bil-lerica, MA). C18 Stagetips were from Proxeon Biosystems (Odense,Denmark). Trypsin was from Promega (Madison, WI). Strong cationexchange andC18 cartridges were fromMichrom (Auburn, CA).MagicC18 material was from (Alltech, Deerfield, IL). Complete proteaseinhibitor mixture tablets were from Roche Applied Science. All othermaterials were obtained from Sigma. Antibodies were kindly providedby Dr. Gwyn Gould (CI-MPR and CD-MPR, University of Glasgow,Glasgow, UK), Dr. ClausMunck Peterson (sortilin,Weizmann Instituteof Science, Rehovot, Israel), Dr. P. Chavrier (Rab5 and Rab7, INSERM-CNRS, Marseille, France), Dr. Robert Parton (Rab11, University ofQueensland, Brisbane, Australia), Dr. Bernad (PKL12, Campus de laUniversidad Autonoma de Madrid, Madrid, Spain), Dr. Castle(SCAMP2 and SCAMP3, University of Virginia, USA), Dr. WanjinHong (syntaxin 16, IMCB, Singapore), and Dr. Julian Mercer (ATP7A,Deakin University, Burwood, Australia). Antibodies were purchasedfrom Santa Cruz Biotechnology (IRS-1; Santa Cruz, CA), CaymanChemical (CD36), BD Biosciences Pharmingen (Vti1b), Synaptic Sys-tems (VAMP2, VAMP8, and mVps45; Goettingen, Germany), ZymedLaboratories Inc.; San Francisco, CA (TfR), Sigma (FLAG M2), Trans-duction Laboratories (syntaxin 6; Lexington, KY), and Berkeley Anti-bodyCo., Inc. (HApeptide (16B12); Richmond, CA). Antibodies againstGLUT4 (17), syntaxin 4 (18), IRAP (5), Rab14 (19), and VAMP3 (18)have been described previously. ALEXA 488 and Cy3-conjugated sec-ondary antibodies were obtained from Molecular Probes (Leiden, TheNetherlands) or Jackson ImmunoResearch (West Grove, PA), respec-tively. Horseradish peroxidase-conjugated secondary antibodies werefrom Amersham Biosciences.

Production of a Rabbit AS160 Antibody—Rabbit polyclonal antibod-ies against human AS160 (TBC1D4) were produced as previouslydescribed (18) using a region of human AS160 from amino acids 621–766 fused with GST. Serum from these rabbits was affinity-purifiedusing the AS160 antigen coupled to a GF-2000 column according tomanufacturer’s instructions.

Cell Culture—3T3-L1 fibroblasts, CHO, and C2C12 fibroblasts werecultured as described previously (5). Briefly, cells were grown inDulbec-co’s modified Eagle’s medium supplemented with 10% newborn calfserum or fetal calf serum, 2 mM L-glutamine, 100 units/liter penicillin,and 100 �g/liter streptomycin at 37 °C in 10% CO2 and passaged at�60% confluence. In the case of 3T3-L1 fibroblasts, confluent cells weredifferentiated into adipocytes and used between days 8 and 10 post-differentiation and between passages 10 and 20. To establish basal con-ditions before use, cells were incubated in serum-free Dulbecco’s mod-ified Eagle’s medium for 2 h at 37 °C in 10% CO2.

Subcellular Fractionation of Adipocytes—3T3-L1 adipocyte fraction-ation was carried out as described previously (5). Briefly, cells wereincubated for 2 h in Dulbecco’s modified Eagle’s medium containing 25mM glucose at 37 °C and lysed using 12 passes through a 22-gauge nee-dle followed by 6 passes through a 27-gauge needle in HES buffer (20mM HEPES, 10 mM EDTA, 250 mM sucrose pH 7.4) containing Com-plete protease inhibitor mixture and phosphatase inhibitors (2 mM

sodium orthovanadate, 1mM pyrophosphate, 1mM ammoniummolyb-date, 10mM sodium fluoride) at 4 °C. The lysate was then centrifuged at

500 � g for 10 min to remove unbroken cells and at 10,080 � g for 12min, 15,750 � g for 17 min, and 175,000 � g for 75 min at 4 °C to obtainthe PM, mitochondria and nuclei high density microsomal pellet con-taining the endoplasmic reticulum and large endosomes, and the lowdensity microsomal (LDM) pellet which contained intracellular trans-port vesicles and the majority of GLUT4 vesicles, respectively. TheLDM was then resuspended in PBS-containing inhibitors for use inimmunoprecipitations.

Cationic Colloidal Silica Plasma Membrane Isolation—Plasmamembranes were purified as per Chaney and Jacobson (21) with modi-fications. Briefly, basal or insulin-stimulated 3T3-L1 adipocytes werewashed twice with ice-cold PBS and twice in ice-cold coating buffer (20mM MES, 150 mM NaCl, 280 mM sorbitol, pH 5.0–5.5). Cationic silica1% (stored as a 30% stock and diluted to 1% in coating buffer) was addedto the cells in coating buffer for 2 min on ice. Excess silica was removedby washing once with ice-cold coating buffer. Sodium polyacrylate (1mg/ml, pH6–6.5)was added to the cells in coating buffer and incubatedat 4 °C for 2 min. Cells were washed once in ice-cold coating buffer andthen washed withmodified HES (20mMHEPES, 250mM sucrose, 1 mM

dithiothreitol, 1 mMmagnesium acetate, 100mM potassium acetate, 0.5mM zinc chloride, pH 7.4) at 4 °C and lysed as described above. Nyco-denz (100%) in modified HES buffer was added to the lysate to a finalconcentration of 50%. The lysate was layered onto 0.5 ml of 70% Nyco-denz in modified HES and centrifuged in a swing-out rotor at 41,545 �g for 20 min at 4 °C. The supernatant was discarded, and the pellet wasresuspended in 0.5 ml of modified HES buffer and centrifuged at 500 �g for 5 min at 4 °C. The pellet was resuspended in SDS-PAGE samplebuffer and heated to 65 °C for 10 min. The sample was then centrifugedat 10,000 � g at 24 °C, and the supernatant was retained for analysis byimmunoblotting.

