©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Mechanisms of Enhanced Transmembrane Signaling by an Insulin Receptor Lacking a Cytoplasmic -Subunit Domain (*)

Toshiyasu Sasaoka , W. John Langlois , David W. Rose , Jerrold M. Olefsky (§)

From the (1) Department of Medicine, Division of Endocrinology and Metabolism, University of California, San Diego, La Jolla, California 92093 and the Veterans Administration Medical Center, Medical Research Service, San Diego, California 92161

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We have recently characterized a mutant insulin receptor (HIR978) in which the insulin receptor -subunit was truncated at amino acid residue 978. Compared with parental Rat1 cells, the cells expressing the truncated receptor exhibited enhanced sensitivity to insulin's biologic actions. All of these effects are now extended to transcriptional events, since we now show enhanced sensitivity to insulin stimulation of c -fos mRNA expression. These effects were insulin-specific, since insulin-like growth factor-1 stimulation of glucose incorporation into glycogen, -aminoisobutyric acid uptake, and thymidine incorporation into DNA were normal. In addition, the truncated receptor exhibited enhanced sensitivity only in vivo, but not in vitro, since the kinase activity of wheat germ agglutinin-purified receptor preparations was comparable between HIR978 and parental Rat1 insulin receptors. Parental rat endogenous insulin-like growth factor-1 receptors and transfected human insulin receptors form hybrid receptors as well as homologous tetrameric receptors. The normal heterotetrameric receptors possess kinase activity in vivo leading to tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and its association with the p85 regulatory subunit of phosphatidyl inositol 3-kinase. Interestingly, preincubation with human-specific anti-insulin receptor antibody abolished the increased insulin sensitivity in glucose incorporation into glycogen in HIR978 cells. Furthermore, microinjection of anti-IRS-1 antibody into HIR978 cells inhibited insulin stimulation of DNA synthesis. In summary: 1) truncated receptors on the cell surface confer enhanced insulin sensitivity in vivo; 2) the normal heterotetrameric receptors are functionally active and couple to IRS-1 efficiently; and 3) IRS-1 is an important molecule transmitting insulin's biological signals in HIR978 cells.


INTRODUCTION

Insulin binding to the extracellular -subunits activates the receptor tyrosine kinase properties of the cytoplasmic portion of the -subunit of the insulin receptor (1) . The receptor undergoes autophosphorylation, which enables the receptor kinase to phosphorylate various substrates. One of the cellular substrates for the insulin receptor tyrosine kinase is IRS-1()(2, 3) . IRS-1 undergoes tyrosine phosphorylation in response to insulin and acts as a multisite docking protein to interact with Src homology 2 (SH2) domains of various signal-transducing molecules to propagate the insulin signal downstream (3) . A variety of studies have indicated that activation of the receptor kinase is necessary for IRS-1 phosphorylation leading to the biological actions of insulin (4, 5, 6, 7) .

Recently, Yamamoto et al.(8) have characterized a mutant insulin receptor, deleting the COOH-terminal 83 amino acids. In the report, Chinese hamster ovary cells overexpressing the truncated insulin receptor exhibited enhanced insulin sensitivity in spite of defective kinase activity of the truncated receptor. Indeed, in earlier studies (9) , we have shown that Rat1 fibroblasts overexpressing a truncated insulin receptor lacking 365 amino acids from the COOH terminus also demonstrated enhanced biological actions of insulin, although the truncated receptor itself lacked tyrosine kinase activity. A common feature of these mutant cell lines is normal IRS-1 phosphorylation, although kinase activity of the transfected receptors per se is markedly decreased (8, 9) . Based on our earlier studies, we suggested that the enhanced insulin action in HIR978 cells was directly mediated by the endogenous rodent insulin receptors, rather than the HIR978 receptors, and that the mutant receptors increased the coupling efficiency between endogenous insulin receptors and downstream signaling molecules. In the current report, we have further explored the cellular mechanisms underlying the enhanced insulin sensitivity of HIR978 cells.


EXPERIMENTAL PROCEDURES

Cell Lines and Materials

The generation of stable clonal cell lines expressing wild type insulin receptors (HIRc) and truncated mutant insulin receptors (HIR978), in which the insulin receptor -subunit was truncated at amino acid residue 978 according to the numbering system by Ullrich et al.(1) , and cell cultures have been previously described (9, 10) . HIRc cells expressing 1.510 insulin receptors and HIR978 cells expressing 1.210 truncated insulin receptors were used for this study, while Rat1 fibroblasts expressed 2000 endogenous insulin receptors. I-Insulin and I-IGF-1 were generously provided by the Lilly Co. p85-SH2 glutathione S-transferase (GST) fusion protein and an anti-IRS-1 antibody were a kind gift from Dr. Alan R. Saltiel (Parke-Davis Pharmaceutical Co., Ann Arbor, MI). An anti-insulin receptor antibody ) was kindly provided by Dr. Kenneth Siddle (University of Cambridge, United Kingdom) (11) . D-[C]Glucose (287 mCi/mmol), -[methyl-H]aminoisobutyric acid (AIB), [H]thymidine (83 Ci/mmol), [-P]dCTP (3000 Ci/mmol), and [-P]ATP (6000 Ci/mmol) were purchased from DuPont NEN. cDNA cycle kit was from Invitrogen (San Diego, CA). 10PCR amplification buffer and Taq-polymerase were from Perkin-Elmer Cetus. Bromodeoxyuridine (BrdU), a monoclonal anti-BrdU antibody, and enhanced chemiluminescence reagents were from Amersham Corp.. A monoclonal anti-phosphotyrosine antibody (pY20) was from ICN (Costa Mesa, CA). Rabbit immunoglobulin G (IgG), fluorescein isothiocyanate- or rhodamine-conjugated anti-mouse and anti-rabbit IgG antibodies were from Jackson Laboratories (West Grove, NY). Electrophoresis reagents were from Bio-Rad. All other reagents were purchased from Sigma.

