Normal Insulin Receptor Substrate - The Journal of Biological Chemistry

Vol. 268, No. 22, Issue of August 5, PP. 16859-16865,1993 Printed in U.S.A.

THEJOURNAL OF BIOLOGICAL CHEMISTRY

Q 1993 by The American Society for Biochemistry and Molecular Biology, Inc.

Normal Insulin Receptor Substrate-1 Phosphorylation in Autophosphorylation-defectiveTruncated Insulin Receptor EVIDENCE THAT PHOSPHORYLATION OF SUBSTRATESMIGHTBESUFFICIENT BIOLOGICAL EFFECTS EVOKED BY INSULIN*

FOR CERTAIN

(Received for publication, December 24, 1992, and in revised form, April 2, 1993)

Ritsuko Yamamoto-HondaS, Takashi KadowakiSg, Kaoru MomomuraSlI, Kazuyuki TobeS, Yoshikazu TamoriSII, Yoshikazu ShibasakiS**, Yasumichi MoriSS, Yasushi KaburagiSS, Osamu KoshiolS, Yasuo AkanumaSS, YoshioYazakiS, and Masato Kasugall From the $Third Department of Internnl Medicine, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan, the SSZnstitute for Diabetes Care and Research, Asahi Life Foundation, 1-6-1 Marunouchi, Chiyoda-ku, Tokvo 100. Jman. and the IISecond Department of Internal Medicine, Kobe University School of Medicine, 7-5-2Kusunoki-cho, ChG-ku, Kode 650, Japan ”

A mutant human insulin receptor that lacked the 8 2 amino acids of the COOH terminus of the &subunit (de182)was studied. Both the wild type insulin receptor (HIR) andthemutantreceptorwereexpressedin Chinese hamster ovary (CHO) cells by stable transfection. Autophosphorylation and tyrosine kinase activities toward exogenous substrates of solubilized and partially purifiedde182 were severelyimpaired. When CHO cells transfected with de182 (CHO-de182) were stimulated with insulin, autophosphorylation was decreased toa great extent compared with cells expressing HIR(CHO-HIR). Nevertheless, tyrosine phosphorylation of an endogenous substrate, ppl85, and 1(IRS- 1)in CHO-de182 was insulin receptor substratecomparable with that in CHO-HIR. Insulin-stimulated activation of phosphatidylinositol 3-kinase activity in CHO-de282 was also equivalent to that in CHO-HIR. Moreover, CHO-de182 exhibited the same insulin sensitivity as CHO-HIR with respect to 2-deoxyglucose uptake and thymidine incorporation into DNA. Insulin-induced internalizationin CHO-de182 was decreased by 46% as compared with that in CHO-HIR. These data suggest that: 1)the COOH-terminal domain of the insulin receptor may play an inhibitory role in the phosphorylation of pp186 and IRS-1; and 2) phosphorylation of substrates such as pp185 and IRS-1, rather than autophosphorylation of the receptor per Be, correlates better with certain biological effects that were mediated by insulin, suggestingthat phosphorylation of the substrates might be sufficient for transducing signals downstream.

* This work was supported by JuvenileDiabetes Foundation International Grant 192125 (to T. K.)and by a grant for Diabetes Research from Ohtsuka Pharmaceutical Co., Ltd. (to T. K. and M. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. § To whom correspondence should be addressed Third Dept. of Internal Medicine, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan. Tel.: 03-3815-5411(ext. 8296); Fax: 03-3815-2087. ll Postdoctoral Fellow of Juvenile Diabetes Foundation International. **Present address: Dept of Cellular and Molecular Physiology, Harvard Medical School, 25 Shattuck St., Boston, MA 02115.

The insulin receptor is a disulfide-linked tetrameric glycoprotein composed of two a-subunits (135 kDa) and two 8subunits (95 kDa) (Kasuga et al., 1982a). Binding of insulin to the extracellular a-subunitsactivatesintrinsic tyrosine kinase activity in the cytoplasmic domain of the P-subunit (Kasuga et al., 198213, 1983; Rothand Casell, 1983). The receptor then undergoes autophosphorylation, which enables the receptor kinase to phosphorylate various substrates. I n vitro‘ and in vivo studies have shown that the major autophosphorylation sites in the insulin receptor are located in two regions of the 8-subunit: the kinase domain including Tyr-1146; Tyr-1150, and Tyr-1151, and the COOH terminus at Tyr-1316 and Tyr-1322. Three tyrosine autophosphorylationsites in the kinase domain seem to be essential for receptor kinase activity and biological effects (Tornqvist et al., 1987, 1988; White et al., 1988a; Ellis et al., 1986; Wilden et al., 1990, 1992a, 1992b). Protein-tyrosine kinases often have a COOH-terminal tail that extends beyond the end of the tyrosine kinase homology region of their primary sequence (Hunter and Cooper, 1985; Hanks et al., 1988). In the human insulin receptor, a pair of leucine residues (1250 and 1251) demarcates this boundary. As so defined, the COOH terminus of this receptor is composed of 92 amino acids. The COOH domain of the insulin receptor contains 1) two tyrosine autophosphorylation sites (1316 and 1322), which may have an inhibitory role in mitogenic signaling (Thies et al., 1989; Takata et al., 1991; Ando et al., 1992); 2) a threonine and at least 2 serine residues, which may be phosphorylated by unidentified serine/threonine kinases (Koshio et al., 1989; Lewiset al., 1990; Rapuano and Rosen, 1991);and 3) turn-acidic helix-turn motifs similar to those of epidermal growth factor receptor (Chen et al., 1989) of unknown function. In addition, insulin binding was reported to induce a conformational change in the receptor COOH terminus(Baron et al., 1990; Levine et al., 1991), suggesting that the receptor COOH terminus may play some role in signaling mechanisms. To assess the role of the COOHterminal domain, we have constructed a truncated mutant that lacked 82 amino acids of the P-subunit (de182). Although the precise molecular links between activation of the insulin receptor and the final cellular responses remain In this report, the term in vivo refers to intact cells, whereas the term in vitro refers to a cell-free system. * Nucleotides and amino acids are numbered according to Ullrich et al. (1985).