Immuno-isolation of GLUT4 Vesicles and Mass SpectrometryAnalysis—GLUT4 vesicles were immuno-isolated using the protocol ofHashiramoto et al. (22) with the exception that either control mouseIgG or the anti-GLUT4 monoclonal 1F8 was covalently coupled toGF-2000 beads according tomanufacturer’s instructions. LDM (2.5mg)was resuspended in 100 �l of PBS and added to 50 �l of both controlmouse IgG and 1F8 beads and incubated overnight with rotation at 4 °Cin the presence of 0.1% bovine serum albumin and PBS in a final volumeof 250 �l. The beads were then washed 5 times with PBS, and the pro-teins were eluted with 100% formic acid. The eluate was removed andvacuum-dried. Ammonium bicarbonate (500 mM) was added to adjustthe pH to 8 and vacuum-dried again. Urea (10 M) and 5 mM dithiothre-itol were then added, and samples were incubated at 37 °C for 1 h.Cysteines were subsequently alkylated using a 10-fold molar excess ofiodoacetamide and incubated at 37 °C for 1 h. Proteins were digested bythe addition of modified trypsin (12.5 ng/�l) in 100 mMNH4HCO3 andincubated overnight at 37 °C. The digestionwas stopped by the additionof 5% formic acid, and peptides were desalted using C18 Stagetips. Pep-tideswere analyzed by two-dimensional liquid chromatography tandemmass spectrometry using a CapLC high performance liquid chromatog-raphy system (Waters, Milford, MA) by binding to a strong cationexchange cartridge and sequential elution of the peptides using saltsteps of 5, 10, 15, 20, 25, 30, 40, 50, 75, 150, 300, 1000 mM ammoniumacetate. After each salt step eluted peptides were desalted on a capillaryC18 cartridge and resolved on a 100-mm � 75-�m C18 Magic reversephase analytical column with a flow rate of 200 nl/min. Peptides wereionized by nanoelectrospray at 2.8 kV from the end of the column,which was pulled to an inner diameter of 5 �m by a P-2000 laser puller(Sutter Instruments Co). Tandemmass spectral analysis was carried outon a Waters (Milford, MA) quadrupole time-of-flight Ultima mass

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spectrometer. A data-dependent acquisition method was used for allexperiments where precursor ions needed to have an intensity higherthan 10 counts and be in the�2,�3, or�4 charge state.MS/MS spectrawere searched against a metazoan data base generated from Swiss-Protand Trembl containing 574734 sequences using Sequest (Thermo Elec-tron Corporation, Waltham, MA). Peptides were counted as valid if inthe�2 charge state they had a Xcorr value�2 or in the�3 charge state,an Xcorr �3.5. All peptides also needed a �CN value greater than 0.08.One missed cleavage per peptide was tolerated, and peptides could bepartially tryptic. Proteins identified by less than two peptides were val-idated manually.

Immunoblotting—All samples were subjected to SDS-PAGE analysison 10% resolving gels according to Laemmli (23). Equal amounts ofprotein were loaded for each sample in a single experiment, with 1–10�g per lane unless otherwise stated. Separated proteins were electro-phoretically transferred to polyvinylidene difluoride membrane,blocked with BB (2% nonfat skim milk in 0.1% Tween 20 in PBS), andincubated with primary antibody in BB. After incubation, membraneswere washed 3 times in BB and incubated with horseradish peroxidase-labeled secondary antibodies in BB. Proteins were visualized usingSupersignal West Pico chemiluminescent substrate and imaged using aVersadoc 5000 imager (Bio-Rad).

Sucrose Gradient Flotation Experiments—LDM (165 �l) obtainedfrom 3T3-L1 adipocytes as described above was mixed with 835 �l of70% sucrose solution inHES to a final concentration of 60% sucrose (24)and placed in an 11 � 60-mm centrifuge tube overlaid with 1 ml of 50,30, and 10% of sucrose and 0.4 ml of 5% sucrose. The sample was thencentrifuged for 18 h at 111,132� g in a Beckman SW61 swing-out rotor.Fractions (0.4 ml) were collected from the bottom of the tube by gravi-tational flow.

Confocal Laser Scanning Microscopy—3T3-L1 adipocytes were cul-tured as described above on glass coverslips. The cells were serum-depleted for 2 h at 37 °C, after which they were incubated in the absenceor presence of 200 nM insulin for 20 min. Cells were then fixed with 3%paraformaldehyde in PBS. Fixed cells were washed with PBS, and freealdehyde groups were quenched with 50 mM glycine in PBS. The cellswere then processed for immunolabeling by permeabilization and label-ing in PBS containing 0.1% saponin and 2% bovine serum albumin usingstandard procedures. Primary antibodies were detected with ALEXA-488 or Cy3-conjugated secondary antibodies. Optical sections wereanalyzed by confocal laser scanning microscopy using a Leica TCS SPsystem. For double labeling, fluorophores were scanned separately andoverlaid using Adobe Photoshop software. Images were generated bythemaximumprojection of a stack sections from themiddle of each cell.

GLUT4 Recycling Experiments—3T3-L1 adipocytes expressing acontrol shRNA or the AS160 shRNA and HA-GLUT4 were incubatedin the presence or absence of 100 nM insulin for 20 min. Anti-HA anti-body (60 �g/ml) was then added for 10 and 60 min. Subsequently thecells were fixed, permeabilized, and incubated with a secondary Cy3-conjugated anti-mouse antibody. Confocal laser scanning microscopywas carried out as described above.

Transfection of C2C12 Cells and CHO Cells—C2C12 and CHO cellswere transiently transfected with DNA constructs for expression ofFLAG-tagged AS160 and the AS160 and control shRNA constructsusing Lipofectamine 2000 according to manufacturer’s instructions.

