©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The B Isoform of the Insulin Receptor Signals More Efficiently Than the A Isoform in HepG2 Cells (*)

(Received for publication, December 23, 1994; and in revised form, April 20, 1995)

Atsushi Kosaki (§) Tahir S. Pillay (¶) Lan Xu Nicholas J. G. Webster (**)

From the Department of Medicine, Division of Endocrinology and Metabolism, University of California, San Diego, La Jolla, California 92093 and the Medical Research Service, Department of Veterans Affairs Medical Center, San Diego, California 92121

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We have demonstrated previously that dexamethasone treatment of HepG2 cells caused an enhancement of insulin's metabolic effects (Kosaki, A., and Webster, N. J.(1993) J. Biol. Chem. 268, 21990-21996). This correlated with increased expression of the mRNA encoding the B isoform of the insulin receptor (IR).

In the present study, we have demonstrated that dexamethasone treatment caused in addition an enhancement in insulin-stimulated immediate-early gene expression (c-fos and egr-1). Dexamethasone treatment caused an increase in in vivo IR autophosphorylation and insulin receptor substrate-1 (IRS-1) phosphorylation both early events in the insulin signaling pathway. Furthermore, the IRS-1 phosphorylation was distinctly left shifted, although the level of IRS-1 protein was unchanged. Total cellular tyrosine phosphatase activity was unaltered when assayed with P-labeled IR and IRS-1. Studies in vitro on wheat-germ agglutinin-purified receptors showed that the B isoform of the IR had increased kinase activity both toward itself and the exogenous substrates poly-glu(4):tyr(1) and recombinant IRS-1 protein. In addition, two-dimensional tryptic phosphopeptide maps indicated that the B isoform has an additional phosphopeptide that is not seen for the A isoform.

In conclusion, it appears that the B isoform of the IR signals more efficiently than the A isoform in HepG2 cells.


INTRODUCTION

The insulin receptor (IR) (^1)protein is a heterotetrameric protein composed of two alpha-subunits that confer the ability to bind insulin and two beta-subunits that contain the membrane spanning and the tyrosine kinase domains(1, 2, 3) . The alpha-subunit is entirely extracellular and is linked by disulfide bounds to the extracellular portion of the beta-subunit. Following binding of insulin to the alpha-subunits, the first observable event is autophosphorylation on the cytoplasmic portion of the beta-subunit leading to an increase in the receptor's intrinsic tyrosine kinase activity toward other substrates(1, 4, 5) . The human IR is encoded by a single gene that is located on chromosome 19 and composed of 22 exons(6) . The mature IR, however, exists as two isoforms, designated A and B, which result from alternative splicing of the primary transcript(6, 7, 8) . The A isoform lacks exon 11, is expressed ubiquitously, and is the only isoform in lymphocytes, brain, and spleen; the B isoform contains exon 11 and is expressed predominantly in liver, muscle, adipocytes, and kidney (9, 10, 11, 12) . Exon 11 is composed of 36 nucleotides that encode a 12-amino-acid segment (residues 717-728) of the carboxyl terminus of the alpha-subunit of IR. It has been reported that the affinity of the A isoform for insulin is higher than that of the B isoform causing a left shift in the insulin dose-response curves for insulin stimulation of autophosphorylation, glycogen synthesis, and thymidine uptake(12, 13, 14) . Two groups found that the A isoform exhibits a higher insulin internalization and recycling rate than the B isoform(14, 15) , whereas a third found no difference in either the rate of internalization or the rate of degradation(13) . However, all of these studies have been performed in Chinese hamster ovary or Rat-1 fibroblast cell lines, which are not good models for insulin-sensitive tissues. The fact that the B isoform is expressed primarily in insulin-sensitive tissues indicates that the B isoform of the IR must play an important role in signaling in insulin-sensitive tissues.

The HepG2 cell is derived from a human hepatoblastoma and has been useful as a model for liver one of the major sites of insulin action (16, 17) . We have shown previously that dexamethasone (dex) causes an increase in sensitivity and responsiveness for insulin's metabolic effects (glucose incorporation into glycogen and 2-deoxyglucose transport) in these cells. This correlated with a switch in expression from the A (-exon 11) to the B (+exon 11) isoform of the IR similar to the ratio seen in adult liver(18) . The aim of the present study was to investigate whether similar enhancements would be seen for insulin's mitogenic effects and to determine whether the expression of the B isoform of the IR is responsible for the enhancement. We show that dex treatment does cause an enhancement in insulin-stimulated immediate-early gene expression (c-fos and egr-1). The B isoform couples more efficiently to the insulin receptor substrate 1 (IRS-1) in vivo than the A isoform. Furthermore, insulin-stimulated autophosphorylation and kinase activity in vitro are greater for the B isoform of the IR. Tryptic peptide mapping identified a novel phosphopeptide in the B isoform that may be involved in the enhanced kinase activity and/or signaling.


EXPERIMENTAL PROCEDURES

Materials

Monocomponent pork insulin was kindly provided by Eli Lilly (Indianapolis, IN). Cell culture reagents were purchased from Life Technologies Inc., and calf serum and fetal calf serum were from Gemini Bioproducts (Calabasas, CA). [alpha-P]dCTP (3,000 Ci/mmol) and [-P]ATP (4,500 Ci/mmol) were purchased from ICN (Costa Mesa, CA). Anti-IR antibodies (83-14 and against COOH-terminal peptide) were kindly provided by Dr. K. Siddle (Cambridge, United Kingdom). Polyclonal anti-IRS-1 antibodies for immunoblotting and immunoprecipitation were kindly provided by Dr. C. R. Kahn (Boston, MA) and Dr. H. Maegawa (Shiga, Japan), respectively. All other chemicals were purchased from Sigma or Fisher Scientific. Taq DNA polymerase (Amplitaq) was purchased from Perkin-Elmer-Cetus.

