©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Insulin Receptor Signaling Is Augmented by Antisense Inhibition of the Protein Tyrosine Phosphatase LAR (*)

(Received for publication, November 18, 1994)

Donald T. Kulas (1)(§) Wei-Ren Zhang (3) Barry J. Goldstein (3) Richard W. Furlanetto (2) Robert A. Mooney (1)(¶)

From the  (1)Departments of Pathology and Laboratory Medicine and (2)Pediatrics, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642 and (3)Dorrance H. Hamilton Research Laboratories, Division of Endocrinology and Metabolic Diseases, Department of Medicine Jefferson Medical College, Philadelphia, Pennsylvania 19107

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Considerable evidence has shown that most physiologic responses to insulin require activation of the intrinsic tyrosine kinase of the insulin receptor. Biochemical studies have also supported the hypothesis that receptor kinase activity can be modulated by cellular protein tyrosine phosphatases (PTPases), which have not yet been identified. To test the hypothesis that the transmembrane PTPase LAR can modulate insulin receptor signaling in vivo, antisense RNA expression was used to specifically suppress LAR protein levels by 63% in the rat hepatoma cell line, McA-RH7777. Hormone-dependent autophosphorylation of the insulin receptor was increased by approximately 150% in the antisense-expressing cells at all insulin concentrations tested. This increase in autophosphorylation was paralleled by a 35% increase in insulin receptor tyrosine kinase activity. Reduced LAR levels did not alter non-hormone-dependent tyrosine phosphorylation nor basal insulin receptor tyrosine phosphorylation and kinase activity. Most significantly, reduced LAR levels resulted in a 350% increase in insulin-dependent phosphatidylinositol 3-kinase activity. These studies provide unique in vivo evidence that LAR is involved in the modulation of insulin receptor signaling in intact cells.


INTRODUCTION

The insulin receptor is a heterotetrameric protein composed of two extracellular alpha subunits linked by disulfide bonds to two transmembrane beta subunits. Upon insulin binding, receptors autophosphorylate on tyrosine residues and increase their intrinsic tyrosine kinase activity (reviewed in (1) ). In vitro studies have shown that protein tyrosine phosphatases (PTPases) (^1)can dephosphorylate the insulin receptor and reduce its tyrosine kinase activity (reviewed in (2) ). When insulin receptors and PTPase 1B, PTPase 1C, or the T-cell PTPase were co-transfected to a level of high transient overexpression, insulin receptor autophosphorylation was inhibited(3, 4) . These studies imply that cellular responses to insulin are the net effect of receptor kinase activity and insulin receptor-selective PTPase activity. An important extension of these investigations would be to identify a physiological insulin receptor PTPase and characterize its effect on insulin receptor signaling.

Our recent work has implicated the transmembrane class of PTPases as likely participants in regulating growth factor receptor signaling (5, 6, 7, 8, 9) . A candidate insulin receptor PTPase is the leukocyte common antigen-related (LAR) PTPase(10, 11) , which has been shown to be a prominently expressed phosphatase in rat liver(12, 13) . In vitro studies have shown that LAR preferentially dephosphorylates the Tyr-1150 domain of the insulin receptor, a site critical for insulin receptor tyrosine kinase activity(8) . Based on this evidence, we employed an antisense strategy to test the hypothesis that LAR is a specific physiological transmembrane PTPase involved in modulating insulin receptor signal transduction.


EXPERIMENTAL PROCEDURES

Materials

McA-RH7777 rat hepatoma cells were purchased from American Type Culture Collection (Rockville, MD). The pRc/CMV eukaryotic expression vector was purchased from Invitrogen (San Diego, CA). The pMEP4b eukaryotic expression vector was a generous gift from Dr. Mark L. Tykocinski (Case Western Reserve University). Anti-phosphotyrosine (4G10), insulin receptor substrate-1 (IRS-1), and Syp/SHPTP2 antibodies were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). [-P]ATP and I-Tyr were from DuPont NEN. Other materials were obtained as described previously(9) .

Cell Culture and Transfection

McA-RH7777 rat hepatoma cells were maintained in a humidified atmosphere of 5% CO(2), 95% air at 37 °C in Dulbecco's modified Eagle's medium (Sigma) supplemented with 20% equine serum and 5% fetal bovine serum (Hyclone, Logan, UT). Since some reports have indicated that the degree of cell confluence can influence LAR levels(14, 15) , the experiments described in the present study were performed under equivalent subconfluent conditions.