c-fos Expression

Cells were grown to confluence and starved for 24 h with serum-free Dulbecco's modified Eagle's medium (DMEM). After stimulation with various concentrations of insulin for 30 min at 37 °C, total cellular RNA was isolated from the cells by the modified method of single-step guanidinium thiocyanate-chloroform-phenol RNA extraction (12) . cDNA from the total cellular RNA was synthesized by using a cDNA cycle kit and the manufacturer's instructions (Invitrogen). Specific cDNA as defined by the c -fos primers was PCR-amplified with L30 primers as an internal standard in the presence of [P]dCTP. PCR products were electrophoresed and visualized by autoradiography. The relative amounts of c -fosversusL30 were quantitated by measuring the incorporated radioactivity on the gels with a Molecular Dynamics PhosphorImager (Sunnyvale, CA) (9) .

Glucose Incorporation into Glycogen

Confluent cell monolayers were incubated in glucose-free DMEM for 3 h. Cells were stimulated with hormones for 2 h at 37 °C with 5 mM glucose and D-[C]glucose. The monolayers were washed with phosphate-buffered saline and solubilized with 30% KOH solution for 30 min at 37 °C. After boiling the samples for 30 min with carrier glycogen, glycogen was precipitated by the addition of ethanol, and radioactivity of the precipitates was counted (13) .

AIB Uptake

Confluent cell monolayers were incubated with Earle's balanced salt solution supplemented with 25 mM NaHCO and 0.1% bovine serum albumin for 2 h at 37 °C. After cells were stimulated with various concentrations of IGF-1 for 3 h, -[methyl-H]AIB and 0.8 µM unlabeled AIB were added for 12 min. AIB uptake was terminated by washing three times with phosphate-buffered saline, and radioactivity was determined (14) .

Thymidine Incorporation

Confluent cell monolayers were incubated in DMEM containing 0.05% fetal calf serum (FCS) for 24 h, then various concentrations of IGF-1 were added. Twenty h later, the cells were pulsed for 4 h with [H]thymidine. The cells were washed twice with phosphate-buffered saline, twice with 10% trichloroacetic acid, and once with 95% ethanol, and radioactivity was measured (15) .

Cross-linking

Cells were incubated with [I]B2(2-nitro-4-azidophenylacetyl)-des-Phe-insulin (NAPA-insulin) for 2 h at 12 °C. UV cross-linking of the insulin to its receptor was then carried out as described previously (16) . The cells were solubilized in a buffer containing 25 mM HEPES, 120 mM NaCl, 5 mM KCl, 1 mM MgCl, 1 mM CaCl, 1% Triton X-100, 8 mM EDTA, 2 mM dichloroacetic acid, 0.5 mg/ml bacitracin, 15 mM benzamidine, 10 µg/ml aprotinin, 2 mM phenylmethylsulfonyl fluoride, 10% (w/v) glycerol, pH 7.4. After removal of insoluble material by centrifugation, the supernatants were immunoprecipitated with specified antibody and analyzed by SDS-polyacrylamide gel electrophoresis under non-reducing conditions, followed by autoradiography.

Autophosphorylation of Partially Purified IGF-1 Receptors

Autophosphorylation of lectin-purified IGF-1 receptors was measured in vitro. The IGF-1 receptor preparations were incubated with various concentrations of IGF-1 for 16 h at 4 °C. Autophosphorylation of the IGF-1 receptor was measured after incubation with 50 µM [-P]ATP for 10 min at 4 °C. Phosphorylated receptors were immunoprecipitated with anti-insulin receptor antibody to remove insulin receptors and insulin receptor-IGF-1 receptor hybrids. The remaining supernatants including homologous tetrameric IGF-1 receptors were subjected to 7.5% SDS-PAGE under reducing condition (9) .

Kinase Activity of Partially Purified Receptor Preparations

Kinase activity to phosphorylate Glu:Tyr polypeptides was measured in vitro. The lectin-purified receptor preparations were incubated with various concentrations of hormones for 16 h at 4 °C. Kinase activity was measured by incubation with Glu:Tyr polypeptides and 50 µM [-P]ATP for 30 min at 4 °C. The reaction was stopped by spotting on Whatman 3MM paper, and washed five times with 10% trichloroacetic acid and counted by -counter (6, 15) .

