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Molecular Endocrinology, doi:10.1210/me.2005-0476
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Molecular Endocrinology 20 (10): 2576-2583
Copyright © 2006 by The Endocrine Society

Interaction of the Akt Substrate, AS160, with the Glucose Transporter 4 Vesicle Marker Protein, Insulin-Regulated Aminopeptidase

Grantley R. Peck, Siying Ye, Vi Pham, Ruani N. Fernando, S. Lance Macaulay, Siew Yeen Chai and Anthony L. Albiston

Department of Medicine (G.R.P.), Royal Melbourne Hospital, Howard Florey Institute (G.R.P., S.Y., V.P., R.N.F., S.Y.C., A.L.A.), Department of Biochemistry and Molecular Biology (S.Y.), Department of Genetics (R.N.F.), and Department of Anatomy and Cell Biology (S.Y.C.), University of Melbourne, Parkville, Victoria 3010, Australia; and Commonwealth Scientific and Industrial Research Organization Molecular and Health Technologies (S.L.M.), Parkville, Victoria 3052, Australia

Address all correspondence and requests for reprints to: Anthony L. Albiston, Howard Florey Institute, University of Melbourne, Parkville, Victoria 3052, Australia. E-mail: a.albiston{at}hfi.unimelb.edu.au.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Insulin-regulated aminopeptidase (IRAP), a marker of glucose transporter 4 (GLUT4) storage vesicles (GSVs), is the only protein known to traffic with GLUT4. In the basal state, GSVs are sequestered from the constitutively recycling endosomal system to an insulin-responsive, intracellular pool. Insulin induces a rapid translocation of GSVs to the cell surface from this pool, resulting in the incorporation of IRAP and GLUT4 into the plasma membrane. We sought to identify proteins that interact with IRAP to further understand this GSV trafficking process. This study describes our identification of a novel interaction between the amino terminus of IRAP and the Akt substrate, AS160 (Akt substrate of 160 kDa). The validity of this interaction was confirmed by coimmunoprecipitation of both overexpressed and endogenous proteins. Moreover, confocal microscopy demonstrated colocalization of these proteins. In addition, we demonstrate that the IRAP-binding domain of AS160 falls within its second phosphotyrosine-binding domain and the interaction is not regulated by AS160 phosphorylation. We hypothesize that AS160 is localized to GLUT4-containing vesicles via its interaction with IRAP where it inhibits the activity of Rab substrates in its vicinity, effectively tethering the vesicles intracellularly.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
GLUCOSE HOMEOSTASIS IS achieved, in part, via the actions of insulin on muscle and fat cells. In these cells, insulin-stimulated translocation of the glucose transporter, glucose transporter 4 (GLUT4), from intracellular locations to the cell surface facilitates glucose uptake. In the absence of insulin (basal state), approximately 50% of total GLUT4 is sequestered to insulin-responsive GLUT4 storage vesicles (GSVs) in the vicinity of the trans-Golgi network (1, 2, 3). Although studies have investigated the mechanisms regulating GSV sequestration, the rate-limiting step in insulin-stimulated GLUT4 translocation remains unknown. A system of interactions between adapter/sorting proteins and specific cytosolic domains of constituent GSV membrane proteins is thought to be involved in the tethering of GSVs intracellularly (3, 4, 5, 6, 7, 8).

GSVs are characterized, in part, by their peripheral and integral membrane protein constituents, including insulin-regulated aminopeptidase (IRAP) (9, 10, 11), vesicle-associated membrane protein 2 (12, 13, 14), syntaxin 6, and syntaxin 16 (15, 16). In the basal state, GSVs are devoid of markers of the constitutively cycling endosomal system, highlighting the specific sequestration of proteins to these specialized vesicles (3, 13, 14, 17). IRAP is the only GSV constituent known to colocalize with GLUT4 throughout the insulin-responsive vesicular trafficking pathway (9, 10, 18, 19, 20).