Cell Culture

HepG2 cells were maintained routinely in minimum essential medium plus Earle's salts with 10% fetal calf serum at 37 °C under 5% CO(2). The cells were plated at a density of 1 10^6 cells/well in 12-well plates. After 3 days, the medium was replaced with differentiation medium (Ham's F-12 plus 0.5% calf serum, 1 µM triiodothyronine, and 20 mM glucose) with and without 1 µM dexamethasone for a further 4 days. Medium was replaced every 2 days.

Reverse Transcription and Amplification of cDNA

Total cellular RNA was prepared using RNAzol B (Tel-Test, Inc., Friendswood, TX) according to the manufacturer's protocol. First-strand cDNA was prepared by reverse transcription using 0.5 µg of total RNA in a volume of 20 µl (250 pmol of random hexamer primers, 1 unit of Inhibit-ACE RNase inhibitor (5`-3`Inc., Boulder, CO), 200 units of Moloney murine leukemia virus reverse transcriptase (Life Technologies Inc.), 50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl(2), 1 mM dithiothreitol, and 1 mM dNTPs) at 42 °C for 1 h. DNA/RNA hybrids were denatured at 95 °C for 2 min.

The c-fos primer pair consisted of oligonucleotides spanning nucleotides 140-169 (sense primer, 5`-GTTCTCGGGTTTCAACGCGGACTACGAGGC-3`) and 276-309 (antisense primer, 5`-GGCACTAGAGACGGACAGATCTGCGCAAAAGTCC-3`) which generate a fragment of 169 base pairs following amplification. The egr-1 primer pair consisted of oligonucleotides spanning nucleotides 539-568 (sense primer, 5`-GAGCCGAGCGAACAACCCTACGAGCACCTG-3`) and 763-791 (antisense primer, 5`-GCGCTGAGGATGAAGAGGTTGGAGGGTTGG-3`) which generate a fragment of 252 base pairs following amplification. The L30 primer pair consisted of oligonucleotides spanning nucleotides 74-98 (sense primer, 5`-GAAAGTACGTGCTGGGGTACAAACAGACTC-3`) and either 285-309 (antisense primer for c-fos measurement, 5`-ATCGGAATCACCTGGGTCAATGATAGCCAG-3`) or 224-254 (antisense primer for egr-1 measurement, 5`-CCACACGCTGTGCCCAATTCAATGTTATTGC-3`) which generate a fragment of 238 and 181 base pairs, respectively. Five µl of the cDNA synthesis reaction was used for polymerase chain reaction (PCR)^1 amplification in a 50-µl final reaction volume (0.5 µM each oligonucleotide primer, 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl(2), 0.1 mM dNTPs, 2 units of Taq DNA polymerase, and 1 µCi of [alpha-P]dCTP). Twenty-five cycles of amplification were performed using a Perkin-Elmer DNA thermal cycler System 9600. Each cycle consisted of a 30-s denaturation at 94 °C, a 30-s annealing at 55 °C, and a 60-s extension at 72 °C. The number of cycles was optimized to ensure that the amplification lay within the exponential phase. The products of the PCR amplification were resolved by electrophoresis on 8% polyacrylamide gels. The gels were dried and exposed to film at room temperature. The band densities were quantified using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). The counts of c-fos and egr-1 were normalized to L30 as a internal standard.

In Vitro Autophosphorylation of IR

To measure in vitro autophosphorylation, IRs from HepG2 cells were partially purified by wheat germ agglutinin (WGA) affinity chromatography(19) . Equal amounts of IR were incubated with increasing concentrations insulin for 16 h at 4 °C, followed by the addition of 30 µCi of [-P]ATP in reaction buffer (25 mM HEPES, pH 7.4, 50 µM ATP, 5 mM MnCl(2), and 50 mM NaF) for 15 min at 4 °C. The reaction was terminated by the addition of equal volume of 50 mM ATP, 4 mM sodium orthovanadate, 200 mM NaF, 20 mM sodium pyrophosphate, 10 mM EDTA, and 25 mM HEPES, pH 7.6. The receptors were immunoprecipitated with anti-IR antibody ) for 16 h at 4 °C followed by precipitation with Pansorbin (Calbiochem). Phosphorylated beta-subunits were visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) and autoradiography and were quantified with the PhosphorImager.

In Vitro Kinase Activity for Exogenous Substrates

The ability of IRs to phosphorylate the exogenous substrates poly-glu(4):tyr(1) and recombinant IRS-1 protein was determined(20) . Briefly, for the poly-glu(4):tyr(1) assay, equal amounts of WGA-purified receptor were preincubated with increasing concentrations of insulin for 16 h at 4 °C. The substrate poly-glu(4):tyr(1) was added for 15 min to a final concentration of 2 mg/ml. Phosphorylation was initiated by the addition of 5 µCi of [-P]ATP in reaction buffer (25 mM HEPES, pH 7.4, 50 µM ATP, 5 mM MnCl(2), and 12 mM MgCl(2), final concentration) for 30 min at 4 °C. The labeling was terminated by the addition of 10 µl of cold ATP (70 mM, final concentration). Then, the mixture was applied to Whatman 3MM paper, washed with 10% trichloroacetic acid, 10 mM sodium pyrophosphate and counted in a liquid scintillation counter.

For the IRS-1 phosphorylation assay, equal amounts of WGA-purified receptor were preincubated with increasing concentrations of insulin for 16 h at 4 °C, followed by the addition of 30 µCi of [P]ATP in reaction buffer (25 mM HEPES, pH 7.4, 50 µM ATP, 5 mM MnCl(2), and 50 mM NaF) for 15 min at 23 °C. Recombinant IRS-1 protein (0.3 µg) was added and the incubation continued for a further 90 min at 23 °C. The reaction was terminated by the addition of an equal volume of 50 mM ATP, 4 mM sodium orthovanadate, 200 mM NaF, 20 mM sodium pyrophosphate, 10 mM EDTA, and 25 mM HEPES, pH 7.6. The IRS-1 proteins were immunoprecipitated with an anti-IRS-1 antibody for 16 h at 4 °C followed by precipitation with Pansorbin. Phosphorylated IRS-1 proteins were visualized by SDS-PAGE and autoradiography and were quantified using the PhosphorImager.