Western Blotting Studies

Cell monolayers were starved for 24 h in DMEM containing 0.05% FCS. The cells were then treated with various concentrations of IGF-1 for 1 min at 37 °C. Cells were then solubilized directly in hot Laemmli buffer, and lysates were separated by SDS-PAGE. For affinity precipitation with p85-SH2 GST fusion protein, after stimulation with hormones, the cells were solubilized in a buffer containing 50 mM HEPES, 150 mM NaCl, 10 mM EDTA, 1% Triton X-100, 4 mM NaVO, 200 mM NaF, 20 mM NaPPO, 10 µg/ml aprotinin, 2 mM phenylmethylsulfonyl fluoride, 10% (w/v) glycerol, pH 7.4. The lysates were incubated with 5 µg of p85-SH2 GST fusion protein and glutathione-Sepharose beads for 90 min at 4 °C. The precipitates were subjected to SDS-PAGE under non-reducing condition. The proteins were then transferred onto Immobilon-P by electroblotting. For immunoblotting, membranes were blocked and probed with anti-phosphotyrosine antibody. Blots were then incubated with horseradish peroxidase-linked second antibody followed by enhanced chemiluminescence detection, according to the manufacturer's instructions (Amersham Corp.) (6, 9) .

Microinjection

Cells were grown on glass coverslips and rendered quiescent by starvation for 24 h in serum-free DMEM. Antibodies, which were solubilized in microinjection buffer consisting of 5 mM NaPO and 100 mM KCl, pH 7.4., were then microinjected using glass capillary needles. Approximately 1 10 liters of the buffer were introduced into each cell. The injection included about 1 10 molecules of IgG. Two h after microinjection, cells were incubated with BrdU plus growth factors for 16 h at 37 °C. The cells were fixed with acid alcohol (90% ethanol, 5% acetic acid) for 20 min at 22 °C and then incubated with mouse monoclonal anti-BrdU antibody for 1 h at 22 °C. The cells were then stained by incubation with rhodamine-labeled donkey anti-mouse IgG antibody and fluorescein isothiocyanate-labeled donkey anti-rabbit IgG antibody for 1 h at 22 °C. After the coverslips were mounted, the cells were analyzed and photographed with an Axiphot fluorescence microscope (Carl Zeiss). Microinjected cell numbers were 250-300/coverslip. Immunofluorescent staining of the injected cells has indicated that about 75% of the cells were successfully microinjected (17, 18) .


RESULTS

Induction of c-fos mRNA

We have previously reported that the ability of insulin to stimulate glucose incorporation into glycogen, AIB uptake, thymidine incorporation, and S6 kinase activity is enhanced in HIR978 cells compared to parental Rat1 cells (9) . Another action of insulin is stimulation of gene transcription, and insulin is known to increase induction of c- fos mRNA (19, 20) . As can be seen in Fig. 1, insulin stimulates c- fos mRNA induction in Rat1 cells with an ED of 15 nM. In contrast, the dose-response curve is left-shifted to the same degree in HIR978 and HIRc cells (ED 1.5 nM). The relative amount of c- fos induction was greater in HIR978 cells than in Rat1 cells, and this further extends our previous observation that HIR978 cells display enhanced insulin signaling although >90% of the HIR978 receptor cytoplasmic -subunit domain is deleted.


Figure 1: Dose-response of c-fos mRNA expression after insulin stimulation. A, cells were serum-starved for 24 h and then stimulated with indicated concentrations of insulin for 30 min at 37 °C. Total cellular RNA was isolated, and cDNA was synthesized from the RNA. Specific cDNA as defined by the c- fos primers was subjected to PCR amplification with L30 primers as an internal standard in the presence of [P]dCTP. PCR products were resolved by polyacrylamide gel electrophoresis and visualized by autoradiography. B, the relative amounts of c- fosversusL30 from HIRc (), HIR978 (), and Rat1 () cells were quantitated by measuring the incorporated radioactivity on the gel with a PhosphorImager. Results are the mean of two separate experiments and are expressed as percentage over basal response.



IGF-I Signaling

To investigate whether enhanced insulin signaling is mediated by IGF-1 receptors in HIR978 cells, and whether the enhanced signaling is specific for insulin, we studied IGF-1 stimulation of glucose incorporation into glycogen, AIB uptake, and thymidine incorporation. The dose-response curves for IGF-1 stimulation of these biologic effects are all comparable among HIRc, HIR978, and Rat1 cell lines (Fig. 2). It should be noted that all cell lines express similar numbers of IGF-I receptors (1.2-1.5 10 receptors/cell). Since IGF-I receptors utilize the same downstream signaling pathways as insulin receptors, including phosphorylation of IRS-I, these results indicate that the enhanced insulin action in HIR978 cells is due to increased signaling at the level of the insulin receptor per se.


Figure 2: Biologic actions of IGF-1 in parental and transfected fibroblasts. A, glucose incorporation into glycogen. Absolute values for basal ( b) and maximal ( m) stimulation were as follows: HIRc, b = 12.1 nmol/200 µg of protein/2 h and m = 19.8; HIR978, b = 12.8 and m = 18.2; Rat1, b = 13.7 and m = 21.2. B, AIB uptake. Absolute values for basal ( b) and maximal ( m) stimulations were as follows: HIRc, b = 0.61 nmol/200 µg of protein/12 min and m = 0.92; HIR978, b = 0.58 and m = 0.88; Rat1, b = 0.54 and m = 0.83. C, thymidine incorporation. Absolute counts of basal levels ( b) and maximal stimulations ( m) were as follows: HIRc, b = 14,850 disintegrations/min and m = 1.83-fold; HIR978, b = 14,342 and m = 1.98; Rat1, 12,898 and m = 2.02. All results are the mean of three separate experiments and are presented as the percentage of maximal IGF-1 stimulation of HIRc (), HIR978 (), and Rat1 () cells.