IRAP has a single transmembrane domain, a large extracellular/intravesicular catalytic domain, and a 109-amino acid cytoplasmic amino terminus containing two dileucine motifs, each with a preceding acidic cluster, implicated in sorting and targeting (11, 21). In support of this, a chimera of the amino terminus of IRAP with the transferrin receptor has been used to demonstrate its requirement for correct sorting to the insulin-responsive pool of GSVs (22, 23). Furthermore, microinjection of the amino terminus of IRAP, or a truncated form containing the second acidic cluster and dileucine motif (IRAP55–82), into 3T3-L1 adipocytes resulted in GLUT4 translocation to the plasma membrane similar to that induced by 100 nM insulin (5).

A number of proteins that interact with the amino terminus of IRAP have been identified. Of these, formin homolog overexpressed in spleen (24), acyl-Coenzyme A dehydrogenase (25), and, more recently, p115 (7) have been implicated in the regulation of GSV transport.

Here, the amino terminus of IRAP was expressed as a glutathione-S-transferase (GST) fusion protein to isolate proteins that interact with IRAP from cell lysates. An interacting protein was identified by tandem mass spectrometry (MS/MS) as AS160. AS160 was recently characterized as a 160-kDa substrate of Akt (AS160) (26), providing the next step in the insulin signaling pathway. AS160 contains several Akt phosphomotifs and a Rab GTPase-activating protein (GAP) domain, suggesting a role in regulating Rab-mediated membrane trafficking (26, 27). GLUT4 translocation was demonstrated to be dependent upon insulin-stimulated phosphorylation of AS160 (27, 28, 29) and inactivation of its GAP activity (27).

This study identified a novel interaction between AS160 and the GSV marker protein, IRAP, providing a direct link between the insulin-signaling and GLUT4-trafficking pathways. The interaction was confirmed and further characterized using IRAP(1–109) to affinity isolate cell-free translated AS160 or truncations thereof and by coimmunoprecipitation from transfected and endogenous cell systems. Furthermore, the IRAP-binding domain of AS160 was determined using a series of AS160 truncation mutants, and the effect of AS160 phosphorylation on its interaction with IRAP was assessed.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Identification of a Novel Interaction between AS160 and IRAP
pETGEXCT-IRAP(1–109) was used to express the cytoplasmic tail of IRAP in the same orientation as it appears in the cell, i.e. with GST fused to its C terminus in place of the transmembrane domain. Protein expressed from this plasmid was used to identify proteins that interact with the amino terminus of IRAP. A number of proteins visible by Coomassie staining interacted specifically with IRAP(1–109)-GST (Fig. 1Go). One of these proteins was identified from six independent tryptic peptides by MS/MS as AS160 with confidence values ranging from 94–99. (Two or more independent peptides matched with confidence >75 is significant.)


Figure 1
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  		Fig. 1. Isolation of Proteins that Interact with the Amino Terminus of IRAP

Approximately equal amounts of GST and IRAP(1–109)-GST used to affinity isolate proteins from cell lysate were resolved by SDS-PAGE and Coomassie stained (see Materials and Methods for details). Proteins bands that bound specifically to the amino terminus of IRAP and were assessed by MS/MS are indicated with their approximate molecular masses. A corresponding autoradiograph showed that several of the proteins bands that appear to be specific to IRAP(1–109) by Coomassie staining have equivalent bands in the GST-alone lane and for this reason were not subjected to mass spectrometry (data not shown). IP, Immunoprecipitation; WB, Western blot.

 
Biochemical Verification
AS160-myc was expressed using a cell-free transcription/translation system and affinity isolated using IRAP(1–109)-GST to assess whether the interaction was direct or dependent on intermediate proteins. Figure 2Go shows that AS160-myc is specifically isolated with IRAP(1–109)-GST, demonstrating a direct interaction between AS160 and IRAP.


Figure 2
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  		Fig. 2. Cell-Free Translated AS160-myc Affinity Isolation by IRAP(1–109)-GST

IRAP(1–109)-GST (lane 2) and GST alone (lane 1) were used to affinity isolate AS160-myc expressed by cell-free translation. An aliquot of the translation reaction is shown (lane 3) (see Materials and Methods for details). Isolated proteins were resolved by SDS-PAGE and immunoblotted with anti-myc monoclonal antibody followed by goat antimouse-HRP-conjugated antibody and developed using ECL. An immunoblot representative of five separate experiments is shown. WB, Western blot.