The pMEP4b antisense vector was constructed by inserting a 546-nucleotide cDNA fragment consisting of 20 upstream nucleotides and the first 526 coding nucleotides of rat LAR message in an inverted orientation downstream of the metallothionein promoter. Transfection of the antisense vector and a null control vector into the McA-RH7777 cell line was performed by calcium phosphate-DNA precipitation(16) . Stable non-clonal populations resistant to 125 µg/ml hygromycin B were obtained.

The pRc/CMV antisense vector was constructed with the same LAR cDNA fragment described above. After a calcium phosphate-DNA precipitation transfection, stable non-clonal populations of cells were selected with 500 µg/ml Geneticin.

Insulin Receptor Binding

Following an overnight starvation in serum-free medium supplemented with 1% bovine serum albumin, subconfluent 24-well plates of null vector- and antisense-expressing cells were incubated overnight at 4 °C with 9.0 pMI-Tyr porcine insulin (2200 Ci/mmol) plus unlabeled insulin competitor as described. Cells were washed three times with ice-cold phosphate-buffered saline and the cell-associated radiolabel quantitated. Each point was performed in triplicate.

Hormone Stimulation of Cells and Immunoprecipitations

Subconfluent 10-cm monolayers containing equal numbers of null vector- and antisense-expressing cells were incubated for 15 h in serum-free Dulbecco's modified Eagle's medium supplemented with 1% bovine serum albumin. Following a 5-min hormone treatment, cells were washed twice with phosphate buffered saline at pH 7.4, harvested in 1.5 ml of lysis buffer A (50 mM Tris, pH 7.4, 100 mM NaCl, 1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 1 mg/ml benzamidine, 1 mM orthovanadate, 50 mM NaF, and 10 mM tetrasodium pyrophosphate) and homogenized in a hand-driven glass homogenizer. After centrifugation at 10,000 times g for 10 min at 4 °C, the clarified supernatants were normalized to protein content (Bio-Rad). Immunoprecipitations and Western blot analysis of phosphotyrosyl proteins were performed as described previously(9) .

In Vitro Insulin Receptor Autophosphorylation

After subconfluent 10-cm monolayers containing equal numbers of null vector- and antisense-expressing cells were starved in serum-free Dulbecco's modified Eagle's medium supplemented with 1% bovine serum albumin, cells were lysed and normalized to protein content, and insulin receptors were isolated by wheat germ agglutinin affinity chromatography. In three experiments, recovery of receptors as determined by tracer binding analysis was within 5% between null vector- and antisense-expressing cells by this technique. Receptors were stimulated with 0 or 100 nM insulin for 30 min at room temperature in the presence of 1 mM orthovanadate, 8.75 mM MnCl(2), and 22 µM [-P]ATP. Reactions were terminated by the addition of SDS-PAGE sample buffer, and proteins were separated by SDS-PAGE and analyzed by autoradiography.

Insulin Receptor Tyrosine Kinase Activity

Insulin receptors were partially purified on wheat germ agglutinin as described above and then subjected to a tyrosine kinase assay using poly(Glu-Tyr) (4:1) as an exogenous substrate. Reactions were assayed for 20 min at room temperature and terminated by applying 30-µl aliquots onto Whatman No. 3MM filter papers. The filter papers were washed in a 10% trichloracetic acid, 10 mM tetrasodium pyrophosphate solution, rinsed with ethanol, and dried before counting.

Phosphatidylinositol 3-Kinase Activity

Null vector- and antisense-expressing cells were hormone treated and harvested as described above. PI 3-kinase activity was measured as described(9) .


RESULTS AND DISCUSSION

To test the hypothesis that the transmembrane PTPase LAR is involved in modulating insulin receptor signal transduction in vivo, an antisense expression system was developed. The LAR antisense vector was constructed by inserting, in an inverted orientation, a 546-nucleotide rat LAR cDNA fragment into the eukaryotic expression vector pMEP4b. This antisense vector, as well as a null vector to serve as a control, was stably transfected into the rat hepatoma cell line McA-RH7777. Western blot analysis of the resulting non-clonal subpopulations of cells demonstrated that LAR protein expression was reduced by 63.0 ± 6.4% (mean ± S.D.) in the LAR antisense cells when compared to the null vector cells (Fig. 1A).