Autophosphorylation-Kinase Activity

To further explore the function of IGF-1 receptors, autophosphorylation was assessed in lectin affinity receptor prepartions. To exclude the influence of truncated insulin receptor-IGF-1 receptor hybrids, a human-specific monoclonal antibody was used to immunodeplete the hybrids from IGF-1-stimulated preparations, and the supernatants were analyzed. As seen in Fig. 3 A, IGF-1 stimulated autophosphorylation was similar in HIRc and HIR978 receptor preparations. IGF-1 stimulation of receptor autophosphorylation was also comparable in Rat1 receptors (data not shown). IGF-1 stimulation of endogenous substrate phosphorylation was also assessed in these cell lines. Cells were incubated with various concentrations of IGF-1, and cell lysates were subjected to Western blotting analysis using anti-phosphotyrosine antibodies. As shown in Fig. 3 B, three prominent IGF-1-stimulated phosphorylated bands can be visualized. The 95 and 105 kDa bands correspond to the -subunit of the IGF-1 receptor, whereas the 185 kDa band represents an endogenous substrate (IRS-1). IGF-1 stimulation of IRS-1 phosphorylation was comparable among HIRc, HIR978, and Rat1 cells. Thus, the enhanced signaling in HIR978 cells is restricted to insulin stimulation.


Figure 3: IGF-1 stimulation of autophosphorylation and endogenous substrate phosphorylation. A, autophosphorylation in vitro. The WGA-purified receptor preparations from HIRc ( lanes 1-5) and HIR978 ( lanes 6-10) cells were incubated with 0 ( lanes 1 and 6), 0.13 ( lanes 2 and 7), 1.3 ( lanes 3 and 8), 13 ( lanes 4 and 9), and 67 ( lanes 5 and 10) nM IGF-1 for 16 h at 4 °C. The receptors were then autophosphorylated. Insulin receptors and insulin receptor-IGF-1 receptor hybrids are immunodepleted by insulin receptor-specific antibody and the remaining sample representing IGF-1 receptors were analyzed by SDS-PAGE under reducing condition. A representative autoradiogram is shown. B, tyrosine phosphorylation of endogenous substrates. Serum-starved HIRc ( lanes 1-4), HIR978 ( lanes 5-8), and Rat1 ( lanes 9-12) cells were stimulated with 0 ( lanes 1, 5, and 9), 1.3 ( lanes 2, 6, and 10), 13 ( lanes 3, 7, and 11), and 67 ( lanes 4, 8, and 12) nM IGF-1 for 1 min at 37 °C. The cells were then lysed in Laemmli buffer. An equivalent number of cells were processed for immunoblot analysis using pY20. A representative autoradiogram is shown. Molecular masses of IRS-1 (185 kDa), p120 (120 kDa), and IGF-1 receptor -subunits (95 and 105 kDa) are shown by arrows.



Insulin stimulation of IRS-1 phosphorylation is enhanced in HIR978 cells. To test the possibility that this might occur by slower dephosphorylation of IRS-1, we examined the time course of IRS-1 phosphorylation. Insulin stimulated rapid tyrosine phosphorylation of IRS-1, with peak phosphorylation by 1 min, declining thereafter in all three cell lines. The amount of insulin-stimulated IRS-1 phosphorylation was similar in HIRc and HIR978 cells and far greater than that seen in Rat1 cells. However, the time course of IRS-1 dephosphorylation was somewhat faster in HIR978 cells compared to HIRc cells and was comparable to Rat1 cells. Clearly, the mechanism of enhanced IRS-1 phosphorylation is not due to impaired dephosphorylation in HIR978 cells (Fig. 4).


Figure 4: Time course of insulin stimulation of IRS-1 phosphorylation. A, serum-starved cells were treated with 17 nM insulin for the indicated times. The cell lysates containing an equivalent number of cells were analyzed by immunoblotting with pY20. A representative autoradiogram is shown and molecular mass of IRS-1 (185 kDa) is shown by an arrow. B, quantitation of time course of IRS-1 phosphorylation. IRS-1 bands were analyzed by densitometry, and the time course of IRS-1 phosphorylation from HIRc (), HIR978 (), and Rat1 () cells is summarized as percentage of maximal tyrosine phosphorylation. Results are the mean of two separate experiments.



Kinase activity of the insulin receptor was determined with Glu:Tyr as substrate. Using lectin affinity purified receptor preparations, containing equal amounts of protein, insulin led to a 3.0 ± 0.1-, 2.9 ± 0.4-, and 2.9 ± 0.3-fold increase in phosphorylation of the exogenous substrate Glu:Tyr with HIRc, HIR978, and Rat1 receptors, respectively. On the other hand, insulin sensitivity was markedly decreased with the mutant receptor, with half-maximal stimulation of 0.8, 4.0, and 4.3 nM in HIRc, HIR978, and Rat1 receptors, respectively (Fig. 5 A). Kinase activity of the IGF-1 receptor was also examined. IGF-1 stimulated a 2.9 ± 0.2-, 3.0 ± 0.3-, and 3.0 ± 0.3-fold increase in phosphorylation of Glu:Tyr with HIRc, HIR978, and Rat1 receptors. Dose-response curves of IGF-1 stimulation of Glu:Tyr phosphorylation were similar between HIRc and Rat1 receptors and slightly decreased at lower concentrations of IGF-1 in HIR978 receptor preparations (Fig. 5 B).