Anti-phosphotyrosine Immunoblotting

Cells were grown to confluence in 12-well plates. Cells were stimulated with increasing concentrations of insulin in Krebs-Ringer phosphate-HEPES buffer (131 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl(2), 1.2 mM MgSO(4), 2.5 mM NaH(2)PO(4), 10 mM HEPES, pH 7.5, 0.1% bovine serum albumin (BSA)) for 1 min at 37 °C. The cells were washed with ice-cold phosphate-buffered saline and solubilized in 2 Laemmli's sample buffer (21) containing 2 mM sodium orthovanadate and 200 mM sodium fluoride. The proteins were denatured by boiling for 5 min, then were separated by electrophoresis on 7.5% SDS-PAGE, and transferred to Immobilon membranes (Millipore, Bedford, MA). The filter was blocked with 3% BSA in T-TBS (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% Tween-20) for 30 min and incubated with mouse monoclonal antibody PY-20 (ICN) in blocking buffer for 2 h. The filter was washed with T-TBS for 30 min and then incubated with a sheep anti-mouse horseradish peroxidase-conjugated antibody (Amersham Corp.) for 30 min. After washing with T-TBS for 60 min, a chemiluminescent detection kit (ECL, Amersham Corp.) was used to visualize the tyrosine-phosphorylated proteins. The band densities were quantified using a Stratascan-7000 densitometer (Stratagene, La Jolla, CA).

Anti-IR and -IRS-1 Immunoblotting

Proteins in WGA-purified receptors or total cellular extracts were separated by electrophoresis on 7.5% SDS-PAGE and transferred to Immobilon membranes. The filters were blocked with 5% BSA in TBS and incubated with polyclonal antibodies against the COOH terminus of either the IR (alpha-IRCt) or IRS-1 (alpha-IRS-1). The filters were washed in T-TBS for 30 min, incubated with sheep anti-rabbit horseradish-peroxidase conjugated antibody (Amersham) in blocking buffer for 60 min, washed for 60 min in T-TBS, then the proteins were visualized using the ECL chemiluminescent kit (Amersham).

Insulin Binding

Insulin binding was measured as described previously(18) . Cells were incubated with 33.3 pMI-insulin in Krebs-Ringer phosphate-HEPES buffer for 3 h at 12 °C. The cells were washed in ice-cold phosphate-buffered saline, solubilized, and counted. Nonspecific insulin binding was determined in the presence of 1 µM insulin. Binding to WGA-purified receptors was performed as published previously(22) . WGA-purified protein was incubated with 33.3 pMI-insulin for 16 h at 4 °C in a solution containing 50 mM HEPES, pH 7.5, 150 mM NaCl, 0.1% Triton X-100, and 0.1% BSA. Free and bound insulin were separated by polyethylene glycol precipitation.

Two-dimensional Tryptic Phosphopeptides Mapping

Polyacrylamide-gel pieces containing P-labeled IR and IRS-1 following in vitro phosphorylation were excised and electroeluted in 20 mM Tris-HCl, pH 8.0, 2 mM EDTA, 0.1% SDS, and 0.1% 2-mercaptoethanol for 4 h. Eluted protein was precipitated with 4 volumes of acetone at -80 °C for 60 min followed by centrifugation at room temperature for 10 min at 10,000 g. The pellet was dried and digested with 10 µg of TPCK-treated trypsin (Worthington Diagnostic Systems, Freehold, NJ) in 100 µl of 100 mMN-ethylmorpholine acetate (NMA), pH 8.2, for 24 h at 37 °C. A further 10 µg of TPCK-treated trypsin was added and digestion continued for 12 h. The peptides were lyophilized, resuspended with water, and relyophilized at least three times. The P-labeled tryptic peptides were then resuspended in 5 µl of electrophoresis buffer and spotted onto thin layer cellulose plates. High voltage electrophoresis was performed in 1:3.5:40.5 formic acid/acetic acid/water, pH 1.9, using a Hunter thin layer electrophoresis system (C. B. S. Scientific, Del Mar, CA). Plates were subjected to ascending thin layer chromatography in the second dimension in 75:15:50:60 n-butanol/acetic acid/pyridine/water, dried, and then subjected to autoradiography at -80 °C(23) .

For the immunoprecipitation study, the P-labeled IR was resuspended in 100 µl of 100 mM NMA, pH 8.3, following trypsin digestion. The sample was boiled for 5 min and 0.5 mM phenylmethylsulfonyl fluoride added to inactivate any remaining trypsin. The digested receptor was incubated with an anti-COOH-terminal peptide (residues 1322-1341) antibody for 16 h and then with Protein A-Sepharose for 2 h in 500 µl of NMA at 4 °C. Sepharose-bound antibody was recovered by centrifugation and washed twice with 1 ml of NMA. Phosphopeptides were eluted by resuspending the complex in 500 µl of 1 M acetic acid containing 10 µg of the cold carrier peptide, and mixing at 4 °C for 30 min. Acetic acid was removed by rotary evaporation, and the phosphopeptide was washed extensively with water before analysis by two-dimensional mapping as described above(24) .