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to be clarified, several candidate molecules including pp185 and PI3 3-kinase have been implicated in thesignal transduction pathways of insulin. In various cell lines, insulin-sensitive tyrosine phosphorylation of pp185 is observed in immunoprecipitates of antiphosphotyrosine antibodies (aPY) immediately after insulin stimulation (White et al., 1985; T. Kadowaki et al.,1987; Momomura et al., 1988;Tashiro-Hashimoto et al., 1989; Tobe et al.,1990; Rothenberg et al.,1991; Sun et al., 1991), and pp185 has been considered one of the putative cellular substrates for the insulin receptor tyrosine kinase. Recently, insulin receptor substrate-1 (IRS-l),which encodes a component of the pp185, was cloned (Sun etal.,1991). The IRS-1 proteinundergoes tyrosine phosphorylation in response to insulin and acts as a multisite docking protein to bind signal-transducing molecules containing Src-homology 2 (SH2)and Src-homology 3 (SH3) domains such as PI 3kinase (Kapeller et al., 1991; Tobe et al.,1993). PI 3-kinase is known to be activated by many oncogenes and receptors for growth factors with tyrosine kinase activities including the insulin receptor (Ruderman et al., 1990; Endemann et al., 1990). During insulin stimulation, products of PI 3-kinase appeared or increased inquantity in uiuo. Theseinsulinstimulated intracellular events and some biological effects were studied and compared in Chinese hamster ovary (CHO) cells transfected with the wild type human insulin receptor (CHO-HIR) and CHO cells transfected with the truncated mutant insulin receptor (CHO-de1821to clarify the role of the COOH-terminal domain in thesignaling pathways of insulin. In the present study, we show that 1) the truncation of COOH-terminal 82 amino acids of the insulin receptor impairs autophosphorylation of the receptor; but 2) phosphorylation of pp185 and IRS-1 aswell as activation of PI 3-kinase remain intact; and 3) certain metabolic and mitogenic effects of insulin are unaffected by this truncation of the insulin receptor.