 
AS160 and IRAP Interact within the Cell
Lysates from human embryonic kidney (HEK)293T cells transiently expressing AS160-V5 and IRAP-myc were immunoprecipitated with V5 antibody and then immunoblotted with myc antibody to assess the association of tagged AS160 and IRAP. Data shown in Fig. 3Go demonstrate IRAP-myc coimmunoprecipitates with AS160-V5. The reciprocal experiment using IRAP-V5 to coimmunoprecipitate AS160-myc was performed and also demonstrated AS160-IRAP binding (data not shown).


Figure 3
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  		Fig. 3. Coimmunoprecipitation of Tagged AS160 and IRAP from Transfected HEK293T Cells

HEK293T cells expressing IRAP-myc and/or AS160-V5 were lysed and then immunoprecipitated using anti-V5 antibody. Immunoprecipitated proteins were transferred to nitrocellulose and probed with anti-myc (A) or anti-V5 (B). Crude cell lysates (10 µl) were also included to show expression of each protein (C and D). Western blot was probed with anti-myc or anti-V5 followed by goat antimouse-HRP or sheep antirabbit-HRP secondary antibodies, respectively. ECL was used to develop all blots. A representative result from four separate experiments, performed as described in Materials and Methods, is shown. WB, Western blot; IP, immunoprecipitation.

 
Endogenous AS160 and IRAP Interact in 3T3-L1 Adipocytes
To determine whether the interaction occurs with endogenous proteins or is the result of overexpression, coimmunoprecipitations of endogenous AS160 and IRAP from 3T3-L1 adipocytes were performed. AS160 coimmunoprecipitated IRAP from these cells (Fig. 4Go).


Figure 4
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  		Fig. 4. Coimmunoprecipitation of Endogenous AS160 and IRAP from 3T3-L1 Adipocytes

3T3-L1 adipocytes were lysed and immunoprecipitated with anti-AS160 (lane 1) or nonspecific goat IgG (lane 2) as described in Materials and Methods. Immunoprecipitates and crude lysate (lane 3) were resolved by SDS-PAGE, transferred to nitrocellulose, and probed with anti-IRAP carboxy terminus antibody (upper panel) before being stripped (30 min at room temperature in 25 mM glycine, pH 2.0; 1% SDS), blocked (30 min in 2% skim milk in TBST), and reprobed with anti-AS160 (lower panel). Similar results were obtained from five independent experiments. WB, Western blot.

 
AS160 (367–439) Interacts with IRAP
To determine the IRAP-binding domain of AS160, IRAP(1–109)-GST was used to isolate in vitro translated amino terminal-truncated forms of AS160 (Fig. 5Go). IRAP(1–109)-GST specifically bound AS160(119–1299), AS160(190–1299), and AS160(367–1299), but did not isolate the shorter truncated AS160 protein, AS160(440–1299).


Figure 5
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  		Fig. 5. Determination of IRAP-Binding Domain of AS160 Using AS160 Truncation Mutants

Diagram of boundaries of AS160 truncation mutants used to identify the IRAP-binding domain of AS160 by affinity isolation and corresponding autoradiographs are shown. Mutagenesis and affinity isolation procedures are described in detail in Materials and Methods. IRAP(1–109)-GST (lane 1) was used to affinity isolate AS160, or truncations thereof, labeled with [35S]met/cys by in vitro translation. GST alone was included to demonstrate IRAP-binding specificity (lane 2). The in vitro translation products reaction (10%) was run on the gel to demonstrate adequate expression of each protein (lane 3) (n = 3). Approximate locations of Akt phosphomotifs are indicated (P). WB, Western blot; TNT, transcription/translation.