Protein Tyrosine Phosphatase Activity

Protein tyrosine phosphatase activity was measured using P-labeled WGA-purified IR and recombinant IRS-1(25) . WGA-purified IR and recombinant IRS-1 were labeled with [-P]ATP as described above. The P-labeled IR and IRS-1 were immunoprecipitated with anti-IR) and anti-IRS-1 antibodies for 16 h at 4 °C followed by precipitation with Pansorbin and washed with EBG-buffer (25 mM HEPES, pH 7.4, 120 mM NaCl, 5 mM KCl, 1 mM MgSO(4), 1 mM MgCl(2), 1 mM CaCl(2), 0.05% Triton X-100, and 10% glycerol). For the preparation of whole cell homogenates, cells were rinsed with phosphate-buffered saline, sonicated for 20 s in 10 volumes of buffer (25 mM imidazole, pH 7.2, 2 mM EGTA, 2 mM EDTA, 0.1% beta-mercaptoethanol, 2 mM MgCl(2), 2.1 mM benzamidine, 0.025% phenylmethylsulfonyl fluoride, 2 µg/ml leupeptin, 20 µg/ml aprotinin, 250 mM sucrose, 10 mM dithiothreitol, and 2% Triton X-100), and set on ice for 30 min. Insoluble proteins were removed by centrifugation at 14,000 g for 10 min. Supernatants (7.1 µg protein/80 µl) were incubated with P-labeled IR and IRS-1 at 30 °C for the indicated period. The reaction was terminated by addition of Laemmli's sample buffer and boiled for 5 min. Dephosphorylated IR and IRS-1 were separated by SDS-PAGE and visualized by autoradiography.


RESULTS

Effect of Dexamethasone on Insulin-stimulated c-fos and egr-1 Gene Expression

We have published previously that dexamethasone treatment of HepG2 cells causes an increase in insulin sensitivity for glucose transport and glucose incorporation into glycogen(18) . To determine whether insulin's mitogenic effects were similarly enhanced, we attempted to measure insulin-stimulated thymidine incorporation. However, we were unable to measure any effect of insulin due to high basal incorporation (data not shown). This appears to be a characteristic of HepG2 cells as they continue to proliferate in serum-free medium. Consequently, we measured insulin-stimulated c-fos and egr-1 expression as components of the mitogenic pathway. Reverse transcription and amplification by PCR (RT-PCR) of total cellular RNA was used to measure c-fos and egr-1 mRNA levels following stimulation by insulin. The ribosomal L30 protein mRNA was co-amplified as an internal control (Fig. 1, A and C). In the cells cultured with dex, maximal insulin stimulation was 1.6-fold higher for both c-fos (p < 0.01, n = 4) and egr-1 (p < 0.02, n = 4) gene expression. Moreover, in both cases the ED for insulin stimulation was distinctly left shifted in the cells cultured with dexamethasone (Fig. 1, B and D; ED 9.8 to 5.6 nM for c-fos, p < 0.05, n = 4; 5.0 to 2.1 nM for egr-1, p < 0.02, n = 4).


Figure 1: Insulin-stimulated c-fos and egr-1 gene expression. HepG2 cells were cultured with and without 1 µM dexamethasone (Dex) in Ham's F-12 mixture for 4 days and stimulated with increasing concentrations of insulin for 45 min at 37 °C. The total cellular RNA was extracted and subjected to RT-PCR to determine c-fos and egr-1 gene expression using the ribosomal protein L30 mRNA as internal standard. Panel A, insulin stimulation of c-fos gene expression. B, insulin dose-response curves for c-fos expression. Results are the mean ± S.E. of four experiments and are normalized to L30 mRNA levels. Maximal insulin stimulation is 1.6-fold higher in the presence of dexamethasone (p < 0.01, n = 4). The ED values were 9.8 and 5.6 nM for cells cultured without and with dex, respectively, and are indicated by arrows (p < 0.05, n = 4). Panel C, Insulin stimulation of egr-1 gene expression. D, insulin dose-response curves for egr-1 expression. Results are the mean ± S.E. of four experiments and are normalized to L-30 mRNA levels. Maximal insulin stimulation is 1.6-fold higher in the presence of dexamethasone (p < 0.02, n = 4). The ED values were 5.0 and 2.1 nM for cells cultured without and with dex, respectively, and are indicated by arrows (p < 0.02, n = 4).



In Vivo Autophosphorylation of IR and Kinase Activity

Results from the study of insulin receptor mutants generated in vitro have suggested that the insulin-stimulated metabolic and mitogenic pathways diverge at a very early point in the signaling pathway, perhaps at the receptor itself. As dex enhances components of both the metabolic and mitogenic pathways, we decided to look at very early steps in the insulin signaling pathway, namely receptor autophosphorylation and endogenous substrate phosphorylation in intact cells. Cells, cultured in the presence or absence of dex, were stimulated with increasing concentrations of insulin, and tyrosine phosphorylation was assessed on whole cell extracts by immunoblotting with an anti-phosphotyrosine antibody. Samples were normalized for insulin binding to allow a direct comparison of receptor autophosphorylation. Insulin stimulated the phosphorylation of two major proteins; one protein at 95 kDa corresponds to the IR beta-subunit and the other at 185 kDa to IRS-1 (Fig. 2A). In spite of loading equal numbers of receptors, maximal insulin stimulation of IR autophosphorylation was 1.5-fold higher in the cells treated with dex (Fig. 2B, p < 0.01, n = 8). A smaller but significant 1.3-fold increase in IRS-1 phosphorylation was observed also (Fig. 2C, p < 0.01, n = 8). Eight independent pair-matched experiments were performed on different passages of cells and at different times to ensure significance. The insulin sensitivity for IR autophosphorylation was unchanged (Fig. 2B). However, the ED for insulin-stimulated phosphorylation of IRS-1 was distinctly left-shifted in the cells treated with dex (5.6 to 2.7 nM) (Fig. 2C, p < 0.04, n = 8). This is most easily seen by comparing lanes 2 and 3 with 8 and 9 in Fig. 2A. Greater phosphorylation is evident at both 1 and 3 nM insulin in the presence of dex.