in Insulin Receptor Mutant

phate-buffered saline and incubated with 0.5miof binding buffer (Ham's F-12, 25 mM HEPES, pH 7.8, and 0.1% bovine serum albumin) containing 20,000 cpm of lZ61-labeledinsulin plus the indicated concentration ;of unlabeled insulin. Cells were washed twice, solubilized with 0.5 ml of 1%Triton X-100, and counted in a y-counter. 126 I-Insulin binding to the solubilized receptor was measured as described (Yamamoto-Hondaet al., 1990). Nonspecific binding was assessed in the presence of 1 p M unlabeled insulin. This value was subtracted to yield specific binding. Purification and Immunoprecipitation of Insulin Receptors-This procedure was performed according to the method of Chou et al. (1987), except that the receptor was immunoprecipitated with a monoclonal antibody in the human insulin receptor, a I R l (purified from culture supernatants of aIR1-producing hybridoma, HB175, American Type Culture Collection) a t a concentration of 30 pg/ml (Kull et aL, 1982). This antibody does not interfere with insulin binding ortyrosine kinase activity. The immune complexes were precipitated with goat anti-mouse IgG preadsorbed to protein ASepharose. The precipitates were then washed extensively with 50 mM HEPES, pH 7.4, 100 mM NaCl, 1%Triton X-100, 0.1% SDS, and 1 mM phenylmethylsulfonyl fluoride. Receptor Tyrosine Kinase Activity-Autophosphorylation was performed in 40 pl of immune complex (which contained 300 fmol of insulin binding capacity) in the presence of buffer A (50 mM HEPES, pH 7.4, containing 0.1% Triton X-100, 10 mM MgCl,, 2 mM MnC12, 1 mM sodium orthovanadate, 10 mM sodium pyrophosphate, 10 mM NaF)containing 50 p~ ATP, 5 pCi of [Y-~'P]ATP, and with or without lo" M insulin. The incorporation of 32Pwas detected by SDS-polyacrylamide gel electrophoresis and autoradiography followed by quantitation by Cerenkov counting. The tyrosine kinase activity of the insulin receptor was measured using Glu-Tyr copolymers (4:l) (Sigma, 0.5 mg/ml) as anexogenous substrate. The reaction 1 on 2 X 2-cm squares of was terminated by spotting the 1 5 ~ aliquots Whatman 3MM paper; the squares were washed four times for at least 10 min each in 10% tricholoacetate containing 10 mM sodium pyrophosphate, washed in acetone, and Cerenkov counted for radioactivity. Both assays were performed for 10 min at 25 "C. Zmmunoblatting of Insulin Receptors-T-29 were polyclonal antibodies raised against the epitopes corresponding to residues 954-969 of insulin receptor. Confluent monolayers of transfected CHO cells in 60-mm plates were lysed in buffer A containing 1%Triton X-100. The lysates were immunoprecipitated with a I R l as described above, and the immune complexes were subjected to SDS-polyacrylamide EXPERIMENTALPROCEDURES gel electrophoresis followed byimmunoblotting with T-29 asa probe. Materials-Porcine insulin was a gift from Eli Lilly Co.; [methylAssay of PI 3-Kinase Actiuity in Immunoprecipitates-The assay (969.4 was performed as described by Fukui and Hanafusa (1989) with some [3H]thymidine (2.7 TBq/mmol) and 2-deoxy-~-[1,2-~H]glucose GBq/mmol) were from Du Pont-New England Nuclear. '261-Insulin modifications. Confluent CHO cells were starved in serum-free Ham's (74 TBq/mmol) and lZ6I-proteinA were from Amersham Corp. All F-12 for 12 h and were incubated with or without insulin for 1 min other materials were obtained from the same sources as described a t 37 "C. The cells were lysed in 500 pl ofbuffer A. Insoluble materials previously (Yamamoto-Honda et al., 1990). were removed with centrifugation, aPY was added, and the lysates Construction of Expression Plusmi&" cDNA encoding the hu- were incubated for 2 h at 4 "C. After incubation with protein Gman insulin receptor was constructed as described (Yamamoto- Sepharose for 45 min, the immunoprecipitates were washed once with Honda et al., 1990) and ligated into pUC19. A part of the COOH- buffer A containing 500 mM NaCl and 1%Triton X-100, twice with terminal human insulin receptor was deleted by digesting the SpeI 20 mM HEPES, pH 7.4, containing 500 mM LiC1, once with PI 3site (at 4334) of the cDNA with exonuclease 111 and mung bean kinase reaction buffer (20 mM Tris-HC1, 100 mM NaCl, 0.5 mM nuclease. After digestion, the cDNA was ligated with Multiframe EGTA). The reaction was started by adding 50 pl of PI 3-kinase TerminatorTM (Pharmacia LKB Biotechnology, Inc.), sequenced, and reaction buffer containing 20 mMMgC12, 10 p M ATP, 3 pci of [ya clone that deleted the COOH-terminal 82 amino acids of the 32P]ATP, and 0.2 mg/ml PI dissolved in dimethyl sulfoxide to the receptor was obtained. The clone has a tag of alanine residue derived immunoprecipitates. After the incubation a t 25 "C for 5 min, the from Multiframe TerminatorTM.This clone was then subcloned into reactions were terminated by the addition of 150 p1 of chloroform, an expression plasmid pSVzneo. methanol, 11.6 N HC1 (100:2002). 100 pl of chloroform was added, Transfection and Establishment of Cell Lines-CHO cells (5 X lo6 and theorganic phase was separated and washed twice with methanol, cells) were transfected by calcium phosphate precipitation with 10 pg 1 N HC1 (1:l).The lipids were concentrated in vacuo, spotted onto a of expression vector and 1 pgof pSVzneo-DNA. After 72 h, G418 Silica Gel 60 plate, and developed in chloroform, methanol, 28% (Geneticin, 600 pg/ml; Life Technologies Inc.) was added to the ammonium hydroxide, water (43:3857) and were visualized by aumedium to select for neomycin-resistant cells (T. Kadowaki et al., toradiography. Antibodies against ZRS-I-a-56 (Tobe et al., 1993) werepolyclonal 1990; Yamamoto-Honda et al., 1990). Cells expressing high levels of the human insulin receptors were identified by 'z61-insulin binding. antibodies prepared against the epitopes corresponding to residues In all cases, the experimental data described below were reproduced 489-505 of IRS-1 (Sun etal., 1991). Immunoblatting of Phasphotyrosine-containing Proteins with Anwith at least two separate clones of transfected cells. Insulin Binding-Assays were performed as described (Russell et tiphasphatyrosine Antibodies-Tyrosine phosphorylation of the inal., 1987). Confluent cells (lo6 cells/well) were washed with phos- sulin receptor and cellular proteins stimulated with insulin in uivo was assessed by immunoblotting using aPY (Momomura et al., 1988). The abbreviations used are: PI 3-kinase, phosphatidylinositol 3- Confluent monolayers of transfected CHO cells in 60-mm plates were kinase; aPY, antibodies specific for phosphotyrosine; CHO, Chinese treated with or without insulin for 1min at 37 "C and lysed in buffer hamster ovary; IRS-1, insulin receptor substrate-1; aIR1, monoclonal A containing 1%Triton X-100. Rabbit polyclonal aPY (1:lOO d i h antibody in the human insulin receptor; HIR, human insulin receptor; tion) preadsorbed to Pansorbin (1:25 dilution) was added to thelysate, and the incubation was continued for 2 h at 4 'C. ImmunoprecipitaSH2, Src-homology 2; SH3, Src-homology 3.