 
Effect of AS160 Phosphorylation on IRAP-Binding
S588A/T642A [AS160(AA)] and S588D/T642D [AS160(DD)] AS160 double mutants were generated to assess the dependence of the interaction between AS160 and IRAP on phosphorylation state. AS160(AA) was designed to mimic AS160 in an unphosphorylated state and is equivalent to that demonstrated to inhibit the majority of GLUT4 translocation (27), and the corresponding AS160(DD) mutant was designed to mimic the phosphorylated form of AS160 with respect to charge. IRAP-myc was successfully coimmunoprecipitated with AS160(AA) or AS160(DD). Moreover, the relative amount of IRAP coimmunoprecipitated by each mutant was not significantly different (Fig. 6Go), suggesting colocalization was not dependent on the phosphorylation state of Ser588 and Thr642 of AS160.


Figure 6
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  		Fig. 6. Effect of Mutations in Akt Phosphorylation Sites of AS160 on IRAP Binding

Lysates of HEK293T cells transiently expressing AS160(AA) alone (A), AS160(DD) alone (D), IRAP alone (I), IRAP-myc and AS160(AA) (A+I), or IRAP-myc and AS160(DD) (D+I) were coimmunoprecipitated with anti-V5 antibody to compare the amount of IRAP bound to each AS160 phosphorylation mutant (see Materials and Methods for details). Each immunoprecipitate was resolved by SDS-PAGE, and Western blots were probed with anti-myc (top panels) or anti-V5 (lower panels) and developed using ECL. To ensure equivalent levels of expression of each protein, cell lysates were also immunoblotted (data not shown). The immunoblot shown is representative of four independent experiments. IP, Immunoprecipitation; WB, Western blot.

 
To support this finding, active recombinant Akt was used to phosphorylate AS160-V5 immunoprecipitated with IRAP-myc (Fig. 7Go). As shown in Fig. 7Go, phosphorylation of AS160 by Akt did not elute AS160 from the immunoprecipitated complex. Therefore, despite the increased level of AS160 phosphorylation over basal, demonstrated by phospho Akt substitute (PAS) immunostaining, the amount of IRAP associated with AS160 remained unchanged.


Figure 7
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  		Fig. 7. Effect of Akt Phosphorylation of AS160 on IRAP Binding

Lysates from transfected HEK293T cells expressing AS160-V5 and IRAP-myc were immunoprecipitated using anti-V5 antibody. Immunoprecipitates were treated with 0.1 µg active recombinant Akt (+Akt), or not (–Akt), for 30 min at 30 C as described in Materials and Methods. Coimmunoprecipitates were resolved by SDS-PAGE and transferred to nitrocellulose for immunoblotting. The representative blot shown was probed with anti-myc for IRAP (upper panel), anti-PAS for phosphorylated AS160-V5 (center panel), and anti-V5 for AS160-V5 (lower panel). The blot was stripped as described in the legend to Fig. 4Go between antibody applications. Similar results were obtained in seven separate experiments. WB, Western blot; IP, immunoprecipitation.

 
Endogenous AS160 and IRAP were coimmunoprecipitated from lysates of 3T3-L1 adipocytes stimulated with or without insulin. Insulin treatment of adipocytes caused a significant increase in the amount of AS160 phosphorylated over basal levels; however, the amount of IRAP coimmunoprecipitated with AS160 remained unaltered (Fig. 8Go).


Figure 8
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  		Fig. 8. Effect of Insulin on the Interaction between AS160 and IRAP in 3T3-L1 Adipocytes

Lysates from 3T3-L1 adipocytes treated, or not, with 100 nM insulin for 20 min at 37 C were immunoprecipitated with anti-AS160 (lanes 2 and 4) or nonspecific goat IgG (lanes 1 and 3) as described in Materials and Methods. Immunoprecipitates were resolved by SDS-PAGE, transferred to nitrocellulose, and probed with anti-IRAP (top panel), anti-PAS (center panel), or anti-AS160 (lower panel). All blots were developed using ECL. The immunoblots shown are representative of three separate experiments. WB, Western blot.