Figure 2: Insulin-stimulated tyrosine phosphorylation in whole cells. HepG2 cells were cultured with and without 1 µM dex (Dex) in Ham's F-12 mixture for 4 days and stimulated with increasing concentrations of insulin for 1 min at 37 °C. Cells were solubilized and aliquots, equalized for either insulin binding (panels A-C) or cell number (panels D and E), were separated by SDS-PAGE, and transferred to an Immobilon-P membrane. Tyrosine phosphorylated proteins, IR, and IRS-1 were visualized by immunoblotting followed by ECL detection. The band densities were quantified by densitometry. Panel A, anti-phosphotyrosine immunoblot. The beta-subunits of the IR and IRS-1 are indicated by arrows. Panel B, insulin dose-response curves for autophosphorylation of IR beta-subunits. Results are the mean ± S.E. of eight experiments. Maximal insulin stimulation is 1.5-fold higher in the presence of dex (p < 0.01, n = 8). The ED values of the curves were 5.5 and 8.8 nM for cells cultured without and with dex, respectively, and are indicated by the arrows (p > 0.05, n = 8). Panel C, insulin dose-response curves for IRS-1 phosphorylation. Results are the mean ± S.E. of eight experiments. Maximal insulin stimulation is 1.3-fold higher in the presence of dex (p < 0.01, n = 8). The ED values of the curves were 5.4 and 2.6 nM for cells cultured without and with dex, respectively, and are indicated by arrows (p < 0.04, n = 8). Panel D, effect of dex on IR protein levels. IRs were visualized using a polyclonal anti-IR antibody. Dex treatment causes a 1.5-fold increase in IR level. Panel E, effect of dex on IRS-1 protein levels. IRS-1 was visualized using a polyclonal antibody to IRS-1. Dex treatment has no effect on IRS-1 levels.



One possible explanation for the increase in sensitivity and maximal phosphorylation of IRS-1 seen in cells treated with dex is that the level of IRS-1 protein has increased. Consequently, we measured IR and IRS-1 levels by immunoblotting. Cell lysates from equal numbers of the cells were immunoblotted with a polyclonal anti-IR antibody (Fig. 2D). The amount of IR was 1.5-fold higher in cells treated with dex similar to the increment in insulin binding that we reported previously(18) . Immunoblotting with an anti-IRS-1 antibody indicated that dex treatment did not alter the amount of IRS-1 protein (Fig. 2E). It should be noted that the increase in IR protein is not the origin of the increased in vivo autophosphorylation as extracts were normalized for insulin binding prior to antiphosphotyrosine immunoblotting (Fig. 2A). However, IRS-1 levels are unaffected by dex treatment so less IRS-1 is loaded in the antiphosphotyrosine immunoblot for cells treated with dex. When adjusted for equal cell number and therefore equal IRS-1 levels, the maximal insulin-stimulated phosphorylation of IRS-1 is 2.2-fold higher in cells treated with dex rather than the 1.3-fold shown in Fig. 2A (data not shown).

In Vitro Autophosphorylation of IR

The increase in maximal autophosphorylation of the IR seen in whole cells could be a result of an increase in the inherent kinase activity of the receptor itself. Therefore, we determined the ability of insulin to stimulate autophosphorylation of purified IRs. Receptors were partially purified from cells, untreated or treated with dex, by WGA affinity chromatography and then equal numbers of IRs, adjusted by insulin binding, were subjected to in vitro autophosphorylation. As shown in Fig. 3A, insulin stimulated autophosphorylation of the IR beta-subunit in a dose-dependent manner. In the cells treated with dex, however, maximal insulin stimulation was 1.7-fold higher (p < 0.01, n = 4) with no change in sensitivity (ED: 9.8 nM for -Dex; 10.2 nM for +Dex, Fig. 3B). This is similar to the results obtained by antiphosphotyrosine immunoblotting of whole cell extracts (Fig. 2B). We verified that we had loaded equal amounts of IR by immunoblotting (Fig. 3C). Previously, we have shown that treatment of HepG2 cells with dex causes a switch in isoform expression from 20:80 to 80:20 A:B by RT-PCR. Assuming that the protein expression mirrors the mRNA ratio, these numbers can be used to determine the activities of each isoform. This analysis predicts that the B isoform incorporates 2.4-fold more P. This is in agreement with the results of Kellerer et al.(26) who documented a 2.5-fold increase in P incorporation for the B isoform IR purified from Rat 1 fibroblasts.


Figure 3: In vitro autophosphorylation of IR isoforms. HepG2 cells were cultured with and without 1 µM dex (Dex) in Ham's F-12 mixture for 4 days. IRs were purified by WGA affinity chromatography. Equal numbers of IRs, normalized by insulin binding, were subjected to in vitro autophosphorylation in the presence of [-P]ATP. The receptors were immunoprecipitated with an anti-IR antibody for 16 h at 4 °C followed by precipitation with Pansorbin. Phosphorylated beta-subunits were visualized by SDS-PAGE and autoradiography and quantified on a PhosphorImager. Panel A, insulin stimulation of IR autophosphorylation. PanelB, insulin dose-response curves for in vitro autophosphorylation. Results are the mean ± S.E. of four experiments. Maximal insulin-stimulated autophosphorylation is 1.7-fold higher in the presence of dex (p < 0.01, n = 4). The ED values are indicated by arrows (ED: 9.8 nM for -Dex; 10.2 nM for +Dex). Panel C, partially purified IRs normalized for insulin binding were separated by SDS-PAGE, transferred to an Immobilon-P membrane and subjected to immunoblotting using an anti-IR antibody to verify equal receptor number.