Immunoprecipitation and GST Pull-down of FLAG-tagged AS160—FLAG-AS160-expressing CHO cells were lysed in extraction buffer (1%Nonidet P-40, 137 mM sodium chloride, 10% glycerol, 25 mM Tris, pH7.4), centrifuged at 18,000 � g for 20 min, anti-FLAG antibody andprotein G beads were added to the supernatant and incubated for 2 h at

4 °Cwithmixing. The beads were then washed extensively and boiled inSDS-PAGE sample buffer. For GST pull-downs, 100 �g of lysate wasincubated with either GST alone or GST-IRAP1–109, GST-IRAP1–58,GST-IRAP1–27, GST-GLUT4466–509, GST-VAMP21–94 coupled toCNBr-activated-Sepharose 4B beads according to the manufacturer’sinstruction (Amersham Biosciences). Beads were washed extensivelyand boiled in sample buffer.

Immunoprecipitation of Endogenous AS160 from 3T3-L1 Adipocytes—The LDM fraction from basal or insulin-stimulated cells prepared asdescribed abovewas resuspended in IP buffer (60mM �-octylglucoside, 1%TritonX-100, 137mM sodium chloride, 10% glycerol, 25mMTris, pH 7.4),incubated for 30min at 4 °C, and centrifuged at 200,000� g for 15min. Analiquot of the supernatant (200 �g) was incubated with either IgG or anti-AS160 or anti IRAP antibodies coupled to CNBr-activated Sepharose 4Bbeads overnight at 4 °C. Beads were washed four times in IP buffer without�-octylglucoside and twice in PBS and boiled in sample buffer.

Retroviral Transfection of shRNA—The shRNA for AS160 was cre-ated using standard procedures with the target sequence 5�-TAAC-GAGGATGCCTTCTAC-3� as described previously (25). 3T3-L1fibroblasts were infected with pBabe-puro-HA-GLUT4 retrovirus aspreviously described (5). After 24 h cells were subsequently infectedusing a similar protocol with pBabe-hygro-shRNA-AS160. Doublyinfected cells were selected in 2 �g/ml puromyocin and 200 �g/mlhygromyocin. The surface HA-GLUT4 was measured in non-perme-abilized adipocytes with anti-HA antibody as described previously (26).Subsequently the cells were permeabilized and incubated with anti-AS160 antibody. The quantification of fluorescence of single cellsinfected with retrovirus expressing an shRNA against AS160 and HA-GLUT4 was performed using the Leica confocal software. The confocalimages of the different conditions and cells were scanned with exactlythe same settings. A region of interest was set around each cell, and theamount of fluorescence per unit area was determined for each channelusing the Leica confocal software.

RESULTS

Identification of GLUT4 Vesicle-associated Proteins in 3T3-L1Adipocytes—To identify proteins that regulate GLUT4 trafficking, weused two-dimensional liquid chromatography tandem mass spectrom-etry to identify all of the proteins associated with immuno-isolatedGLUT4 vesicles. The immuno-isolation of GLUT4 vesicles was per-formed using antibodies covalently attached to acrylamide beads (27).To ensure that we ignored proteins that were isolated non-specifically,we used a subtractive experimental design where we compared the pro-tein composition of eluates from the control IgG versus that obtainedwith the GLUT4 antibody. Only peptides identified using the GLUT4antibody and not the control IgG in at least three separate experimentswere classified as valid.The consensus of these experiments in 3T3-L1 adipocytes yielded 48

proteins (TABLE ONE), many of which are known to be involved invesicle trafficking. Details of peptides identified from each protein aregiven in Supplemental Table 2. The proteins identified fall into fiveclasses; they are previously knownGLUT4 vesicle proteins (e.g. sortilin),vesicle transport regulatory proteins (e.g. SNAREs), proteins from thesecretory pathway (e.g. cystatin c), recycling membrane proteins (e.g.low density lipoprotein receptor), and proteins with unknown GLUT4vesicle transport functions (e.g. ubiquitin, V-ATPase). Samples wereisolated from basal and insulin-stimulated adipocytes to determinewhether we could identify additional proteins that associated withGLUT4 vesicles in an insulin-specific manner. However, no additionalproteins were identified in samples isolated from insulin-treated cells.

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TABLE ONE

Summary of GLUT4 vesicle proteins identified by mass spectrometry from 3T3-L1 adipocytesProteins shown were the consensus of three separate experiments. �, no significant translocation; �, �2-fold translocation; ��, �2-fold translocation. Summary oflocalization of protein identified in GLUT4 vesicles was based on either the present study (†) or other published data.

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We next wanted to determine which of these proteins might play afunctional regulatory role in GLUT4 trafficking or indeed determinewhich proteins may be targeted with GLUT4 into insulin-responsivevesicles. To accomplish this we divided these proteins into integral ver-sus peripheral membrane proteins.

Characterization of Integral Membrane Proteins Found in GLUT4Vesicles—It has previously been shown that GLUT4 is found in GSVs,endosomes, and the TGN (28–30). Therefore, it was not surprising tofind a large number of endosomal and TGN-derived proteins inimmuno-isolated GLUT4 vesicles. To determine which of these werelocalized to GSVs, we used two separate criteria. First, we examinedinsulin-responsive movement to the PM, as this is a distinguishing fea-ture of GSV constituents (31). Second, we performed co-localizationexperiments to compare the intracellular distribution of each integralmembrane protein with GLUT4.To quantify the insulin-dependent movement of proteins to the PM,