Figure 5: Kinase activity of WGA-agarose purified receptor preparations. Kinase activity of insulin receptors ( panelA) and IGF-1 receptors ( panelB) in vitro. The cells were solubilized in the presence of 1% Triton X-100. The lysates were partially purified on WGA-agarose. The receptor preparations with same amount of insulin ( panelA) or IGF-1 ( panelB) binding capacity were incubated with various concentrations of insulin ( panelA) or IGF-1 ( panelB) for 16 h at 4 °C and were allowed to phosphorylate Glu:Tyr polypeptides in the presence of [P]ATP for 30 min at 4 °C as described under ``Experimental Procedures.'' Absolute values for basal ( b), maximal insulin ( m1), maximal IGF-1 ( m2) stimulations were as follows: HIRc, b = 0.91 pmol/10 min, m1 = 2.70, and m2 = 2.65; HIR978, b = 0.87, m1 = 2.56, and m2 = 2.59; Rat1, b = 0.88, m = 2.51, and m2 = 2.60. Results are the mean of three separate experiments and presented as the percent of maximal hormone response for HIRc (), HIR978 (), and Rat1 () cells.



In addition, WGA preparations were obtained from all three cell types and immunoprecipitated with a human-specific monoclonal anti-insulin receptor antibody. With this approach, the endogenous rodent receptors remain in the supernatants, and the human receptors are in the immunoprecipitates. Kinase activity toward Glu:Tyr was then measured in both supernatants and precipitates (Fig. 6). Kinase activity was readily detected in the precipitates from the HIRc cells, but not from HIR978 or Rat1 cells. In addition, kinase activity was detected in the supernatants from all three cell lines, representing the contribution of endogenous rodent receptors. Thus, a marked decrease in autophosphorylation and kinase activity was noted with the mutant receptor, and the kinase activity toward Glu:Tyr can be attributed to the contribution of the endogenous rodent receptors in the HIR978 cells in vitro.


Figure 6: Immunodepletion of kinase activity by anti-insulin receptor antibody. Effect of immunodepletion in HIRc ( panelA), HIR978 ( panelB), and Rat1 ( panelC) cells. WGA-agarose-purified receptor preparations were incubated with 83-14 (1:200 dilution) for 5 h at 4 °C. The entire precipitates () and all of the remaining supernatants () were incubated with various concentrations of insulin for 16 h at 4 °C, and then the sample were allowed to phosphorylate Glu:Tyr polypeptides in the presence of [P]ATP for 30 min at 4 °C. The reaction was stopped by spotting on Whatman 3MM filter paper, and washed five times with 10% trichloroacetic acid and counted by -counter. Results are the mean of three separate experiments and presented as the percent of maximal insulin response.



Autophosphorylation of Cross-linked Receptors

Previously, we have shown that insulin stimulation of -subunit phosphorylation is markedly decreased, but still detectable, in HIR978 cells (9) . To examine which receptor species are autophosphorylated after insulin stimulation, cells were incubated with NAPA-[I]insulin, a photoaffinity insulin derivative, which was then cross-linked to the receptors. Following cross-linking, the cell lysates were immunoprecipitated by anti-insulin receptor antibody or phosphotyrosine antibody and analyzed by SDS-PAGE under non-reducing conditions (Fig. 7). With this approach, the intersubunit disulfide bonds were not disrupted and the heterotetrameric 22 structure was maintained. In HIRc cells, wild type insulin receptors were demonstrated by the band at 430 kDa (Fig. 7, lane 1). The presence of a small number of endogenous Rat1 insulin receptors was demonstrated by a faint band of 430 kDa (Fig. 7, lanes 3-6). Approximately 70% of the wild type or endogenous insulin receptors were tyrosine phosphorylated, as shown by anti-phosphotyrosine antibody precipitation (Fig. 7, lanes 2 and 6). In HIR978 cells, three distinct bands migrating at 430, 380, and 330 kDa, molecular masses which correspond to endogenous rodent receptors, HIR978:IGF-1 receptor hybrids, and HIR978 homodimers, respectively, were demonstrated (Fig. 7, lane 3). Among these species, only endogenous insulin receptors were autophosphorylated, as shown in the anti-phosphotyrosine immunoprecipitates (Fig. 7, lane 4). Thus, in HIR978 cells, only intact heterotetrameric endogenous receptors are functionally active in terms of autophosphorylation as well as kinase activity.