Figure 1: LAR antisense vector and its effect on LAR expression. A, Western blot analysis of LAR antisense and null vector cell lysates probed with a recombinant rat LAR antibody (F. Ahmad and B. J. Goldstein, submitted for publication). Error bars represent mean ± S.D. from four experiments. Autoradiographs were densitometrically scanned(9) . Inset is from a representative experiment. B, Western blot analysis of LAR antisense and null vector whole cell lysates (matched to those used in A) probed with an antibody against Syp/SHPTP-2 (Upstate Biotechnology Inc.). Error bars represent mean ± S.D. from three experiments. Inset is from a representative experiment. C, Zn-dependent LAR suppression. Subconfluent LAR antisense cells were cultured for 3 days in Dulbecco's modified Eagle's medium supplemented with 1% bovine serum albumin and the Zn concentrations noted, followed by Western blot analysis of LAR expression. Errorbars represent mean ± one-half the range from two experiments.



To demonstrate specificity, null vector and LAR antisense cell lysates were probed with an antibody against the PTPase Syp/SHPTP-2. As shown in Fig. 1B, Syp/SHPTP-2 protein levels were equivalent between the two cell subpopulations. Additionally, in spite of their difference in LAR expression, the null vector- and LAR antisense-expressing cells possessed equivalent morphology, nuclear/cytoplasmic ratios, growth rates, and total protein per cell (data not shown).

Since the pMEP4b vector contains a metallothionein promoter, maximum LAR suppression was attained with either serum-containing medium or with serum-free medium when the latter was supplemented with 50 µM Zn. Culturing the LAR antisense cells in low Zn or Zn-free medium allowed LAR protein levels to return to those of null vector control cells (Fig. 1C), indicating that the suppression of LAR protein was mediated by the transcriptional product of the antisense construct.

The next series of experiments were designed to investigate whether reduced LAR protein levels altered insulin receptor signal transduction. The first parameter examined was insulin binding and affinity. As shown in Fig. 2, insulin binding and affinity were equivalent in the two subpopulations of cells. Scatchard analysis of these data demonstrated approximately 50,000 receptors/cell with a K(d) of 0.3 nM.


Figure 2: Insulin receptor binding. Insulin binding was performed as described under ``Experimental Procedures'' A, competitive insulin binding. B, Scatchard analysis of data in A. Opensquares, null vector. Closedcircles, antisense vector.



If LAR exerted a negative modulating effect on insulin receptor signal transduction, then suppression of LAR protein levels would be expected to increase cellular responses to insulin. When intact null vector- and LAR antisense-expressing cells were stimulated with insulin, reduced LAR levels resulted in approximately a 150% increase in insulin receptor beta subunit (95 kDa) autophosphorylation at all hormone concentrations tested (Fig. 3A). In contrast, basal insulin receptor tyrosine phosphorylation was unaffected by altered LAR levels. An insulin-dependent phosphotyrosyl protein was also observed at 120 kDa. This may be the liver-specific receptor substrate pp120/HA4(17) . Insulin-dependent phosphorylation of the 120-kDa band was increased in cells containing suppressed levels of LAR. Hormone-dependent phosphorylation of IRS-1/pp185 was not observed. This may be due in part to a hormone-independent band at approximately 185 kDa. This hormone-independent phosphotyrosyl band was unchanged when LAR levels were reduced. The latter data would suggest that reducing LAR levels does not result in a generalized increase in cellular tyrosine phosphorylation. Furthermore, the observed difference in receptor autophosphorylation was not due to an irreversible alteration in the intrinsic tyrosine kinase activity of these receptors. When insulin receptors from null vector- and antisense-expressing cells were purified by wheat germ agglutinin affinity chromatography and then stimulated with insulin in vitro, autophosphorylation levels were equivalent (Fig. 3B). This control experiment demonstrates that by removing the influence of cellular PTPases from the analysis, the observed effect of LAR antisense expression is abolished.