we developed a method to isolate highly purified plasma membranes(21). Electron microscopy studies of these membranes revealed thatthey contained large PM sheets with very little contamination fromother organelles (data not shown). The only other organelle we coulddetect by electron microscopy was mitochondria. We were unable todetect Golgi or endoplasmic reticulummembranes proteins in the puri-fied PM fraction by immunoblotting (data not shown), which suggeststhat this is a highly purified fraction. In contrast PM markers such assyntaxin 4 or caveolin 1 were highly enriched in this fraction (data notshown). We observed very little GLUT4 in the PM in the absence ofinsulin, whereas after insulin stimulation there was a time-dependentmovement of GLUT4 to the PM, and this effect was inhibited by thephosphatidylinositol 3-kinase inhibitor wortmannin (Fig. 1A). The levelof the t-SNARE syntaxin 4 in the PM remained constant under theseconditions (Fig. 1A). Among the integral membrane proteins examinedGLUT4, IRAP and VAMP2 exhibited the largest insulin-dependentincrease at the PM, showing 10-, 6-, and 2.5-fold increases, respectively(Fig. 1B) (n 4). The protein that exhibited the next most significantchange with insulin was the TfR, showing a 1.5-fold increase in cellsurface levels (Fig. 1B). In contrast, other endosomal proteins such asVAMP3 did not undergo any significant change in PM localization withinsulin. Consistent with previous studies (22, 32–35) we observed aslight effect of insulin on PM levels of CI-MPR, CD-MPR, and sortilin,but this was much lower than that observed for GLUT4, IRAP, orVAMP2 (Fig. 1b). Intriguingly, the copper-transporting ATPase(ATP7A), which was identified in GLUT4 vesicles and can translocateto the plasmamembrane with copper-stimulation (36), did not translo-cate with insulin-stimulation.

We next compared the intracellular localization of these proteinswith GLUT4. A defining feature of the intracellular localization ofGLUT4 is its concentration in peripheral vesicles that are thought torepresent the insulin-responsive vesicles (37). Our laboratory has pre-viously described quantitative immunoelectron microscopic labeling ofbasal versus insulin-treated 3T3-L1 and primary rat adipocytes (4, 37).We observed a 53% reduction in GLUT4 labeling of cytosolic vesicles inresponse to insulin, whereas only an 18% reduction in GLUT4 labelingwas seen for theTGNor perinuclear region. These data indicate that theperipheral cytosolic vesicles correspond to the insulin-responsiveGSVs. In agreement with this conclusion, we observed significant co-localization between GLUT4, IRAP, and VAMP2 in cytosolic vesicles(Fig. 2A). However, this was not the case for any of the other integralmembrane proteins examined. For example some proteins such as sor-tilin (Fig. 2A) and syntaxin 16 (data not shown) were highly concen-trated in the perinuclear area, presumably corresponding to TGN label-ing, butwere not concentrated in the peripheral cytosolic vesicles. Someproteins like the copper-transporting ATPase ATP7A (Fig. 2A) orVAMP3 (data not shown) exhibited both perinuclear and peripheralcytosolic vesicular staining (Fig. 2A). However, the peripheral cytosolicvesicular staining did not correspond to that containing GLUT4, indi-cating that these proteins reside in separate vesicles. Therefore, thesestudies indicate that the major integral membrane proteins in GSVs areGLUT4, IRAP, and VAMP2.

Characterization of Peripheral Membrane Proteins Found in GLUT4Vesicles—Several novel peripheral membrane proteins were discoveredassociated with GLUT4 vesicles in this study by mass spectrometry.Perhaps the most interesting among these were 3 Rab proteins Rab10,Rab11, Rab14, and the RabGAPAS160. Rab11 has previously been stud-ied in the context of GLUT4 trafficking, but there is little evidence toindicate a role for this protein in the insulin-regulated trafficking ofGSVs to the PM (11). In contrast, Rab10 and Rab14 have not previouslybeen described in GLUT4 vesicles. To verify our mass spectrometrydata, we obtained antibodies against Rab11 and Rab14; however, noRab10-specific antibodies are currently available. We immunoblottedGLUT4 vesicles isolated from adipocytes that had been incubated in theabsence or presence of insulin and found that both Rab11 and Rab14remained associated with the GLUT4 vesicles with and without insulinstimulation (Fig. 3). Rab7, which had been found to non-specificallyassociate with GLUT4 vesicles in themass spectrometry analysis, is alsoshown (Fig. 3). Rab5, which has previously been shown not to associatewith GLUT4 vesicles (38), was neither detected in the control nor inGLUT4 immuno-isolations (Fig. 3). We next wanted to examine theintracellular localization of the Rabs using confocal immunofluores-

TABLE ONE—CONTINUED

A Entry name as shown in the Swiss-prot and Trembl databases.B Accession number as shown in the Swiss-prot and Trembl databases.C Overlap of protein with GLUT4 in the perinuclear area of unstimulated 3T3-L1 adipocytes.D Overlap of protein with GLUT4 in the peripheral cytosolic area of unstimulated 3T3-L1 adipocytes.E Translocation of proteins to the plasma membrane of 3T3-L1 adipocytes treated with 100 nM insulin for 30 min.

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cence microscopy. To accomplish this we attempted to localize theendogenous Rab proteins because overexpression of Rab proteins hasbeen shown to impair endogenous trafficking pathways (39). OnlyRab14 antibodies gave specific labeling in adipocytes. Rab14 displayedpunctate staining in the periphery of the cell and perinuclear staining aspreviously described in other cell types (19) (Fig. 2B). When the co-localization of Rab14 with GLUT4 was studied we found some overlapin peripheral GLUT4 vesicles and in the perinuclear region correspond-ing to the Golgi/TGN (Fig. 2B). However, co-localization in peripheralGLUT4-containing vesicles was not as significant as that seen for IRAPand VAMP2.The identification of AS160 on the samemembranes as the three Rab