Figure 7: Insulin stimulation of autophosphorylation in vivo. HIRc ( lanes 1 and 2), HIR978 ( lanes 3 and 4), and Rat1 ( lanes 5 and 6) were serum-starved for 16 h. An equivalent number of cells were incubated with [I]NAPA-insulin for 2 h at 12 °C. UV cross-linking of the insulin to its receptor was carried out, and the cells were solubilized. After removal of insoluble material by centrifugation, the supernatants were immunoprecipitated with anti-insulin receptor antibody ( lanes 1, 3, and 5) or anti-phosphotyrosine antibody ( lanes 2, 4, and 6). The precipitates were then analyzed by 5-12% SDS-PAGE under non-reducing condition. A representative autoradiogram is shown.



Interaction with p85 Subunit of PI3K

Insulin receptor and IRS-1 interact with the SH2 domain of the p85 regulatory subunit of PI3-kinase (21, 22) . Lysates from insulin-stimulated cells were incubated with the NH-terminal p85-SH2 domain expressed as a GST fusion protein bound to glutathione-agarose beads. Associated proteins were separated by SDS-PAGE under non-reducing conditions and analyzed by immunoblotting with anti-phosphotyrosine antibody (Fig. 8 A). Phosphorylated IRS-1 was affinity precipitated by the p85-SH2 domain from all three cell types; quantitatively the amount of IRS-I precipitated was HIRc > HIR978 > Rat1 cells. In addition, in HIRc and Rat1 cells, the 430 kDa band, corresponding to 22 heterotetrameric human or rodent insulin receptors, respectively, was associated with the p85-SH2 domain. Interestingly, among the three types of receptors shown in Fig. 7 ( lane 3), only intact endogenous rodent receptors were associated with the p85-SH2 domain in HIR978 cells (Fig. 8 A, lane 4). Furthermore, insulin increased the association of a 120-kDa protein with the p85-SH2 domain, although the identity of this 120-kDa protein is unknown. To more directly determine which types of insulin receptors were associated with the p85 SH2 domain, cells were incubated with NAPA-[I]insulin, and the ligand was cross-linked. Cell lysates were affinity precipitated by the p85-SH2 domain GST fusion protein bound to glutathione-agarose beads and analyzed by SDS-PAGE under non-reducing conditions. By this analysis, the association of 22 heterotetrameric insulin receptors with the p85-SH2 domain was visualized in both HIRc and Rat1 cells. Importantly, of the three receptor species in the HIR978 cells, only the 430-kDa endogenous rodent receptors were associated with the p85-SH2 domain (Fig. 8 B).


Figure 8: Association of the insulin receptor with p85 subunit of PI 3-kinase. A, association of p85 with tyrosine-phosphorylated protein. HIRc ( lanes 1 and 2), HIR978 ( lanes 3 and 4), and Rat1 ( lanes 5 and 6) were serum-starved for 16 h. An equivalent number of cells were stimulated without ( lanes 1, 3, and 5) or with 17 nM ( lanes 2, 4, and 6) insulin for 1 min at 37 °C. The cells were then solubilized and insoluble materials were removed by centrifugation. The supernatants were affinity precipitated with GST-p85 SH2 fusion protein. The precipitants were subjected to 5-12% SDS-PAGE under non-reducing condition and analyzed by immunoblotting with anti-phosphotyrosine antibody. A representative autoradiogram is shown. B, association of p85 with the insulin receptor. After the cells were serum-starved, an equivalent number of cells were incubated with 0.2 nM [I]NAPA-insulin for 2 h at 12 °C. UVcross-linking of the insulin to its receptor was carried out, and the cells were solubilized. The lysates were affinity precipitated with GST-p85 SH2. The precipitates were then analyzed by 5-12% SDS-PAGE. A representative autoradiogram is shown.



Endogenous Rat Receptors Mediate the Insulin Signaling in HIR978 Cells

In previous studies with HIR978 cells, we have found evidence that the enhanced insulin signaling is conveyed from the endogenous rat receptors and that the overexpression of HIR978 receptors, in some way, enhances the coupling efficiency of the rodent receptors (9) . Thus, when cells were acutely stimulated with an agonistic human specific monoclonal antibody, we found that the antibody had marked agonistic effects in HIRc cells, but was without biologic effect in HIR978 cells, just as it was in Rat1 cells (9) . This indicated that insulin binds to both human and rodent receptors in the HIR978 cells, but that biologic signaling occurs only through the rodent receptor. To further explore this concept, we sought to determine whether binding of insulin to the truncated HIR978 receptors at the cell surface was necessary to confer enhanced signaling properties to the endogenous receptors. To answer this question, we incubated HIR978 cells with a saturating concentration of the human-specific monoclonal antibody, which competes with insulin for receptor binding, for 60 min at 37 °C. This led to antibody occupancy of the human insulin receptors, and 80% of them remained at the cell surface, whereas 20% internalized (data not shown). Similar experiments were also performed withHIRc cells and Rat1 fibroblasts. When the antibody preincubated cells were subsequently stimulated with insulin and glucose incorporation into glycogen was assayed (Fig. 9), the dose-response curves for both the HIRc cells and HIR978 cells were right-shifted, whereas no effect was observed in Rat1 cells. The insulin sensitivity of the antibody preincubated HIR978 cells was comparable to that seen in parental Rat1 fibroblasts. This suggested that inhibiting the binding of insulin to the truncated receptors, or clearing the truncated human receptor from the cell surface, prevented their ability to enhance the signaling properties of the endogenous receptors.