 
Confocal microscopy was used to investigate the subcellular localization of AS160 with respect to IRAP and GLUT4 in basal and insulin-stimulated 3T3-L1 adipocytes (Fig. 9Go). IRAP and GLUT4 demonstrate typical punctate staining in the cytoplasm with areas of concentrated staining adjacent to the nucleus. Insulin caused a marked increase in expression of these proteins at the cell membrane. AS160 produced a diffuse staining pattern throughout the cytoplasm with areas of concentrated staining adjacent to the nucleus in both basal and insulin-stimulated cells. AS160 shows a high degree of colocalization with both IRAP and GLUT4 in the juxtanuclear region and some overlap in the cytoplasmic puncta in both unstimulated and stimulated cells.


Figure 9
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  		Fig. 9. Effect of Insulin on Colocalization of AS160 with IRAP or GLUT4

3T3-L1 adipocytes were treated with 150 nM insulin (for 15 min at 37 C) and then immunostained with mouse anti-IRAP (A), or rabbit anti-GLUT4 (B) and goat anti-AS160 antibodies. Images were merged to demonstrate colocalization (yellow) of AS160 and IRAP (A) or AS160 and GLUT4 (B). Secondary antibody conjugates were antimouse-488 (green), antigoat-Cy5 (red), antigoat-488 (green), and antirabbit-Cy5 (red). A detailed description of the procedure is in Materials and Methods.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
This study identified an interaction between the Akt substrate, AS160, and IRAP, a marker of GLUT4 vesicles, providing a direct link between the insulin signaling cascade and GLUT4 storage vesicles. Until recently, activation of Akt was the most distal step in the signaling cascade by which insulin stimulates GLUT4 translocation and glucose uptake (30). More recently, however, Lienhard and co-workers (26, 27) identified a substrate of Akt, they called AS160, that provides an attractive candidate protein for mediating the effects of Akt on glucose transport. Data presented showed that AS160 contains several Akt phosphomotifs (RXRXXS/T) and has a Rab GAP domain (26, 27). Furthermore, mutation of AS160 in two or more of its Akt phosphorylation sites inhibits GLUT4 translocation, and this inhibition is abrogated by mutation of an arginine residue essential for Rab GAP activity (27, 28, 31). This suggests that phosphorylation of AS160 inhibits GAP activity and that these events are required for GLUT4 translocation. In addition, given the integral involvement of Rab proteins in membrane traffic (for review see Ref. 32), it was postulated that phosphorylation of AS160 might inhibit Rab GAP activity, thereby facilitating Rab-mediated GSV translocation to the plasma membrane (27). A number of in vitro Rab substrates of AS160 associated with GSVs have been identified, although the in vivo substrate of AS160 remains unknown (31).

Recent studies demonstrate that GLUT4 exocytosis, but not endocytosis, is dependent upon AS160 and that the Rab GAP activity of AS160 is essential for the intracellular retention of GLUT4 in basal 3T3-L1 adipocytes (28). Insulin stimulation results in AS160 phosphorylation in fat and muscle in a phosphatidylinositol 3-kinase-dependent manner (26, 27, 28, 29, 33) and has been demonstrated to cause the translocation of AS160 from the low-density microsome fraction to the cytosol in 3T3-L1 adipocytes. However, we demonstrate that insulin stimulation does not result in the dissociation of AS160 from IRAP. In addition, we found that phosphorylation of AS160 did not disrupt its interaction with IRAP, consistent with the absence of AS160 Akt phosphomotifs within the IRAP-binding sequence, AS160(367–439) (27, 28). Furthermore, the amount of AS160 colocalized with IRAP observed using confocal microscopy was unaltered after insulin treatment, although this may be due to the diffuse distribution of AS160 masking any change.

In support of our findings are recent results suggesting that insulin stimulation does not alter the level of AS160 found associated with GSVs (31). During the preparation of this manuscript Larance et al. (34) reported the dissociation of AS160 from GSVs after insulin stimulation. However, they did not demonstrate the movement of AS160 from the low-density membrane fraction to the cytosol upon insulin stimulation (34).