proteins was particularly interesting and raises the possibility that theymay be in vivo substrates for the GAP activity of AS160. To examine thefunction of AS160 in GLUT4 trafficking, we generated a rabbit poly-clonal antibody against a central region of AS160. Consistent with pre-vious studies by Sano et al. (40), our antibody against AS160 specificallyrecognizes immunoprecipitated FLAG-tagged AS160 (Fig. 4A). Thisantibody also immunolabeled a protein of 160 kDa in adipocytes thatwere enriched in the LDM fraction (Fig. 4B), which contains the major-ity of GLUT4 vesicles (22). We have previously shown that the LDMfraction is comprised of bothmembrane vesicles and large protein com-plexes (24). To clarify which structures AS160 is associated with in theLDM, we subjected this fraction to sucrose density flotation analysis.Intriguingly, although themajority of AS160 was found at the bottom ofthe gradient, a significant amount was found in the vesicle-enrichedfraction, migrating at a similar position toGLUT4. To confirm themassspectrometry data showing the association of AS160 with GLUT4 ves-icles, we immunoblotted purified GLUT4 vesicles with the AS160 anti-body. Consistent with the mass spectrometry data, AS160 was detectedin the GLUT4 vesicles but was not detected in control IgG samples. Inaddition, we found that insulin stimulation induced a marked decreasein the amount of AS160 associated with GLUT4 vesicles (Fig. 4D). Thedissociation of AS160 from GLUT4 vesicles was blocked by treatmentwith the phosphatidylinositol 3-kinase inhibitor wortmannin beforeinsulin stimulation (Fig. 4E).

Identification of IRAP as the Binding Partner for AS160 on GLUT4Vesicles—The above data are consistent with a model whereby theinteraction of AS160 with GLUT4 vesicles plays a key role in their intra-cellular sequestration in the basal state. Therefore, we next wanted toidentify the molecular basis for this interaction. In view of our datashowing that GLUT4, IRAP, and VAMP2 are the major integral mem-brane proteins in GLUT4 vesicles, we next explored the possibility thatAS160 may interact with one or more of these proteins. To test this weperformed in vitroGSTpull-down experiments using lysates fromCHOIR/IRS-1 cells that had been transiently transfected with FLAG-taggedAS160. As shown in Fig. 5A, whereas we were unable to observe anyspecific binding of AS160 to GST alone, GST fused to the cytosolic tailof VAMP2, or GST fused to the carboxyl tail of GLUT4, there was asignificant interaction between AS160 and the GST-IRAP1–109 fusionprotein (data not shown) and aGST-IRAP1–58 fusion protein (Fig. 5A).In contrast, we were unable to observe an interaction between AS160and a truncated IRAP cytosolic tail comprising the first 27 amino acids(Fig. 5A). Moreover, the interaction between AS160 and the IRAP tailwas significantly higher using basal compared with insulin-treated celllysate. To further confirm this interaction, we next endeavored to showan interaction between AS160 and IRAP in vivo. LDM was preparedfrom basal or insulin-stimulated 3T3-L1 adipocytes and solubilized in60 mM �-octylglucoside, 1% Triton X-100, and the soluble fraction wasincubated with either control antibodies or antibodies against AS160 or

FIGURE 1. Translocation of GLUT4 vesicle proteins identified by mass spectrometryto the plasma membrane with insulin-stimulation in 3T3-L1 adipocytes. Plasmamembranes were isolated for each condition using the cationic silica isolation method.A, translocation of GLUT4 and syntaxin 4 to the plasma membrane after 2 or 30 min ofstimulation with 100 nM insulin or 30 min with 100 nM wortmannin before a 30-minstimulation with 100 nM insulin. Protein levels were detected by immunoblotting. B,translocation of proteins to the plasma membrane after 30 min of stimulation with 100nM insulin. C, quantification of protein translocation to the plasma membrane with 100nM insulin stimulation for 30 min. Error bars are S.E. Immunoblots are from a representa-tive experiment (n 4).

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IRAP coupled to CNBr beads. As shown in Fig. 5B, IRAP co-immuno-precipitated with AS160, and this interaction was only observed in basalbut not in insulin-stimulated cells. Similarly, AS160 co-immunoprecipi-tated with IRAP from adipocyte lysate, but in contrast to that observedin the AS160 immunoprecipitate, this interaction was only slightlyreduced by insulin (Fig. 5C). The explanation for this difference is notclear; however, these data suggest that the effect of insulin on the inter-action between AS160 and IRAP may be more complex than a simpledissociation event.

Co-localization of AS160withGLUT4andCharacterization of AS160shRNA Knockdown—To further characterize AS160 in 3T3-L1 adipo-cytes, we determined the co-localization of AS160 with GLUT4 by con-focal immunofluorescence microscopy. AS160 showed a predomi-nantly peripheral vesicular staining pattern (Fig. 2B). Co-localization ofAS160 with GLUT4 indicated some overlap of the two proteins inperipheral vesicles and the perinuclear area (Fig. 2B). The insulin-de-pendent release of AS160 from vesicles suggests a model whereby thetargeting of AS160 to GLUT4 vesicles in the absence of insulinmay play

an important role in the intracellular sequestration of this compart-ment. To test this we used RNA interference to suppress the expressionof AS160. We constructed a hairpin vector specifically targeting AS160that was able to suppress AS160 levels by greater than 80% after tran-sient transfection in C2C12 cells (Fig. 6A). To express the shRNA ininsulin-responsive adipocytes we employed the pBabe retrovirus. Asindicated in Fig. 6A adipocytes infected with this retrovirus demon-strated a 40% reduction in AS160 protein levels. To measure GLUT4translocation in cells in which AS160 levels were suppressed, we co-infected cells with retroviruses expressing HA-GLUT4 and AS160shRNA. Because of the variable reduction in AS160 levels, we used asingle cell analysis to study the relationship between total AS160 expres-sion and cell surface levels of HA-GLUT4. This analysis revealed thatoverall, cells infected with the AS160 shRNA had an 8-fold increase insurface HA-GLUT4 labeling compared with control cells (p � 0.005).Analysis of the individual data points clearly indicates that there is athreshold level of AS160 expression, below which surface levels of HA-GLUT4 are significantly elevated. Importantly, the increase in PM levels

FIGURE 2. Localization of integral and periph-eral membrane proteins with GLUT4. 3T3-L1adipocytes were serum-starved for 2 h and fixedand processed for immunofluorescence. GLUT4was detected using a Cy3-conjugated secondaryantibody, and the other proteins shown weredetected using ALEXA 488-conjugated secondaryantibodies. A, co-localization of GLUT4 vesicleintegral membrane proteins with GLUT4 vesiclesin the basal state. B, co-localization of GLUT4 vesi-cle peripheral membrane proteins with GLUT4vesicles in the basal state. Images are from a rep-resentative experiment (n 3).