In Vitro Kinase Activity of IR

To test whether the B isoform of the IR has enhanced kinase activity in vitro, phosphorylation assays with equal numbers of WGA-purified IRs, adjusted by insulin binding, were performed using either the exogenous substrate poly-glu(4):tyr(1) or recombinant IRS-1 protein. Insulin stimulated phosphorylation of both poly-glu(4):tyr(1) and recombinant IRS-1 protein in a dose-dependent manner (Fig. 4). In the cells treated with dex, however, maximal insulin-stimulated phosphorylation was 1.6- and 1.5-fold higher for poly-glu(4):tyr(1) (p < 0.01, n = 4; Fig. 4A) and recombinant IRS-1 protein (Fig. 4B), respectively, with no difference in sensitivity. Using the isoform ratios mentioned earlier, the contributions of the individual isoforms can be calculated. Thus the B isoform has 2.2- and 2.0-fold higher activity toward poly-glu(4):tyr(1) and IRS-1, respectively. These results parallel the increase in maximal phosphorylation of IRS-1 in vivo (Fig. 2C). However, no differences in sensitivity were observed for either substrate. Again these results agree with those of Kellerer et al.(26) who observed a 2.0-fold increase in GluTyr phosphorylation for the B isoform.


Figure 4: In vitro kinase activity of IR isoforms. HepG2 cells were cultured with and without 1 µM dex (Dex) in Ham's F-12 mixture for 4 days. WGA-purified IRs normalized for insulin binding were subjected to an in vitro kinase assay using poly-glu(4):tyr(1) or recombinant IRS-1 protein as a substrate. Panel A, insulin dose-response curves for phosphorylation of poly-glu(4):tyr(1) plotted as phosphorylation above basal versus insulin concentration. Samples were normalized for insulin binding. Results are the mean ± S.E. of four experiments. Maximal insulin-stimulated phosphorylation is 1.6-fold higher in the presence of dex (p < 0.01, n = 4). The ED values are 21.7 and 19.7 nM for -Dex and +Dex, respectively, and are indicated by the arrows. Panel B, insulin-stimulated phosphorylation of recombinant IRS-I. Equal numbers of IRs were used to phosphorylate recombinant IRS-1 in vitro. Phosphorylated IRS-1 proteins were immunoprecipitated then visualized by SDS-PAGE and autoradiography. A representative autoradiogram is shown. Maximal insulin-stimulated phosphorylation is 1.5-fold higher in the presence of dex. Panel C, insulin-stimulated of IR autophosphorylation. Following immunoprecipitation of the IRS-1 proteins, the supernatants were subjected to SDS-PAGE and autoradiography to visualize the IR autophosphorylation.



Two-dimensional Tryptic Phosphopeptide Maps of IR Isoforms

To determine whether the increased autophosphorylation of the IR following dex treatment is due to utilization of additional phosphorylation sites in the B isoform, two-dimensional tryptic phosphopeptide mapping was performed (Fig. 5). Equal numbers of WGA-purified IRs, adjusted by insulin binding, were allowed to autophosphorylate in the presence of [-P]ATP, purified by gel electrophoresis, digested with trypsin, and separated by electrophoresis and then ascending chromatography on a thin layer cellulose plate. Phosphopeptides A-D have been assigned by Tavare and Denton (23, 27) based on charge and digestion by V8 protease and are derived from the triple tyrosine region of the IR (tyrosines 1158, 1162, and 1163). Peptides from the tyrosine 960 region are more hydrophobic and run at a position higher than peptide B in the chromatographic axis. Peptide F has been assigned as the peptide containing the two tyrosines from the COOH terminus of the beta-subunit. Peptide G remains unassigned. The intensity of phosphopeptides A, B, C, D, and F were all increased in cells treated with dex. Moreover, an additional phosphorylated peptide E (Fig. 5B) was observed for the B isoform of the IR.


Figure 5: Two-dimensional tryptic phosphopeptide maps of IR isoforms. WGA purified IRs from cells cultured without (panel A) and with (panel B) dex were incubated with 100 nM insulin for 16 h at 4 °C, followed by the addition of 30 µCi of [P]ATP in reaction buffer for 30 min at 4 °C. The reaction was terminated by the addition of excess cold ATP with phosphatase inhibitors. The IRs were immunoprecipitated with an anti-IR antibody for 16 h at 4 °C followed by precipitation with Pansorbin and subjected to SDS-PAGE. Phosphorylated beta-subunits were eluted from gel and digested with TPCK-treated trypsin. P-Labeled tryptic phosphopeptides were separated on thin layer cellulose plates by electrophoresis at pH 1.9 followed by ascending chromatography. A representative autoradiogram is shown.



The position of phosphopeptide E suggested that it may be derived from the carboxyl terminus of the beta-subunit. We attempted to confirm this assignment by immunoprecipitation with an antibody that had been raised against the COOH-terminal peptide (residues 1322-1341) that contains the two known auto-phosphorylation sites (tyrosines 1328 and 1334) (Fig. 6). Peptide F was precipitated along with peptide G and a fraction of peptide D (Fig. 6B). However, none of fragment E was precipitated so it is unlikely the E is derived from the COOH terminus. Peptide D contains the doubly phosphorylated tryptic peptide (amino acids 1156-1168) having an approximate charge of +1.5 at pH 1.9. The marker dye dinitrophenyl lysine has a charge of +1.7 at pH 1.9. The doubly phosphorylated COOH-terminal peptide (amino acids 1327-1341) is predicted to have an approximate charge of +1.5 whereas the singly phosphorylated form has a charge of +2.5. Thus these two peptides should migrate either side of the dinitrophenyl lysine marker. Only a fraction of D is immunoprecipitated by the antibody, so D most likely represents a mixture of the doubly phosphorylated COOH terminus peptide (amino acids 1327-1341) and the doubly phosphorylated peptide from the triple tyrosine region (amino acids 1156-1168). The singly phosphorylated peptide derived from the COOH-terminal peptide is thus G by analogy with peptides A and B and C and D(27) . Phosphopeptide F is likely to be a doubly phosphorylated tryptic peptide that contains a single additional lysine or arginine residue due to incomplete cleavage (amino acids 1326-1341 or 1327-1342). This would cause the peptide to have an additional +1 charge. The identity of peptide E is unknown.