Substrate Phosphorylation in Insulin Receptor Mutant tion with aPY was performed as described (Ando et al. 1992). Total cell lysates or immunoprecipitates were boiled in Laemmli’s (1970) sample buffer (with dithiothreitol) for 5 min. To quantitate tyrosine phosphorylation of the insulin receptor, samples with maximal tyrosine phosphorylation (CHO-HIR stimulatedwith lo-’ M insulin) were subjected to immunoblots a t different concentrations. Quantitation was performed by excising labeled bands and counting in a y-counter. In anotherexperiment,immunoprecipitationwith a-56 was performed instead of aPY. The samples were electrophoresed on 7% polyacrylamide-SDS gels, transferred to polyvinylidene difluoride membranes (Millipore), and probed with aPY and 12sI-proteinA to detect phosphotyrosine of IRS-1. 2-Deoxyglucose Uptake and Thymidine Incorporation-2-Deoxyglucose uptake assays were performed as described previously (Chou et al., 1987). Measurements of thymidine incorporation into DNA were performed as described (Livneh et al., 1986). Confluent monolayers of cells in 24-well plates were maintained for 36 h a t 37 “C in RPMI 1640 containing 0.05% fetal calf serum. Insulin was then added to the wells for 15 h. The cells were then pulsed with 18 kBq/well [methyl-3H]thymidine for 2 h, washed three times with phosphatebuffered saline, and cold 10% trichloroacetic acid was added. After 60 min on ice, the wells were washed once with 10% trichloroacetic acid, solubilized with 0.5% SDS, and counted for radioactivity. Insulin Internalization-This procedure was performed as described (H. Kadowaki et al., 1990) with some modification (Ando et al., 1992). ’”I-Insulin (-0.1 ng/ml) in 1 ml of binding buffer (Ham’s F-12 containing 0.1% bovine serum albumin, pH 7.8) was added to cells in each well of six-well plates, and theincubation was continued for 4 h a t 4 “C. Cells were incubated in the binding buffer a t 37 “C for 5 min. The medium was removed, and the radioactivity in the The cells were rapidly chilled on ice, and medium was measured (S). 2 ml of acid-binding buffer (binding buffer adjusted to pH 3.5) was added to the cells. After 3 min, the medium was removed in order to measure its radioactivity (C1). Another 2 ml of acid binding buffer (pH 3.5) was added to cells, the incubation was continued for an additional 3 min, the medium was removed, and radioactivity was measured (C2). Following the acid wash, the cells were solubilized with 1 ml of 2% Triton X-100 for 5 min, and the contentof radioactivity was measured ( E ) . The datawere analyzed according to the following formula: internalized 1261-insulin= ( E / @ + Cl+ C, E ) ) X loo(%).

In:rL Concentration(M) FIG. 1. Insulin binding to transfected CHO cells. Binding of ‘%I-insulinto thecell lines was measured as described under “Experimental Procedures.” Results were presented as the percentage of tracer binding. The number of the receptors on the cell surface was 3.0 X 10’ in CHO-HIR and3.7 X lo6 in two clones of the CHO-de182, respectively. 0, CHO-HIR A,CHO-de182a; CHO-deB2b.

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Insulin Binding-To estimate the number of receptors expressed on the cell surface and the affinity of the different insulin receptors, we measured the binding of 1251-insulinto the cells expressing wild type or mutant insulin receptors by displacement of 1251-insulin by cold insulin. The cells expressing the wild type receptor (CHO-HIR) expressed 3.0 X los receptors/cell, whereas the cells transfected with the de182 mutant (CHO-de1821expressed 3.7 x lo6 receptors/cell. Since untransfected cells bound 1-2% of added insulin, which corresponded to approximately 2,000 receptors/cell, more than 99% of the receptors on the surface of the transfected cells were recombinant human insulin receptors encoded by the transfected cDNA. In addition, de182 receptors bound insulin with almost the same affinity as wild type receptors (Fig. 1). Half-maximal competition of tracer binding was seen at 0.4 nM insulin for HIR and at 0.3 nM insulin for the two clones of the de182 mutant, respectively. Autophosphorylatwn of Solubilized, Lectin-purified Transfected Receptors-Insulin stimulated the phosphorylation of the &subunit of the wild type human receptor in vitro. Autophosphorylation derived from the parental CHO cells was only faintly detectable in the aIR1immunoprecipitate. Under the conditions used in Fig. 2A, insulin-stimulated autophosphorylation of de182 was hardly detectable. Insulin-dependent Tyrosine Kinase Activities of the Transfected Receptors in a Cell-free System-The protein tyrosine kinase activity of the insulin receptors was examined by their ability to phosphorylate Glu-Tyr copolymers (4:l) in a cellfree system. The phosphorylation of Glu-Tyr copolymers was