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of HA-GLUT4 upon reduced AS160 expression were similar to thatobserved in control cells treated with insulin (Fig. 6B). It has previouslybeen shown that overexpression of the AS160 4P mutant in adipocytesprincipally affects GLUT4 translocation by perturbing the exocytosis ofGLUT4 (41). We wanted to determine whether the AS160 shRNA wasfunctioning in a similar manner. To determine this, recycling experi-ments were performed in which either control cells or cells expressingtheAS160 shRNAwere incubatedwith anti-HA antibody at 37 °C for 10min or 60min. As shown in Fig. 6C in non-stimulated control cells therewas little antibody uptake over the course of the 60-min incubation,consistent with previous studies showing that the basal recycling rate ofHA-GLUT4 is very slow (6, 42). In contrast, in cells expressing theAS160 shRNA there was a significant increase in antibody uptake evenin the absence of insulin, and labeling was detected at an intracellularlocalization consistent with increased exocytosis of GLUT4 uponAS160 reduction.

DISCUSSION

In this study we have made several novel observations about insulin-regulatedGLUT4 trafficking in adipocytes. First, using a comprehensiveproteomic and biochemical analysis of affinity-purified GLUT4 vesicleswe have found that the major membrane protein constituents of GSVsare GLUT4, IRAP, and VAMP2. The remaining constituents are prob-ably endosomal or TGN-derived proteins. Second, we have identifiedthree Rab proteins associated with GLUT4 vesicles, Rab10, Rab11, andRab14, two of which have not previously been reported to associate withGLUT4 vesicles. Third, we have shown that the putative RabGAPAS160 is associated with GLUT4 vesicles in 3T3-L1 adipocytes anddissociates from GLUT4 vesicles with insulin stimulation. Fourth, wehave demonstrated that the binding of AS160 to GLUT4 vesicles in thebasal state is mediated at least in part via an interaction with the integralmembrane protein IRAP and that this interaction is insulin-regulated.Finally, we show that knockdown of AS160 in adipocytes with shRNAcauses insulin-independent movement of GLUT4 to the PM.Previous studies attempting to identify proteins associated with

GLUT4 vesicles used analytical techniquesmuch less sensitive than thatused in the present study and have, therefore, only identified the mostabundant proteins. The proteins identified previously include GLUT4,IRAP (43), VAMP2 (44), TfR (45), CD-MPR (37), CI-MPR (46), sortilin(35), copper amine oxidase (34), SCAMPs (47), andVAMP3 (for review,see Bryant et al. (1)). We have identified all of these proteins and manyother novel proteins not previously identified in GLUT4 vesicles. The

large number and the diverse localizations of the proteins identifieddemonstrate that we have achieved a comprehensive analysis of themajority of proteins associatedwithGLUT4 vesicles. Based on ourmor-phological and biochemical analysis of a large number of these proteins,we have concluded that GLUT4, IRAP, and VAMP2 are the major pro-teins that are uniquely targeted to peripheral GLUT4 vesicles in adipo-cytes, and these studies likely explain the unique responsiveness of these

FIGURE 3. Rab proteins associate with immuno-isolated GLUT4 vesicles. The LDMfraction and a control and GLUT4 vesicle immuno-isolation eluate purified from the LDMfraction of 3T3-L1 adipocytes were immunoblotted for the presence of Rab proteins.These fractions were purified from 3T3-L1 adipocytes that had been incubated in theabsence (�) or presence (�) of 100 nM insulin for 30 min. Protein levels were detected byimmunoblotting with specific antibodies and visualized by chemiluminescence. Immu-noblots are from a representative experiment (n 4). IP, immunoprecipitated.

FIGURE 4. AS160 associates with GLUT4 vesicles in the basal state and dissociateswith insulin stimulation in 3T3-L1 adipocytes. A, FLAG-tagged AS160 was overex-pressed in CHO cells and immunoprecipitated (IP) using a monoclonal anti-FLAG anti-body. Control and FLAG-AS160 lysates and FLAG immunoprecipitates were immuno-blotted with the AS160 antibody. B, 3T3-L1 adipocytes incubated in the absence (�) orpresence (�) of 100 nm insulin for 30 min were fractionated to generate cytosol (Cyto),PM, LDM, high density microsome, mitochondria, and nuclei (M/N). AS160 levels weredetected by immunoblotting with the AS160 polyclonal antibody. C, 3T3-L1 adipocytesin the basal state were fractionated to obtain the LDM fraction, which was then analyzedby sucrose density flotation. The pellet and fractions 1–11, which represent the bottomand the top of the sucrose gradient, respectively, were then analyzed by immunoblot-ting. D, the LDM fraction and a control and GLUT4 vesicle immuno-isolation eluate puri-fied from the LDM fraction of 3T3-L1 adipocytes were immunoblotted for the presenceof GLUT4, IRAP, VAMP2, and AS160. These fractions were purified from 3T3-L1 adipo-cytes that had been incubated in the absence (�) or presence (�) of 100 nm insulin for 30min. E, GLUT4 vesicle immuno-isolation eluate purified from the LDM fraction of 3T3-L1adipocytes were immunoblotted for the presence of GLUT4 and AS160. These fractionswere purified from 3T3-L1 adipocytes that had been incubated in the absence (�) orpresence (�) of 100 nM insulin for 30 min or incubated with 100 nM wortmannin for 30min before incubation with 100 nM insulin for 30 min. Protein levels were detected byimmunoblotting with specific antibodies and visualized by chemiluminescence. Immu-noblots are from a representative experiment (n 4).