In conclusion, we demonstrate that AS160 interacts with the GSV marker, IRAP, identifying a direct link between insulin signaling and GLUT4 vesicles. Furthermore, we provide evidence that this interaction is not affected by insulin or phosphorylation of AS160 by Akt. Given these findings we propose the following: AS160 is associated with GSVs via its interaction with IRAP in the basal and insulin-stimulated states. This interaction facilitates GSV tethering in the basal state and/or GSV translocation to the plasma membrane after insulin stimulation by ensuring AS160 remains proximal to its substrate Rab proteins at the GSV.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Materials
All chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise stated

Antibodies
Primary and HRP-conjugated secondary antibodies were purchased from Chemicon (Temecula, CA) with the following exceptions: Mouse anti-IRAP amino terminus was a gift from M. Birnbaum (University of Pennsylvania, Philadelphia, PA) (35). Rabbit anti-IRAP carboxy terminus was a gift from S. Mizutani (RIKEN, Saitama, Japan) (36); goat anti-TBC1D4 (Anti-AS160) and rabbit anti-GLUT4 were purchased from Abcam (Cambridge, UK); rabbit anti-phospho-(Ser/Thr) Akt substrate (anti-PAS) was purchased from Cell Signaling Technology (Beverly, MA); goat antimouse-HRP conjugate was purchased from Bio-Rad Laboratories (Hercules, CA); mouse monoclonal anti-myc (Clone 9E10) was a gift from M. Morfis (Howard Florey Institute, Parkville, Australia); and anti-rabbit-Cy5, antigoat-488, antimouse-488, and antigoat-Cy5 were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA).

Cell Culture
3T3-L1 fibroblasts (37, 38, 39), HEK293T cells, and SN56 cells (40) were grown in maintenance medium (DMEM; Thermo Electron Corp., Melbourne, Australia) containing 10% fetal bovine serum (Invitrogen, Carlsbad, CA), 2 mM L-glutamine (Thermo), 50 U/ml penicillin (Thermo), and 50 mg/ml streptomycin (Thermo) in a humidified incubator at 37 C in 5% CO2.

3T3-L1 fibroblasts were differentiated as described previously (41) except that maintenance media contained fetal bovine serum, cells were grown in 5% CO2, 0.5 mM 3-isobutyl-1-methylxanthine and 0.25 µM dexamethasone were removed after 3 d, and 5 U/ml insulin (Novo Nordisk, Bagsvaerd, Denmark) media was maintained and changed daily for 3 d. Cells were used between d 8 and 10.

Plasmids
pGEX-2T was purchased from Amersham Biosciences (Buckinghamshire, UK); pcDNA3.2/V5-DEST and pENTR/D-TOPO were purchased from Invitrogen; pGEX-5X-3-IRAP (2–109) cDNA was a gift from S. Keller (11) and was used to clone IRAP(1–109) into pETGEXCT (42) by L. Castelli (Commonwealth Scientific and Industrial Research Organization Molecular and Health Technologies, Parkville, Australia); pBluescript II SK(+)-AS160 was a gift from T. Nagase (Kazusa DNA Research Institute, Chiba, Japan) (43); pCR3.1/2myc-DEST was a gift from G. Christopoulos (Howard Florey Institute, Parkville, Australia), and pIRAP-myc was a gift from H. Lodish (Massachusetts Institute of Technology, Cambridge, MA) (44). Plasmid preparations were performed using a Promega Wizard Plus SV Minipreps DNA Purification System (Promega Corp., Madison, WI) or a Qiagen QIAfilter Plasmid Maxi Kit (QIAGEN, Valencia, CA) according to manufacturer’s instructions.

Cloning and Mutagenesis
Gateway technology (Invitrogen) was used according to manufacturer’s instruction. Briefly, the entry clone, pENTR/D-AS160, was generated using pBluescript II SK(+)-AS160 as template to generate a CACC-tagged PCR product for cloning into pENTR/D-TOPO using a pENTR/D-TOPO cloning kit (Invitrogen). The destination vectors pcDNA3.2/V5-DEST (Invitrogen) or pCR3.1/2myc-DEST (in-house) were used for expression of AS160 and all mutations thereof fused C-terminally to V5 or 2myc, respectively. AS160 phosphorylation mutants were generated using a QuikChange Site-Directed mutagenesis kit (Stratagene, La Jolla, CA) to mutate Ser588 and Thr642 to alanines [AS160(AA)] or aspartic acids [AS160(DD)], respectively. AS160 truncation mutants (119-, 189-, 367-, and 439-1299) were produced by PCR.