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FIG. 2. Receptor tyrosine kinase activity in vitro. Panel A, autophosphorylation of the wild type insulinreceptor and the mutant receptor (de182). Insulin receptors on parental CHO, CHO-HIR, or CHO-de182 purified by wheat germ agglutinin-agarose (300 fmol of insulin binding capacity) were immunoprecipitated by aIRl andwere subjected to autophosphorylation. The assay was performed as described under “Experimental Procedures.” Autoradiograms were exposed a t -70 “C for 48 h. Panel E , phosphorylation of Glu-Tyr copolymers by the wild type insulinreceptor and the mutantreceptor (de182). Partially purified HIR and mutant receptors (300 fmol of insulin binding capacity) were exposed to insulin and then allowed to phosphorylate Glu-Tyr copolymers in the presence of [y3*P]ATP as described under“Experimental Procedures.” The results were presented as the (means ? S.D.) percentage over basal.

evaluated in the immune complex (whichcontained 300 fmol of insulin binding capacity) in the presence of 50 pM [y”P] ATP, 10 mM MgC12,2 mM MnC12with or without insulin for 10 min at 25 “ C . As shown in Fig.2B, aIR1-purified HIR exhibited an insulin-stimulatable kinase activity by 3.2-fold, whereas the insulin-stimulatable kinase activity of aIR1purified de182 was severelyimpaired.


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Tyrosyl Phosphorylation in Intact Cells-Insulin is known to induce tyrosyl phosphorylation of several cellular proteins in intact cells. As shown in Fig. 3A, by immunoblot using aPY antibodies as a probe, a previously described 185-kDa phosphoprotein (pp185) was phosphorylated on tyrosine residues in response to insulin (White et al., 1985; T. Kadowaki et al., 1987; Momomuraet al., 1988; Tashiro-Hashimoto et al., 1989; Tobe et al., 1990). The phosphorylation of pp185 and the insulin receptor was evaluated in the aPY immunoprecipitates (Fig. 3A, upper panel). The phosphorylation of pp185 in CHO-de182 was almost equal to that in CHO-HIR cells. Very similar results were obtained when total cell lysates instead of aPY immunoprecipitates were used (Fig. 3A, lower panel). In contrastto the phosphorylation level of pp185, decreased insulin-stimulated autophosphorylation of the mutantinsulin receptor (de182) wasobserved (Fig.3A, upperpanel), although the amount of the mutant insulin receptor detected by immunoblot using T-29 as a probe was the same as thatof the wild type receptor (Fig. 3B). The amount of autophosphorylated de182 was then compared with that of autophosphorylated HIR by varying the amount of the aPY immunoprecipitates subjected to SDS-polyacrylamide gel electrophoresis, followed by immunoblots with aPY antibodies and counting the band with a y-counter. Autophosphorylation of de182 was decreased by 75% as compared with that of HIR (Fig. 3C). An in uiuo labeling experiment has also revealed that phosphorylation of the del82 mutant receptor was undetectable in the basal state under our assay conditions, and theamount of insulin-stimulatable phosphorylation of the mutant receptor was very small (data not shown). Next we examined whether IRS-1 in CHO-deB2, as well as pp185 in the mutant cells, was as efficiently phosphorylated in response to insulin as that in CHO-HIR cells. Tyrosyl phosphorylation of IRS-1 was evaluated in the a-56 (polyclonal antibodies against the epitopes of IRS-1) immunoprecipitates and compared with pp185 in the aPY immunoprecipitates (Fig. 3 0 ) . In CHO cells, a significant amount of the pp185 seemed to be derived from phosphorylated IRS-1. Insulin-induced tyrosyl phosphorylation of IRS-1 in CHO-de182 evaluated by immunoblots using aPY antibodies as a probe was almost equal to thatin CHO-HIR cells (Fig. 3 0 ) .

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FIG. 3. Insulin-dependent tyrosine phosphorylation of an endogenous substrate (pp185 and IRS-1) in CHO cells. A,

insulin-induced tyrosine phosphorylation in total cell lysates and in aPY immunoprecipitates. Cells (confluent monolayers from two 100mm plates) were incubated with the indicatedconcentrations of insulin for 1 min. Extracts were prepared, and total lysates (lower p a n e l ) or aPY immunoprecipitates (upper panel) were subjected to immunoblots as described under “Experimental Procedures.” Autoradiograms were exposed a t -70 ‘C for 48 h. B, immunoblot of insulin receptor. Equal amount of extracts used in A, lower p a n e l , were immunoprecipitated with aIR1, and immunoblots against T-29 were performed as described under “Experimental Procedures.” Autoradiograms were exposed a t -70 “C for 48 h. C, quantitation of phosphorylated insulin receptor. Samples with maximal tyrosine phosphorylation (CHO-HIR stimulated with M insulin) were subjected to immunoprecipitation with aPY.These samples were subjected to immunoblots a t different concentrations. Quantitation was performed by excising labeled bands or unlabeled squares (as background) and countinginay-counter. Data are presented as percentage of maximal stimulation and the means k S.D.of two separate experiments. 0, CHO-HIR 0, CHO-deB2. D, insulin-induced phosphorylation of IRS-1. Cells (confluent monolayers from two 100-mm plates) were incubated with or without M insulin for 1 min. Extracts were prepared as described under “Experimental control Procedures” and immunoprecipitated by aPY, a-56 (aZRS-I), nonimmune immunoglobulin (C),or a I R l (IR).The immunoprecipitates were then subjected to immunoblots against aPY as described under “Experimental Procedures.” Autoradiograms were exposed for -70 “C for 48 h. Lanes I , 4, 7, and 10, parental CHO; lanes 2, 5 , 8 , 11,13, and 14,CHO-HIR lanes 3,6,9, and 12,CHO-deB2.