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proteins. Notably, insulin also has a smaller effect on the translocationof other proteins including the TfR (Fig. 1b) and GLUT1 (48). There areat least three possible explanations for this effect (1). First, insulin mayinhibit general endocytosis. This is unlikely because it has been shownthat insulin has no effect on TfR endocytosis in adipocytes (49). Second,a small proportion of the TfR may be targeted to GSVs and undergoexocytosis together with GLUT4, VAMP2, and IRAP. However, Akt isnot involved in TfR exocytosis, which suggests that the mechanism forits regulated release is different compared with these other proteins(50). Third, insulinmay increase endosomal recycling. This seems likelybecause a number of endosomal recycling proteins including MPR,GLUT1, andTfR have been shown to undergo a relatively small increasein translocation to the PM with insulin (1). We cannot rule out thatsome of the other proteins identified for which we do not have antibod-ies may be in GSVs. However, studies of synaptic vesicle proteins havealso shown that synaptic vesicles are quite simple in composition, astheir sole role is the stimulated release of neurotransmitter (51). How-ever, similar to this study, low levels of other proteins not crucial forsynaptic vesicle exocytosis, such as Vti1A and SCAMPs, are also foundin purified synaptic vesicles. This suggests the idea that such factorsarise from the recycling of small synaptic vesicles through endosomes orthe TGN (52, 53). Two proteins that we have identified that may beworthy of future study are the marvel domain-containing proteins pro-teolipid protein 2 and the myeloid-associated differentiation marker.Proteins containing this domain have been shown to regulate vesicletrafficking, but their exact role is not clear (54). Most of the other pro-teins found in the purified GLUT4 vesicles are involved or associatedwith GLUT4 recycling within endosomes or the TGN. This minimalGSV translocation system is consistent with previous electron micros-

copy studies of GLUT4 vesicles in adipocytes (37), which showed themajority of GLUT4 is recruited to the PM from vesicles that do notcontain recyclingmembrane proteins such as CD-MPR. It also supportsa growing number of studies that suggest the majority of regulatoryfactors involved in vesicle transport, aside from cargo and SNAREs, areperipherally associated cytosolic factors such as coat proteins or Rabproteins.Previous studies have tried to examine which Rab proteins play a role

in GLUT4 trafficking, but only Rab4 and Rab11 have been implicated ininsulin-stimulated GLUT4 vesicle translocation (11, 14, 15). Expressionof a dominant negative mutant of Rab4 in adipocytes inhibits GLUT4translocation by�50%, and the only direct evidence of a role for Rab4 inGLUT4 translocation is its binding to syntaxin 4 (14). However, thesignificance of this observation is not clear, as Rab proteins have notbeen found to bind directly to t-SNAREs in other vesicle transport path-ways. Moreover, we failed to observe Rab4 in our GLUT4 vesicle frac-tion, suggesting that the majority of Rab4 probably does not co-localizewith GLUT4 in adipocytes. Rab11 is known to be involved in trafficthrough recycling endosomes, and it has previously been shown to beassociated with GLUT4 vesicles (11). Rab11 translocates to the PMslightly with insulin stimulation (11). However, Rab11 does not translo-cate to the PMwithGTP�S stimulation, indicating that the PMmay notbe the Rab11 target organelle (13). Rab11 function has been implicatedin insulin-stimulated GLUT4 translocation through interactions withRab11bp (55). However, themechanism of action of Rab11 is unknown.The discovery in this study that Rab10, Rab11, and Rab14 are associatedwithGLUT4 vesicles is exciting, because one ormore of these three Rabproteins may be substrates for the RabGAP AS160 and play an integralrole in insulin action. Rab14 has been shown to be involved in TGN toendosomal trafficking (19); however, significant amounts of Rab14 arefound at the PM. Therefore, Rab14 may have other more specializedroles in exocytosis such as insulin-stimulated GLUT4 translocation.The role of Rab10 has yet to be elucidated. Intriguingly, it is one of theclosest mammalian homologues of the yeast sec4 protein (56) and,therefore, may function in the post-Golgi secretory pathway. Sec4 hasbeen shown to regulate assembly of the tethering complex known as theexocyst (57). This is of interest because the exocyst has been implicatedin insulin-regulated GLUT4 trafficking in adipocytes (58). Amino acidsequence alignment of the three Rab proteins identified in this studyshows that Rab14 and Rab11 are much more closely related to eachother than to Rab10, which may indicate that Rab10 has a more uniquerole in GLUT4 exocytosis. Further work will be required to examine therole of each Rab in GSV translocation and their association with AS160.Intriguingly, Lienhard and co-workers (59) have recently shown in an invitro GAP assay that the GAP domain of AS160 shows GAP activitytoward Rab10 and Rab14.Recently, we and others have suggested that themovement ofGLUT4

vesicles toward the PM is Akt-independent, whereas a step close to thePM, likely involving docking and/or fusion of GLUT4 vesicles, is themajor Akt-dependent step (7, 9, 40, 41, 60). In this study we have shownthat AS160 dissociates from GLUT4 vesicles with insulin stimulation.Furthermore, we have shown that there is a specific association betweenAS160 and the cytosolic tail of IRAP. This observation is intriguing for anumber of reasons. First, it has previously been shown that microinjec-tion of a fusion protein comprising the cytosolic N terminus of IRAPinto adipocytes was sufficient to triggerGLUT4 translocation to the PM(61). A similar effect was observed using a fusion protein encompassingthe first 58 amino acids of the IRAP tail, and we have also observed thatthis peptide interacts with AS160 in vitro. Hence, it is tempting to spec-ulate that overexpression of the IRAP cytosolic tail in adipocytes blocks

FIGURE 5. Identification of IRAP as the binding partner for AS160 on GLUT4 vesicles.A, CHO cells overexpressing FLAG-AS160 were incubated in the absence or presence ofinsulin (100 nM) for 15 min. Cells were lysed and incubated with GST alone, GST-GLUT4CT,GST-VAMP21–94, GST-IRAP1–27, and GST-IRAP1–58 coupled to CNBr-activated Sepharose4B Beads. AS160 in the GST pull-down was detected with anti-FLAG antibody. B and C,3T3-L1 adipocytes were incubated in the absence or presence of insulin for 15 min, anda LDM fraction was prepared. LDM was dissolved in 1% Triton X-100 and 60 mM �-octyl-glucoside, centrifuged at 200,000 � g for 15 min, and used for immunoprecipitation (IP)with IgG control, IRAP, or AS160 antibodies. Immunoprecipitates were then immuno-blotted with antibodies specific for both AS160 and IRAP. Ø indicates beads-only control.Immunoblots are from a representative experiment (n 3).