Figure 9: Effect of anti-insulin receptor antibody on glucose incorporation into glycogen. Serum- and glucose-starved HIRc ( panel A), HIR978 ( panelB), and Rat1 ( panelC) cells were incubated without () or with () 83-14 (1:500 dilution) for 2 h at 37 °C. Glucose incorporation into glycogen was then measured as described under ``Experimental Procedures.'' Results are the mean ± S.E. of three separate experiments and are presented as percent of maximal insulin stimulation. Absolute values for basal ( b) and maximal ( m) stimulation were as follows: without incubation; HIRc, b = 12.0 nmol/200 µg of protein/2 h and m = 20.1; HIR978, b = 12.7 and m = 19.2; Rat1, b = 13.4 and m = 21.0: with incubation; HIRc, b = 12.7 and m = 19.5; HIR978, b = 13.4 and m = 18.9; Rat1, b = 13.6 and m = 21.3.



Microinjection of IRS-I Antibody

To evaluate the functional role of IRS-1 in HIR978 cells, in transmembrane signaling by insulin and IGF-1, we performed single cell microinjection studies in which affinity purified anti-IRS-1 antibody was introduced into HIR978 cells. Following this, the cells were stimulated with insulin or IGF-I and cell cycle progression was monitored by measuring BrdU incorporation into newly synthesized DNA. In the basal state, 34.0 ± 2.1% of cells incorporated BrdU. Insulin, IGF-1, and FCS stimulated BrdU incorporation into 71.2 ± 1.9, 67.8 ± 1.8, and 75.0 ± 2.2% of cells, respectively. Microinjection of preimmune control IgG did not alter this stimulatory effect (data not shown). In contrast, microinjection of anti-IRS-1 antibody markedly inhibited the ability of insulin and IGF-1 to induce DNA synthesis by 80 and 76%, respectively, whereas, the stimulatory effects of FCS were not impaired (Fig. 10).


Figure 10: Inhibition of DNA synthesis by microinjection of anti-IRS-1 antibody in HIR978 cells. Serum-starved cells were microinjected with anti-IRS-1 antibody. BrdU incorporation in the injected cells ( open bars) and uninjected cells ( hatched bars) on the same coverslip was determined as described under ``Experimental Procedures.'' Cumulative data are shown and results are the mean ± S.E. of three separate experiments.




DISCUSSION

We have previously shown that overexpression of truncated 978 insulin receptors confers enhanced insulin sensitivity in Rat1 fibroblasts (9) . This is the case despite the fact that the truncated receptor has no intrinsic kinase activity and is missing 90% of the intracellular cytoplasmic domain. Our previous studies indicated that although the truncated insulin receptors do not directly mediate insulin action by themselves, they increase the signaling efficiency of the endogenous insulin receptors. Thus, the increased insulin sensitivity in HIR978 cells is due to enhanced signaling properties of the endogenous insulin receptors (9) . We have termed this phenomenon dominant/positive potentiation, and in the current studies we have extended these observations and have further explored the mechanisms underlying this process.

Insulin stimulates the transcription of a series of early response genes, an example of which is c- fos(19, 20) . To quantitatively examine this biologic effect, we have utilized the reverse transcriptasePCR method to measure insulin-induced induction of c- fos mRNA and have compared the dose response effects of insulin in HIRc cells, HIR978 cells, and untransfected parental Rat1 fibroblasts. The results indicated that insulin leads to a dose-responsive increase in c- fos mRNA in all cell lines, but that the dose-response curves are comparably, and markedly, left-shifted in both the HIRc and HIR978 cells. Thus, expression of the kinase inactive truncated insulin receptor leads to enhanced insulin sensitivity for this biologic effect of insulin.

To understand the mechanism of this enhanced insulin sensitivity, it is important to determine whether the increased biologic signaling is specific for insulin. For example, if overexpression of HIR978 receptors leads to augmentation of the insulin signaling pathway at a post-receptor site, then one might anticipate increased signaling properties of other growth factors. This is particularly true in the case of IGF-I, which has a similar range of biologic effects as insulin, and, beyond the receptor itself, appears to engage the same set of signaling molecules, including IRS-I. Consequently, we measured a variety of biologic effects of IGF-I in the various cell types and found that the dose-response curves for IGF-I stimulation of glucose incorporation into glycogen, AIB uptake, and DNA synthesis, were comparable across the cell lines. Thus, HIR 978 receptors did not lead to enhanced IGF-I signaling, in marked contrast to its effects on insulin action. Since IGF-I and insulin signaling pathways are thought to converge immediately after receptor activation, these results strongly argue that the augmented insulin action in HIR978 cells is due to increased signaling properties of the insulin receptors themselves. We have previously shown that homodimeric HIR978 receptors are incapable of directly transmitting biologic signals, since a human-specific agonistic monoclonal antibody had no stimulatory effect in these cells, whereas, it led to marked stimulation of insulin action in cells expressing intact human insulin receptors (9) . Taken together, these results indicate that the HIR978 receptors confer increased signaling properties to the endogenous rodent insulin receptors in these cells. The current results also indicate that the continuing presence of occupied HIR978 receptors at the cell surface is necessary to sustain this effect. Thus, HIR978 cells were incubated with a human-specific monoclonal antibody directed against the insulin receptor ectodomain, and the cells were subsequently stimulated with insulin. Receptors bound to the antibody no longer can bind insulin (data not shown), and the truncated receptors became occupied and 20% of them internalized. In contrast, the endogenous rodent receptors did not interact with the antibody and remained at the cell surface. Blockade of subsequent insulin binding to receptors on the cell surface prior to insulin stimulation abbrogated the enhanced insulin signaling properties of these cells.