Nucleotide Sequencing
Fidelity of all plasmid constructs was confirmed by sense and antisense sequencing performed by the Australian Genome Research Facility (Parkville, Australia).

SDS-PAGE and Western Blotting
SDS-PAGE was performed as previously described (45) except a Bio-Rad Mini-PROTEAN II Electrophoresis Cell Assembly (Bio-Rad Laboratories, Hercules, CA) was used and gels were poured with a 4% stacking gel. Gels were run at 100 V for 2.5 h. Western transfers were performed at 100 V for 2 h using a Bio-Rad Mini Trans-blot Cell Assembly (Bio-Rad Laboratories) as described previously (46). Blots were developed using an enhanced chemiluminescence (ECL) kit (Amersham Biosciences), and chemiluminescence was recorded either on Hyperfilm enhanced chemiluminescence (ECL) (Amersham Biosciences) or using a Fujifilm LAS-1000plus system (Fujifilm, Tokyo, Japan).

GST/IRAP(1–109)-GST Expression and Purification
GST and IRAP(1–109)-GST were expressed in BL21 (DE3) Escherichia coli and purified on Glutathione Sepharose 4B (Amersham Biosciences) as per manufacturer’s instructions.

Preparation of Cell Lysates for Identification of IRAP-Binding Proteins
Maintenance media was removed from 25 T175 flasks of SN56 cells at 80% confluence, and cells were washed twice with PBS. Cells were scraped into 5 ml of ice-cold PBS and pelleted at 200 x g for 2 min at 4 C. Cells were lysed by resuspending in 2 ml hypotonic buffer (50 mM Tris, pH 7.5) and homogenizing for 10 sec on ice using an IKA-WERK Ultra-Turrax fitted with an IKA S25N-10G dispersing tool. Lysate was centrifuged at 600 x g for 10 min at 4 C to remove cell nuclei. Two milliliters of 2x concentrated solubilization buffer [50 mM Tris (pH 7.5) 200 mM NaCl, 2 mM EDTA, 2 mM EGTA, 10 mM dithiothreitol, 0.2 mM Na3VO4, 2 mM phenylmethylsulfonylfluoride, and 40 mM NaF containing 2% Triton X-100 and Complete protease inhibitors (Roche, Mannheim, Germany)] was added to the supernatant and incubated on ice for 15 min. Cell debris was sedimented by centrifugation at 30,000 x g for 10 min at 4 C, and the supernatant was retained on ice until required.

IRAP(1–109)-GST Affinity Isolation of Interacting Proteins
All steps were performed at 4 C or on ice. Glutathione Sepharose-bound GST and IRAP(1–109)-GST (200 µl) were blocked for 1 h in 2% BSA in Tris-buffered saline-Tween (TBST). Two 12.5-ml lots of cell lysate were precleared for 1 h with Glutathione Sepharose 4B and then added to either Glutathione Sepharose-bound GST (negative control) or IRAP(1–109)-GST blocked with 2% BSA in TBST. After binding at 4 C for 3 h, Glutathione Sepharose-bound proteins were washed five times with ice-cold solubilization buffer, eluted in 200 µl of sodium dodecyl sulfate (SDS) sample buffer, and resolved by SDS-PAGE. Coomassie-stained protein bands specific to IRAP(1–109) were excised from dried gels and sent for identification by tandem mass spectrometry.

For cell-free translated proteins, the reaction product was diluted to 300 µl with solubilization buffer and 100-µl aliquots were added to blocked GST or IRAP(1–109)-GST. Binding was at room temperature for 1 h, and bound proteins were transferred to nitrocellulose (AS160-myc) or dried and phosphorimaged (Fig. 5Go).

MS/MS
MS/MS was performed by Proteomics International P/L (Perth, Australia).

Cell-Free Translation
Cell-free translation was performed using a Quick Coupled Transcription/Translation Systems kit (Promega) as per manufacturer’s instructions.