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the interaction of AS160 with the GLUT4 vesicles, thus allowing theirconstitutive translocation to the PM. Second, it has been shown thatGLUT4 trafficking is unaffected in adipocytes from IRAP knock outmice, suggesting that IRAP is not necessary for the intracellular seques-tration of the vesicles (62). A likely explanation for this is that AS160may be capable of interacting with other proteins associated withGLUT4 vesicles, possibly even its putative Rab substrate. Cargo bindingmay be a general function of RabGAPs in view of the large number ofRabGAPs in the human genome, and one could envisage that if eachmember of this family encoded unique cargo binding specificity, thiscould represent a novel mechanism for regulating protein trafficking atdifferent locations in eukaryotic cells. Each cargo-RabGAP interactioncould be controlled by discrete signaling inputs. In the case of AS160,this appears to involve phosphorylation by Akt, and as described above,this would place the action of AS160 close to the PM. In this way thedissociation of AS160 fromGSVsmay trigger GTP loading of a relevantRab protein. Although Rabs have been implicated at several steps invesicle transport, one of their major functions appears to involve regu-lation of vesicle docking at the target membrane (63). The fact that thedown-regulation of AS160 led to insulin-independent movement ofGLUT4 to the PM indicates the involvement of AS160 before fusion. Arecent study by Zeigerer et al. (41) has also shown that a step beforeGLUT4 vesicle fusion is AS160-dependent. These studies also suggestthat AS160 plays an important role in the intracellular sequestration ofGLUT4 in the absence of insulin. As reported previously (6, 26), GLUT4has a very low rate of cell surface recycling in the absence of insulin asindicated in Fig. 6C. Suppression of AS160 levels enhance this basalrecycling rate (Fig. 6C). This is unlikely due to an effect of AS160 on

endocytosis because we observed rapid internalization of GLUT4 incells expressing the AS160 shRNA. This is consistent with experimentsperformed by Zeigerer and co-workers (41) showing that overexpres-sion of the AS160 4P mutant inhibited GLUT4 exocytosis with noeffects on GLUT4 endocytosis. These studies are consistent with a roleof AS160 in GLUT4 exocytosis. This demonstrates that AS160 is a spe-cific regulator for the exocytosis of insulin-responsive GLUT4 vesiclesin adipocytes.We propose amodel of GLUT4 translocation such that inthe basal state AS160 is associated with GSVs by interacting with IRAPand possibly other vesicle cargo, and its GAP activity maintains a keyGTPase(s) in an inactive GDP-bound form. Upon stimulation withinsulin the GSVsmove toward the PM, and AS160 is phosphorylated byAkt, leading to its dissociation from GSVs. This allows GTP loading ofone or more Rab proteins, enabling the docking and fusion of the GSVswith the PM.

Acknowledgments—We thank JonathanDavey for help with the shRNA exper-iments, Donna Stolz for providing the cationic silica particles, Gus Lienhardfor the AS160 constructs, and all those colleagues who provided antibodiesused in this study.

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FIGURE 6. Knockdown of AS160 expression byshRNA induces basal GLUT4 translocation. A,C2C12 cells and 3T3-L1 adipocytes transientlytransfected with a control shRNA or the AS160shRNA were lysed, and protein levels of AS160 andsyntaxin 4 were determined by immunoblotting.B, 3T3-L1 fibroblasts were infected with HA-GLUT4and AS160 shRNA and differentiated into adipo-cytes. Cell surface HA-GLUT4 levels were deter-mined by incubation of fixed non-permeabilizedcells with a mouse anti-HA antibody followed byincubation with a Cy3-conjugated secondary anti-body. AS160 was then labeled by permeabilizingthe cells and incubating with the AS160 antibodyfollowed by ALEXA 488-conjugated secondaryantibody. The confocal images of the differentconditions and cells were scanned with exactly thesame settings. A region of interest was set aroundeach cell, and the total fluorescence per unit areawas determined for Cy3 and ALEXA 488 for eachcell shown as arbitrary units. The fluorescence val-ues for each cell type are shown as control shRNAbasal (�), control shRNA insulin (�), AS160 shRNAbasal (�), AS160 shRNA insulin (E). C, 3T3-L1 fibro-blasts were infected with a retrovirus expressingHA-GLUT4 and the AS160 shRNA and differenti-ated into adipocytes. Cells were incubated in thepresence or absence of 100 nM insulin for 20 minfollowed by the addition of HA antibody for 10 or60 min. Cells were subsequently fixed, permeabi-lized, incubated with Cy3-conjugated secondaryantibody, and processed for immunofluores-cence. Images are from a representativeexperiment.

AS160 and GLUT4 Trafficking

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AS160 and GLUT4 Trafficking

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Guilhaus and David E. JamesValerie Wasinger, Fiona Simpson, Michael Graham, Jagath R. Junutula, Michael

Mark Larance, Georg Ramm, Jacqueline Stöckli, Ellen M. van Dam, Stephanie Winata,Insulin-regulated GLUT4 Trafficking

Characterization of the Role of the Rab GTPase-activating Protein AS160 in

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