Figure 6: Immunoprecipitation of tryptic fragments of IR. IRs were labeled and digested as in Fig. 5. The tryptic-phosphopeptides were incubated with an anti-COOH-terminal peptide antibody for 16 h and then with Protein A-Sepharose for 2 h in 500 µl of 100 mM NMA at 4 °C. Sepharose-bound antibody was recovered by centrifugation and washed twice with 1 ml of NMA. Phosphopeptide was eluted by resuspending the complex in 500 µl of 1 M acetic acid containing 10 µg of the cold carrier peptide at 4 °C for 30 min. Acetic acid was removed by rotary evaporation and the phosphopeptide was washed extensively with water. Then P-labeled tryptic phosphopeptides were separated on thin layer cellulose plates by electrophoresis at pH 1.9 followed by ascending chromatography. A representative autoradiogram is shown. Panel A, total phosphopeptides. Panel B, immunoprecipitated phosphopeptides.



Two-dimensional Tryptic Phosphopeptide Maps of IRS-1

To determine whether IRS-1 is phosphorylated on different sites by the two receptor isoforms, we generated two-dimensional phosphopeptide maps of IRS-1 that had been phosphorylated in vitro by either isoform of the IR as above (Fig. 7). Phosphorylation by the B isoform of the IR causes greater P incorporation into IRS-1 (Fig. 4), and this is reflected by the increased intensity of the phosphopeptide spots (Fig. 7, A and B). However, no differences in the pattern of spots are observed suggesting that the receptors interact with IRS-1 in the same manner in vitro.


Figure 7: Two-dimensional tryptic peptide maps of IRS-1. Equal numbers of IRs from cells cultured without (panel A) and with (panel B) dex were preincubated with 100 nM insulin for 16 h at 4 °C, followed by the addition of 30 µCi of [P]ATP in reaction buffer for 20 min at 23 °C. Recombinant IRS-1 protein (0.3 µg) was added and the incubation continued for a further 90 min at 23 °C. The reaction was terminated by the addition of excess cold ATP with phosphatase inhibitors. The IRS-1 proteins were immunoprecipitated with anti-IRS-1 antibody for 16 h at 4 °C followed by precipitation with Pansorbin. Phosphorylated IRS-1 proteins were separated by SDS-PAGE, eluted from gel, and digested with TPCK-treated trypsin. P-Labeled tryptic phosphopeptides were separated on thin layer cellulose plates by electrophoresis at pH 1.9 followed by ascending chromatography. A representative autoradiogram is shown.



Protein Tyrosine Phosphatase Activity

The increase in insulin-stimulated whole cell phosphorylation in cells treated with dex could result from decreases in the activities of the tyrosine phosphatases that act on the IR or IRS-1. At the present time it is not known which phosphatases are involved. Given this limitation, we attempted to determine whether dex treatment induced any alterations in total tyrosine phosphatase activity using IRs and IRS-1 as substrates. Tyrosine phosphatase activity was measured on Triton X-100-soluble cellular extracts. B isoform IRs and recombinant IRS-1 were phosphorylated in vitro in the presence of [-P]ATP and immunoprecipitated. Cellular extracts were incubated with the P-labeled proteins for the indicated periods, boiled in sample buffer to stop the reaction, and the extent of dephosphorylation measured by SDS-PAGE and autoradiography (Fig. 8). Dex treatment had no effect on the extent of IR and IRS-1 dephosphorylation. This result suggests but does not prove that tyrosine phosphatase activity is not grossly altered by dex treatment. Identification of the phosphatases that can act on the IR and IRS-1 will be required before this question can be answered definitively.


Figure 8: Effect of dexamethasone on phosphatase activity in HepG2 Cells. Cells cultured with and without dex (Dex) were homogenized in lysis buffer (see ``Experimental Procedures''). After centrifugation, the Triton X-100-soluble total fraction was incubated with in vitroP-labeled IR and IRS-1 for indicated period at 30 °C. The reaction was terminated by boiling, and samples were separated by SDS-PAGE to determine dephosphorylation of IR and IRS-1. A representative autoradiogram is shown.




DISCUSSION

We have published previously that dexamethasone treatment of HepG2 hepatoma cells causes a switch in insulin receptor isoform expression from A to B. This alteration is accompanied by an increase in responsiveness and insulin sensitivity for two of insulin's metabolic effects, namely glucose incorporation into glycogen and 2-deoxyglucose transport. Changes in isoform expression and insulin response are also observed during the differentiation of 3T3-L1 adipocytes. In the present study, we demonstrate that similar enhancements are observed for two of insulin's mitogenic effects, namely induction of the c-fos and egr-1 genes. This suggested to us that it was expression of the B isoform of the IR upon dexamethasone treatment that caused the enhanced signaling as the metabolic and mitogenic pathways are thought to diverge at a very early point, perhaps at the insulin receptor itself. Consequently, we examined two of the earliest events in the signaling pathway, IR autophosphorylation and IRS-1 phosphorylation in vivo. Maximal insulin-stimulated phosphorylation of both proteins was elevated in cells treated with dexamethasone. Furthermore, the sensitivity for IRS-1 phosphorylation, but not for IR autophosphorylation, was increased. This increase in sensitivity for IRS-1 phosphorylation is likely to be the origin for the observed enhancement in signaling as it has been demonstrated that IRS-1 is involved in both insulin-stimulated DNA synthesis in Rat-1 fibroblasts and Glut-4 translocation in 3T3-L1 cells(28, 29) .