Mammalian Cell Transfection
HEK293T cells were transfected using Lipofectamine Transfection Reagent (Invitrogen) according to manufacturer’s instructions.

Coimmunoprecipitations
All cell and lysate manipulations were performed on ice or at 4 C. Cells were solubilized in lysis buffer [40 mM HEPES (pH 7.5), 150 mM NaCl, 2 mM EDTA, 50 mM NaF, 1% Triton X-100, and Complete protease inhibitors (Roche)] for 15 min, clarified by centrifugation at 30,000 x g for 10 min, and the resulting supernatant was precleared for 1 h with Protein G Sepharose (Amersham Biosciences). Immunoprecipitations were performed at 4 C for 2 h with Protein G coupled to 5 µg antibody blocked with 2% BSA in TBST. Sepharose-bound proteins were washed three times with lysis buffer, resuspended in 40 µl of SDS sample buffer, resolved by SDS-PAGE, and transferred to nitrocellulose.

Phosphorylation of AS160 with Active Recombinant Akt
AS160-V5 and IRAP-myc were coimmunoprecipitated from transfected HEK293T cell lysates. Immunoprecipitates were equilibrated in 40 µl kinase reaction buffer (25 mM Tris, pH 7.4; 5 mM glycerol 2-phosphate; 2 mM dithiothreitol; 0.1 mM Na3VO4; 10 mM MgCl2; 0.1 mM ATP). Active recombinant Akt (0.1 µg) (Upstate Cell Signaling Solutions, Lake Placid, NY) in Akt buffer (50 mM Tris pH 7.5, 100 mM NaCl, 0.1% 2-mercaptoethanol, 0.1 mM EGTA, 270 mM sucrose, 1 mM benzamidine, 0.2 mM phenylmethylsulfonylfluoride, 150 mM imidazole, 0.03% Brij-35) or Akt buffer alone was added to separate immunoprecipitates. The reaction was incubated at 30 C for 30 min and then placed on ice before washing three times with kinase reaction buffer. Protein A Sepharose-bound proteins were resuspended in 40 µl SDS sample buffer resolved by SDS-PAGE and transferred to nitrocellulose.

Confocal Microscopy
3T3-L1 adipocytes on ethanol-washed, glass coverslips in six-well plates were washed twice with PBS and then incubated in serum-free maintenance media for 3 h. Cells were treated with or without 100 nM insulin for 20 min at 37 C, 5% CO2 and then placed on ice and washed twice with ice-cold PBS. Cells were fixed in methanol at –20 C for 5 min, washed three times with PBS, and blocked with 10% donor horse serum in PBS. Immunostaining and confocal microscopy were performed as described previously (47). Primary antibody dilutions used were 1:1000 for mouse anti-IRAP and rabbit anti-GLUT4 and 1:500 for goat anti-AS160. Secondary fluorophore-conjugated antibodies were used diluted 1:1000.


   ACKNOWLEDGMENTS
 
We thank M. Birnbaum, M. Tsujimoto, and M. Morfis for gifts of antibodies; G. Christopoulos for pCR3.1/2myc-DEST; T. Nagase for AS160 cDNA; and S. Keller and H. Lodish for IRAP plasmid constructs.


   FOOTNOTES
 
This research was supported by the Robert J. Kleberg, Jr., and Helen C. Kleberg Foundation (San Antonio, TX) and Deutsche Bank Australia (Sydney, Australia).

Disclosure statement: The authors have nothing to disclose.

First Published Online June 8, 2006

Abbreviations: AS160, Akt substrate of 160 kDa; ECL, enhanced chemiluminescence; GAP, GTPase-activating protein; GLUT4, glucose transporter 4; GST, glutathione S-transferase; GSV, GLUT4 storage vesicle; HEK, human embryonic kidney; IRAP, insulin-regulated aminopeptidase; MS/MS, tandem mass spectrometry; PAS, Phospho Akt Substrate; SDS, sodium dodecyl sulfate; TBST, Tris-buffered saline-Tween.

Received for publication November 28, 2005. Accepted for publication May 26, 2006.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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