Figure 3: Effect of LAR expression on insulin receptor autophosphorylation and tyrosine kinase activity. A, in vivo receptor autophosphorylation. After incubating cells for 5 min with insulin at the concentrations described, whole cell lysates were immunoprecipitated with an anti-phosphotyrosine antibody, separated by SDS-PAGE, and transferred onto polyvinylidene difluoride paper. Protein bands were located by autoradiography after incubation with an anti-phosphotyrosine antibody (4G10). B, in vitro receptor autophosphorylation. Insulin receptors were partially purified from cell lysates on wheat germ agarose affinity columns. Partially purified receptors were autophosphorylated with [-P]ATP in the presence of 100 nM insulin for 30 min at room temperature. C, in vivo tyrosine kinase activity. After incubating cells for 5 min with insulin at the concentrations described, insulin receptors were partially purified on wheat germ agarose affinity columns. Partially purified receptors were then subjected to a tyrosine kinase assay using poly(Glu-Tyr) (4:1) as an exogenous substrate. Data represent mean ± S.E. from three experiments performed in duplicate (p < 0.05 for 1 nM insulin treatment and p < 0.0001 for 100 nM insulin treatment; p values generated from a Student's t test). D, in vitro tyrosine kinase activity. Experiment was identical to C with the exception that insulin was added after wheat germ agarose purification. Student's t test confirmed the lack of significant differences between null and antisense cells in this control experiment. In C and D, openbars represent null vector cells and hatchedbars represent antisense cells. * denotes a S.E. of less than 5%.



Augmentation of insulin receptor autophosphorylation in cells containing reduced LAR protein levels provides evidence that LAR might be involved in insulin receptor signaling. The increased insulin-dependent phosphorylation of the 120-kDa substrate in Fig. 3A suggested that LAR altered in vivo insulin receptor tyrosine kinase activity. By isolating insulin receptors from insulin-treated cells in the presence of phosphatase inhibitors, the in vivo tyrosine kinase activity of the insulin receptors can be preserved and analyzed directly. Using poly(Glu-Tyr) (4:1) as a substrate, in vivo tyrosine kinase activity of insulin receptors within LAR antisense-expressing cells was found to be increased by 29 ± 4 and 39 ± 1 at 1 and 100 nM insulin (n = 3), respectively, when compared to the null vector-expressing cells (Fig. 3C). These differences were statistically significant as determined by a Student's t test and demonstrate that the activity of intact cell insulin receptors is increased by reduced LAR levels. No difference in basal insulin receptor tyrosine kinase activity was detected between null vector- and antisense-expressing cells. As an additional control, insulin receptors were purified from each subpopulation without prior insulin stimulation. When these latter receptors were stimulated with insulin in vitro, they possessed equivalent tyrosine kinase activities (Fig. 3D). These data indicate that the number of insulin receptors purified from null vector- and antisense-expressing cells, under the conditions of the tyrosine kinase assay, is the same. Additionally, the results support the interpretation from the autophosphorylation experiments that the observed effect of LAR antisense expression is abolished by removing the influence of cellular PTPases from the analysis.

Increases in insulin receptor autophosphorylation and tyrosine kinase activity, both receptor level effects, suggest that LAR is a physiological PTPase that either directly or indirectly acts on the insulin receptor. To determine if LAR is capable of exerting its effect on insulin receptor signaling distal to the receptor, insulin-dependent phosphatidylinositol 3-kinase (PI 3-kinase) activation was examined. PI 3-kinase may be involved in insulin stimulation of glucose transport and insulin-dependent anti-lipolysis(18) , as well as in insulin stimulation of mRNA synthesis(19) . Therefore, any PTPase that alters PI 3-kinase activity demonstrates a downstream receptor site of action and potentially links that PTPase to a biological response. When this parameter was tested, insulin-dependent activation of PI 3-kinase averaged 350% greater in LAR antisense-expressing cells when compared to null vector-expressing cells (Fig. 4A). This effect was not due to differences in the total cellular pool of PI 3-kinase since these levels were the same between the two subpopulations of cells (data not shown).


Figure 4: Effect of LAR on insulin-dependent phosphatidylinositol 3-kinase activity. Following treatment with insulin at the concentrations described, PI 3-kinase was immunoprecipitated from null vector- and antisense-expressing cells with either an anti-phosphotyrosine antibody (A) or an anti-IRS-1 antibody (B) and assayed as described(9) . A, results were obtained by the densitometric scanning of autoradiographs from at least three experiments. Results are plotted as percent increases in insulin-dependent PI 3-kinase activity normalized to null vector cells at 100 nM insulin (mean ± S.D.). Openbars represent null vector cells. Hatchedbars represent antisense cells. * denotes a S.D. of less than 5% (p < 0.0001 for each hormone concentration tested; p values generated from a Student's t test). B, autoradiogram of the P-labeled product of the PI 3-kinase assay separated by TLC.