Tyrosine phosphorylation is regulated in vivo by a balance of kinase and phosphatase activities. Alteration in either activity could cause the observed increase in phosphorylation. Therefore, we undertook experiments on receptors purified by wheat-germ agglutinin affinity chromatography. In vitro, the B isoform of the IR autophosphorylates to a greater extent than the A isoform and has increased kinase activity toward the synthetic substrate poly-glu(4):tyr(1) and recombinant IRS-1 protein. Conversely, we were not able to detect any alterations in total cellular tyrosine phosphatase activity when assayed with in vitroP-labeled IR and IRS-1. Thus it appears that the enhanced phosphorylation seen in vivo is due to differences in the intrinsic kinase activities of the IR isoforms. There is still a discrepancy between the in vitro and in vivo results, however, as there is no difference in insulin sensitivity for IRS-1 phosphorylation in vitro. Although IRS-1 is a direct substrate for the IR kinase in vitro, there may be additional factors in vivo that enhance the interaction between the two molecules causing the left-shift in the insulin dose-response curve. Alternatively, the receptor may require the intact plasma membrane for correct function.

The two receptor isoforms differ by 12 amino acids in the carboxyl terminus of the extracellular alpha-subunit. The intracellular kinase domains of the two isoforms are identical. How is it possible that the two isoforms have different kinase activities? Kellerer and co-workers (26) found that if the receptors are activated by trypsin cleavage rather than insulin stimulation then no differences in activity are observed. So how is insulin able to activate one kinase more than the other? The B isoform autophosphorylates to a greater extent than the A isoform both in vivo and in vitro. There are two possible explanations for this finding. It has been shown that following insulin stimulation only 43% of insulin receptors isolated from human adipocytes are phosphorylated on tyrosine(30) . This has lead to the proposal that there are distinct pools of kinase competent and incompetent receptors. In non-insulin-dependent diabetes mellitis, fewer than 15% of IRs are phosphorylated on tyrosine suggesting that the proportion of incompetent receptors is greater leading to a kinase defect(30) . However, these findings can be interpreted differently as the phosphorylation reaction is inherently reversible. The observed autophosphorylation is a balance between the forward phosphorylation and the reverse dephosphorylation reactions. So an increase in the forward reaction due to an increase in V(max) would shift the equilibrium toward a more fully phosphorylated state.

An alternative explanation could be that the B isoform may be utilizing autophosphorylation sites that are unavailable in the A isoform due to conformational restraints imposed by the extracellular alpha-subunits. This latter alternative can be tested by tryptic phosphopeptide mapping. Kellerer et al.(26) used HPLC analysis of the tryptic peptides but were not able to observe any differences. However, direct comparison of HPLC and thin layer peptide mapping of insulin receptor phosphopeptides has shown that HPLC lacks the sensitivity and resolution of two-dimensional thin layer mapping(27) . Therefore, we utilized two-dimensional tryptic phosphopeptide mapping (Fig. 5). The intensity of all phosphopeptides was higher in the B isoform suggesting that the equilibrium has been shifted to a more highly phosphorylated state consistent with the increase in V(max). More interestingly, an additional phosphopeptide E was detected in the B isoform that is not seen in the A isoform. The exact identity of peptide E remains to be determined, but two possibilities are the peptides spanning amino acids 1086-1092 and 1102-1127, which include tyrosines 1087 and 1122, respectively. Both these peptides are predicted to have a charge of +1.5 at pH 1.9 if phosphorylated on tyrosine. Interestingly, both peptides are derived from domains that are conserved among tyrosine kinases; however neither are known autophosphorylation sites(31) .

In conclusion, dexamethasone causes an increase in responsiveness and sensitivity for insulin-stimulated immediate early gene expression in HepG2 cells. The results presented here indicate that it is the expression of the B isoform of the IR upon dex treatment that is responsible for this enhancement. The B isoform has a greater insulin-stimulated kinase activity, phosphorylates IRS-1 more efficiently in vitro, and couples to IRS-1 more efficiently in vivo. Autophosphorylated tyrosine residues on tyrosine kinases are the docking sites for signaling proteins containing SH2 domains, and the identification of a new phosphopeptide in the B isoform raises the possibility that this isoform could interact with signaling molecules that are not available to the A isoform(32, 33, 34) . It will be interesting to identify this new autophosphorylation site and determine whether the B isoform couples to novel SH2-containing proteins. It should be stated, however, that we cannot rule out the possibility that dexamethasone could be having other effects on the cell that could contribute to the enhanced signaling. For example, it has been shown that dex treatment of IM-9 and Fao cells causes changes in the carbohydrate composition of the IR, but it is not known whether this effects signaling(35) . Alternatively, dex may effect membrane fluidity which could alter the function of the IR. Although these are possibilities, we believe that the major determinant for enhanced signaling is the switch in expression of IR isoforms from A to B. A method of modulating the alternative splicing in vivo that does not require hormonal treatment will be required to rule out these alternatives definitively.


FOOTNOTES

*
This work was supported by Grant DK44643 from the National Institute of Diabetes, Digestive, and Kidney Diseases, National Institutes of Health and a Merit Review Award from the Department of Veterans Affairs. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Clinical Research Unit, Diabetes Center, Kyoto National Hospital, 1-1 Fukakusa-Mukaihata, Fushimi-ku, Kyoto 612, Japan.

Supported by a Juvenile Diabetes Foundation international fellowship.

**
Faculty member of the UCSD Biomedical Sciences Graduate Program. To whom correspondence should be addressed: Dept. of Medicine 0673, University of California, San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0673. Tel.: 619-534-6275; Fax: 619-534-7181.

(^1)
The abbreviations used are: IR, insulin receptor; dex, dexamethasone; IRS-1, insulin receptor substrate 1; PCR, polymerase chain reaction; RT, reverse transcription; PAGE, polyacrylamide gel electrophoresis; WGA, wheat germ agglutinin; BSA, bovine serum albumin; TPCK, [l]-1-tosylamido-2-phenylethyl chloromethyl ketone; NMA, N-ethylmorpholine acetate.


ACKNOWLEDGEMENTS

We thank Dr. Jerrold Olefsky for helpful advice and encouragement and Drs. K. Siddle, C. R. Kahn, and H. Maegawa for antibodies.


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