We have previously shown that insulin-stimulated IRS-1 phosphorylation is greater in HIR978 cells compared to Rat1 fibroblasts and is comparable to the degree of IRS-1 phosphorylation observed in HIRc cells. Since IRS-1 is an immediate substrate of the insulin receptor (3) , it seems likely that enhanced transduction of IRS-1 phosphorylation by endogenous insulin receptors explains the increased insulin action observed in these cells. These results are fully consistent with the data presented in Fig. 4 which show greater IRS-1 phosphorylation in HIR978 cells compared to Rat1 fibroblasts, and comparable peak IRS-1 phosphorylation between HIR978 cells and HIRc cells. This line of reasoning is strengthened by the current results on IGF-I signaling. Thus, we find that IGF-I receptor kinase activity, and, more importantly, stimulation of IRS-I phosphorylation is comparable across the three cell lines. Thus, augmented stimulation of IRS-I phosphorylation is only observed for the insulin signaling system. This further points to the coupling step between endogenous insulin receptors and IRS-I phosphorylation as the site of the enhanced signaling efficiency in HIR978 cells. Along these lines, the net amount of tyrosine phosphorylated IRS-I is a function of insulin receptor kinase activity as well as the rate of tyrosine dephosphorylation. Thus, it seemed possible that increased IRS-I phosphorylation in HIR978 cells might be due to decreased IRS-I dephosphorylation. To assess this, we performed time course studies to determine whether dephosphorylation of IRS-I was delayed or decreased in HIR978 cells. The results showed that, if anything, the dephosphorylation of IRS-I was slightly greater in HIR978 cells compared to HIRc cells. Although inferential, these results indicate that increased dephosphorylation is not the mechanism for the increase in the IRS-I phosphorylation state.

We also assessed the functional importance of IRS-I in the augmented insulin effects by performing signal cell microinjection studies in which anti-IRS-I antibodies were directly introduced into living HIR978 cells, followed by stimulation with insulin and measurement of DNA synthesis. The results showed that intracellular blockade of IRS-I led to an 80% inhibition of insulin action, demonstrating the critical role of IRS-I phosphorylation in the biological signaling properties of insulin in these cells.

It is known that overexpression of insulin receptors in fibroblasts can lead to the formation of hybrid receptors consisting of insulin receptor and IGF-I receptor halves (13) . Since the insulin receptor in HIR978 cells contains a large truncation, this allows us to visualize the different homodimeric and hybrid receptor species which are formed in these cells. Immunoprecipitation of I-NAPA cross-linked insulin receptors followed by SDS-PAGE on non-reducing gels revealed the three distinct insulin binding species which exist in these cells. These include the homodimeric endogenous insulin receptors (430 kDa), the hybrid receptors (380 kDa), and the homodimeric HIR978 receptors (330 kDa). Only the intact endogenous insulin receptors are capable of autophosphorylation, and both the homodimeric HIR978 and hybrid receptors were kinase inactive. Since the hybrid receptors contain an intact IGF-I receptor half, these results indicate that the major intramolecular signal transduction is trans, rather than cis, in mediating autophoshorylation.

In summary, the current studies demonstrate that the expression of truncated HIR978 insulin receptors at the cell surface imparts enhanced signaling properties to the endogenous rodent insulin receptors. This effect is specific for the insulin receptor system, since IGF-I action is not increased in these cells. The site of this positive-dominant effect appears to involve an increased coupling efficiency between the endogenous rodent receptors and IRS-I, leading to a generalized increase in insulin's biologic effects.


FOOTNOTES

*
This work was supported in part by Grant DK33651 from the National Institute of Diabetes and Digestive Diseases, National Institutes of Health, by the Veterans Administration Medical Research Service, by the Sankyo Diabetes Research Fund, by a Research Fellowship Grant from the Medical Research Council of Canada, and by an American Diabetes Mentor-Based Fellowship Award. 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.

§
To whom correspondence should be addressed: Dept. of Medicine (0673), University of California, San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0673. Tel.: 619-534-6651; Fax: 619-534-6653.

The abbreviations used are: IRS-1, insulin receptor substrate-1; IGF, insulin-like growth factor; GST, glutathione S-transferase; AIB, -[methyl-H]aminoisobutyric acid; BrdU, bromodeoxyuridine; DMEM, Dulbecco's modified Eagle's medium; PCR, polymerase chain reaction; FCS, fetal calf serum; PAGE, polyacrylamide gel electrophoresis; WGA, wheat germ agarose.


ACKNOWLEDGEMENTS

We thank Dr. Alan R. Saltiel for p85-SH2 GST fusion protein and anti-IRS-1 antibody, Dr. Kenneth Siddle for anti-insulin receptor antibody, and Dr. Byung H. Jhun for advice on the c-fos induction studies. We are grateful to Elizabeth Hansen for her assistance in the preparation of this manuscript.


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