Insulin in

PIS-Kinase Actiuity in the Immunoprecipitate-PI 3-kinase activity was evaluated in the aPY immunoprecipitates. As shown in Fig. 4, insulin-stimulatable PI 3-kinase activation was observed in CHO-HIR and CHO-del82 cells. Maximal activation of PI 3-kinase as well as sensitivity to insulin in this assay between the two celllines showed no difference. 2-Deonyglucose Uptake and Thymidine Incorporation into DNA-Insulin is able to stimulate 2-deoxyglucose uptake in these cell lines (Fig. 5). Half-maximal stimulation occurred at 5 x 10"' M insulin in parental CHO cells, at 2 x 10"' M in CHO-HIR, and at 2 X 10"' M in two clones of CHO-del82, respectively. In a representative experiment, basal and maximally stimulated 2-deoxyglucose uptake were: CHO-HIR, 3,767 + 6,797 dpm; CHO-del82a,4,044 + 11,785 dpm; CHOdel82b,4,013 + 11,649 dpm; and parental CHO,4,768 + 9,423 dpm. Thus CHO-del82 exhibited the same insulin sensitivity as CHO-HIR with respect to 2-deoxyglucose uptake. Insulin, like other growth factors, is able to stimulate thymidine incorporation into DNA (Fig. 6). Half-maximal stimulation occurred at 3 X lo-' M insulin in parental CHO cells, at 6 X lo-" M in CHO-HIR, and at 10"' M and 3 X lo-" M in two clones of CHO-del82, respectively.In one representative experiment, incorporation of thymidine into DNA was stimulated to 405% in parental CHO cells (10,327 + 41,867 cpm), 637% in CHO-HIR (6,830 + 43,510 cpm), 548% in CHO-del82a (6,344+ 34,763 cpm), and 321% in CHO-del82b (18,187 cpm+58,469cpm) of unstimulated cells, respectively. Thus, CHO-del82 also exhibited the same insulin sensitivity as CHO-HIR with respect to thymidine incorporation into DNA. Insulin Internalization-Insulin internalization was assessed by allowing '251-insulinto bind to thecells at 4 "C and then shifting the temperature to 37 "C. After 5 min a t 37 "C,

Receptor Mutant

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FIG.6. Stimulation of thymidine incorporation into DNA. Cells were incubated with the indicated concentrations of insulin. After incubation with[methyL3H]thymidine (18 kBq/well) for 2 h, trichloroacetic acid-precipitable radioactivity was measured. Allof the assays were performed in duplicate. The results are expressed as the (means& S.D.) percentage of maximal stimulation by insulin and are themeans of three separateexperiments. 0, CHO-HIR, A,CHOde182a; H, CHO-deB2b; 0,parental CHO.

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FIG.4. PI 3-kinase activity. Cells were incubatedwith the indicated concentrations of insulin for 1 min a t 37 'C, lysed and immunoprecipitated with aPY. PI 3-kinase activity was analyzed as described under "Experimental Procedures." Autoradiograms were exposed a t -70 "C for 5 min.

FIG.7. Insulin internalization. Cells were incubated for 5 min with '?-insulin, and the amount of internalized 'aI-insulin was measured as described under "Experimental Procedures." The acidresistant radioactivity was normalized to total cell-associated radioactivity for each cell line. This value (acid-resistant radioactivity/ total radioactivity X 100) is termed percent internalized. The results are themeans of three separateexperiments (means f S.D.). 0,CHOH I R A, CHO-deB2.

CHO-HIR internalized 37% of labeled insulin (Fig. 7). The amount of insulin internalized by CHO-del82 was smaller than CHO-HIR (20% after 5 min). Consistent with previous reports (Hari et al., 1987; McClain et al., 1987; Russell et al., 1987), the amount of insulin internalized by cells expressing kinase-defective insulin receptor-mutated Arg for Lys-1018 (CHO R1018K) was much lowerthan thatby CHO-HIR (5% after 5 min, data not shown). DISCUSSION

In thepresent study, we have characterized a mutant insulin receptor that lacked 82 amino acids of the B-subunit (del82). This del82 mutant hasunique characteristics. Insulin binding to the mutant receptor was normal; however, autophosphorInwlln ConcmtratlMI (M) ylation and tyrosine kinase activity of del82 toward exogenous FIG.5. 2-deoxyglucose uptake. Cells were incubated with the substrates were markedly decreased in vitro. In fact, we could 1,2-3H]glu- not detect autophosphorylation or tyrosine kinase activities indicated concentrations of insulin and with 2-deoxy-~-[ cose as described under "Experimental Procedures." The results are expressed as the (means f S.D.) percentage of maximal stimulation against Glu-Tyr copolymers of del82 in vitro under our assay and themeans of three separate experiments. 0,CHO-HIR A,CHO- conditions (Fig. 2). It is interesting to note that in intact cells, this mutant deB2a; H,CHO-deB2b; 0 ,parental CHO.