The increase in PI 3-kinase activity found in phosphotyrosine immunoprecipitations after insulin stimulation is thought to be due to an association of PI 3-kinase with phosphorylated IRS-1(20, 21) . Although the presence of phosphorylated IRS-1 was not demonstrated in Fig. 3A, we investigated the role of IRS-1 in PI 3-kinase activation by directly examining PI 3-kinase activity in IRS-1 immunoprecipitates. As shown in Fig. 4B, reduced LAR levels led to dramatic increases in IRS-1 immunoprecipitable PI 3-kinase activity. This result would suggest that the 350% increase in PI 3-kinase activation described in Fig. 4A is mediated by IRS-1.

Depending on LAR's site of action, there are several potential mechanisms by which a 63% reduction in LAR levels can lead to a 150% increase in receptor autophosphorylation and a 350% increase in PI 3-kinase activation but only a 35% increase in receptor tyrosine kinase activity. First, poly(Glu-Tyr) may not be an ideal substrate for the insulin receptor; the use of this substrate in an in vitro assay may not reflect in vivo insulin receptor tyrosine kinase activity. Second, the 350% increase in PI 3-kinase activity could simply represent a downstream amplification of the LAR-dependent 35% increase in receptor kinase activity. Third, tyrosine kinase activity may be lost under the conditions of the assay; the 35% increase may underestimate the difference seen within intact cells. More intriguing, however, is the possibility that LAR could specifically dephosphorylate tyrosine residues on either the insulin receptor itself or a downstream substrate that are critical for PI 3-kinase activation. This latter mechanism would imply that LAR functions as an active component in determining the manner in which a cell responds to insulin. Whether LAR performs this function in a direct or indirect manner remains to be elucidated.

To demonstrate the sensitivity and reproducibility of the antisense approach, a second antisense construct was synthesized by inserting the described LAR cDNA fragment into the pRc/CMV eukaryotic expression vector. As shown in Fig. 5, studies performed with the pRc/CMV antisense vector confirmed the results found with the pMEP4b vector. Suppressed LAR protein levels resulted in an increase in the responsiveness of early insulin receptor signaling, including 100% increases in insulin receptor autophosphorylation (Fig. 5A) and increases of up to 250% in insulin-dependent phosphatidylinositol 3-kinase activation (Fig. 5B) at all insulin concentrations tested.


Figure 5: Demonstration of antisense sensitivity and reproducibility using the pRc/CMV antisense vector. A, effect of LAR expression on in vivo insulin receptor autophosphorylation. Cells were treated as described in Fig. 3A. B, effect of LAR expression on insulin-dependent PI 3-kinase activity. Cells were treated as described in Fig. 4. Errorbars represent mean ± one-half the range from two experiments. Results are plotted as in Fig. 4. Openbars represent null vector cells. Hatchedbars represent antisense cells. * denotes a range of less than 5%.</.



This work represents the first evidence that manipulation in situ of a natively expressed PTPase can have profound effects on insulin receptor signaling in an intact cell and further supports the hypothesis that LAR is a physiological regulator of insulin action. The fact that a 63% reduction in LAR protein levels results in dramatic increases in early insulin receptor signaling indicates that LAR is a critical component of insulin receptor signaling. Defects in LAR expression and/or activity could have profound effects on a cell's ability to respond to insulin.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants R01-DK43396 (to B. J. G.), R01-CA38981 (to R. W. F.), and R01-DK38138 (to R. A. M.). 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.

§
Student in the Medical Scientist Training Program at the University of Rochester.

To whom reprint requests should addressed: Dept. of Pathology and Laboratory Medicine, University of Rochester School of Medicine and Dentistry, 601 Elmwood Ave., Rochester, NY 14642. Tel.: 716-275-7811; Fax: 716-273-1101.

(^1)
The abbreviations used are: PTPase, protein tyrosine phosphatase; IRS-1, insulin receptor substrate-1; PAGE, polyacrylamide gel electrophoresis; PI 3-kinase, phosphatidylinositol 3-kinase.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.