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receptor was able to phosphorylate itself, although the extent of autophosphorylation was greatly decreased (Fig. 3). There are some mutant insulin receptors whose autophosphorylation is impaired either by the mutations within the ATP binding domains (R1018K) (Chou et al., 1987) or by the mutations within the autophosphorylation sites (the mutants Phe for Tyr-1150 (F1150Y), Phe for Tyr-1150 and 1151 (F1150Y, F1151Y) (Ellis et al., 1986; Zang et al., 1991), and Phe for Tyr-1146, 1150, and 1151 (F1146Y, F1150Y, F1151Y) (Murakami and Rosen, 1991)). In these mutant insulin receptors, a good correlation is observed between the extent of insulinstimulatable autophosphorylation in vitro and that in uiuo. Nevertheless, it seems possible that in the cases of some mutant insulin receptors like de182, the extent of autophosphorylation in vitro does not always represent thoseactivities in uiuo. The 8-subunit of insulin receptor undergoes autophosphorylation on several tyrosine residues immediately after binding of insulin. The major autophosphorylation sites inthe insulin receptor are located in two @subunit regions; the region in the kinase domain (at Tyr-1146, Tyr-1150, and Tyr-1151) and that in COOH terminus (at Tyr-1316 and Tyr-1322) (Tornqvist et al., 1987, 1988; White et al., 1988a). In intact cells, de182 receptors displayed decreased (25% of HIR, Fig. 3) autophosphorylation. In the present study, tyrosines 1316 and 1322 were deleted; however, the reduced level of autophosphorylation of the de182 mutant is not fully explained by the removal of these tyrosines because the maximal response of insulin-stimulatable autophosphorylation was decreased to a smaller extent in the mutants lacking these tyrosines(Myers et al., 1991; Andoet al., 1992).4This is in contrast to thecase of epidermal growth factor receptors, in which the autophosphorylation and the kinase activities remain intact by the deletion of the entire turn-acidic helix-turn motifs (Chen et al., 1989). A COOH-terminal domain adjacent to the kinase domain seems to be necessary for.full autophosphorylation of the receptor induced by insulin. There have been controversies on how the autophosphorylated insulin receptor transduces signals downstream. Some put emphasis on the conformational changes of the insulin receptor caused by autophosphorylation stimulated with insulin (Hawley et al., 1989; Maddux and Goldfine, 1991), whereas others believe that the insulin-stimulated tyrosine phosphorylation of cellular substrates such as IRS-1 by the insulin receptor is an initial step required for insulin action (KahnandWhite, 1988). Studies on themutant insulin receptors by site-directed mutagenesis give some clue to this question. In general, an insulin-induced increase in autophosphorylation of the receptor determines the level of substrate phosphorylation such as that of pp185 and IRS-1, and correlation of somebiological effects of insulin with the phosphorylation of both of insulin receptor and pp185 has been observed ina variety of mutant insulin receptor such as R1018K (Chou et al., 1987), Phe for Tyr-1146 (F1146Y) (Backer et al., 1992), F1150Y, F1151Y (Yonezawa and Roth, 1991), F1146Y, F1150Y, and F1151Y (Wilden et al., 1992a). These studies, however, did not answer whether autophosphorylationorsubstrate phosphorylation is more directly linked to insulin's biological effects. In this context, studies with the mutant insulin receptors with substitution of Phe for Tyr-960 (F960Y) areimportant. This mutant displays normal autophosphorylation of insulin receptor, yet phosphorylation of pp185 is reduced. Thus, Tyr-960of the insulin receptor may be necessary for interacting insulin receptor with pp185 and IRS-1. Interestingly, many kinds of the bio-

'K. Momomura and T. Kadowaki, unpublished observation.

in Insulin Receptor Mutant logical effects of insulin are impaired in this mutant, suggesting that autophosphorylation of the receptor is not sufficient, and pp185 is required for some metabolic and mitogenic signaling (White et al., 1988b).6We have now shown that de182 receptors displayed decreased autophosphorylation, yet they displayed a normal ability to phosphorylate pp185 and IRS-1, a normal ability to activate PI 3-kinase, and some normal metabolic and mitogenic signaling properties. Thus, the present study of the de182 mutant has proven that substrate phosphorylation such as that of pp185 and IRS-1, rather thanautophosphorylation of the receptor per se, might be closely correlated with some metabolic and mitogenic signaling, suggesting that substrate phosphorylation could be sufficient for some signaling evoked by insulin in a situation in which the extent of phosphorylation of substrates such as pp185 and IRS-1 does not correlate with that of autophosphorylation of the receptor. PI 3-kinase is known to be activated by many oncogenes and growth factor receptors which have tyrosine kinase activities (Cantley et al., 1991). PI 3-kinase is composed of an 85-kDa regulatory subunit that contains one SH3 and two SH2 domains and a 110-kDa catalytic subunit (Escobedo et al., 1991; Skolnic et al., 1991; Otsu et al., 1991). When cells are stimulated with insulin, this enzyme activity is found to be increased in antiphosphotyrosine immunoprecipitates (Ruderman et al., 1990; Endemann et al., 1991). An earlier reporthas proposed the direct association of PI 3-kinase activity with the autophosphorylated receptor (Ruderman et al., 1990) potentially via the interaction of the SH2 domains of the 85-kDa subunit with YTHM (residues 1322-1325) of the insulin receptor, a homolog of the YMXM motif (Cantley et al., 1991). More recently, the association of PI 3-kinase activity with IRS-1 via the interaction of the SH2 domains of the 85-kDa subunit with the YMXM motifs of IRS-1 was proposed (Sun et al., 1991). Normal insulin-stimulatable PI 3-kinase activity and normal IRS-1 phosphorylation of de182 mutant insulin receptors (which lacked the YTHM motif and also showed severely decreased autophosphorylation)are clearly consistent with the latter proposal. PI 3-kinase activation is impaired in spite of normal autophosphorylation in the mutant insulin receptor F960Y or the mutant lacking 12 amino acids including Tyr-960, both of whichshowed decreased tyrosine phosphorylation of pp185. Moreover, PI 3kinase activation is normal in the mutantlacking the homolog of YMXM motif (Phe for Tyr-1316, 1322 or themutant lacking COOH-terminal 36 or 43 amino acids) which showed normal tyrosine phosphorylation of IRS-1 (Backer et al., 1992; Yonezawa et al., 1992).5These earlier observations also support the latter proposal. Ligand-induced internalization was moderately decreased in CHO-de182 cells. Thus, in contrast to epidermal growth factor receptor (Chen et al., 1989), in which breakdown of helix-turn motifs in the COOH-terminal domain of the epidermal growth factor receptors was detremental, the breakdown of turn-acidic helix-turn motifs of the insulin receptor seems to affect ligand-induced internalization to a small degree. In summary, the following can be stated. 1) The COOHterminal region adjacent to thekinase domain may be necessary for full autophosphorylation of the insulin receptor induced by insulin. 2) In contrast to a moderate decrease in insulin-stimulatable autophosphorylation of the insulin receptor in vivo,de182 exhibited severely impaired insulinKaburagi, Y., Momomura, K., Yamamoto-Honda, R., Tobe, K., Tamori, Y., Sakura, H., Akanuma, Y., Yazaki, Y., and Kadowaki, T. (1993) J. Bid. Chern. 268, 16610-16622.


Substrate Phosphorylation induced autophosphorylation and kinase activities of the insulin receptor in vitro. 3) The COOH-terminal domain of the insulin receptor may play an inhibitory role for phosphorylation of pp185 and IRS-1. 4) In the case of this mutant receptor deB2, insulin-induced phosphorylation of the substrates can occur without full autophosphorylation of the insulin receptor, and phosphorylation of the substratesmight be sufficient for activating PI 3-kinase and certain biological effects such as 2-deoxyglucose uptake and thymidine incorporation into DNA. Acknowledgments-We thank Dr. Kinori Kosaka, Dr. Ryoko Hagura, Dr. Narihito Yoshioka, Dr. Hiroko Kadowaki, and Director Hajime Kawashima (The Institute for Diabetes Care and Research, Asahi Life Foundation) for valuable support. We also appreciate the support offered by Dr. Akifumi Ando (Kobe University School of Medicine) for biological assay. Furthermore, Kimiko Toyoshima is acknowledged for skillful typing of this manuscript. Finally, we greatly appreciate the generous gift of insulin receptor cDNA from Dr. G . I. Bell. REFERENCES Ando, A., Momomura, K., Tobe, K., Yamamoto-Honda, R., Sakura, H., Tamori, Y., Kaburagi, Y., Koshio, O., Akanuma, Y., Yazaki, Y., Kasuga, M., and Kadowaki, T. (1992) J. Biol. Chem. 267,12788-12796 Backer, J. M., Schroeder, G. G., Kahn, C. R., Myers, M. G., Jr., Wilden, P. A,, Cahill, D. A., and White, M. F. (1992) J. Biol. Chem. 267,1367-1374 Baron! V., Gautier, N., Komoriya, A,, Hainaut, P., Scimeca, J. C., Mervic,, M., Lavielle, S., Dolais-Kitabgi, J., and Van-Obberghen, E. (1990) Blochemutry 29,4634-4641 Cantlev, L. C., Auaer. K. R.. Camenter. C.. Duckworth. B.. Graziani.. A... Kapeiler, R.,andsoitoff, S. (1991) Cell 64,281-302 Chen, W. S., Lazer, C. S., Lund, K. A., Welsh, J. B., Chan ,C P , Walton, G. M., Der, C. J., Wiley, H. S., Gill, G. N., and Rosenfeld, G: (1989) Cell 6 9 , 33-43 chOU,c. K., Dull, T. J., Russell, D. S., Gherzi, R., Lebwohl, D., Ullrich, A., and Rosen, 0. M. (1987) J. Biol. Chem. 2 6 2 , 1842-1847 Ellis, L., Clauser, E., Morgan, D. O., Marc, E., Roth, R. A., and Rutter, W. J. (1986) Cell 4 6 , 721-732 Endemann, G., Yonezawa, K., and Roth,R. A. (1990) J. Bioi. Chem. 266,396'

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