An Extracellular Domain of the Insulin Receptor - The Journal of ...

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eter. PSIMethionine Labeling of the Insulin Receptor-Confluent monolayers of Fao or Hep G2 cells in 100-mm dishes were incubated with methionine-free MEM ...
Vol. 264, No. 15, Issue of May 25,pp. 8627-8635,1989 Printed in U.S.A.

THEJOURNAL OF BIOLOGICAL CHEMXSTRY 0 1989 by The American Society for Biochemistry and Molecular Biology, Inc.

An Extracellular Domain of the Insulin Receptor @-Subunit with Regulatory Function on Protein-Tyrosine Kinase* (Received for publication, August 16, 1988)

Roberto Gherzi, Giorgio SestiS, Gabriella Andraghetti,Roberto De Pirrop, RenatoLauroS, Lucian0 Adezati, and Renzo Corderall From the Department of Internal Medicine, University of Genoua, 16132 Genoua, Italy, the $Department of Internal Medicine, 2nd University of Roma, Roma, Italy, and the §Division of Endocrinology, University of Ancona, Ancona, Italy

Anti-insulin receptor monoclonal antibody MA- 10 inhibits insulin receptor autophosphorylation of purified rat liver insulin receptors without affectinginsulin binding (Cordera, R., Andraghetti, G., Gherzi, R., Adezati, L., Montemurro, A., Lauro, R., Goldfine, I. D., and De Pirro, R. (1987)Endocrinology 121,20072010). The effect of MA-10 on insulin receptor autophosphorylation and on two insulin actions (thymidine incorporation intoDNA and receptordown-regulation) was investigated in rat hepatoma Fao cells. MA-10 inhibits insulin-stimulated receptor autophosphorylation, thymidine incorporation into DNA, and insulininduced receptor down-regulation without affecting insulin receptor binding. We show that MA-10 binds to a site of rat insulin receptors different from the insulin bindingsite in intact Fao cells. Insulin does not inhibit MA-10 binding, and MA-10 does not inhibit insulin binding rat to Fao cells. Moreover, MA-10 binding to down-regulated cells is reduced to the same extent as insulin binding. In rat insulin receptors the MA-10 binding site has been tentatively localized in the extracellular partof the insulin receptor @-subunit based on the following evidence: (i) MA-10 binds to insulin receptor in intact rat cells; (ii) MA-10 immunoprecipitates isolated insulinreceptor B-subunits labeled with both [“S]methionine and ”P; (iii)MA-10 reactswith rat insulin receptor@-subunits by the method of immunoblotting, similar to an antipeptide antibody directed against the carboxyl terminus of the insulin receptor @-subunit.Moreover, MA- 10 inhibits autophosphorylation and protein-tyrosine kinase activity of reduced and purified insulin receptor @-subunits. The finding that MA-10 inhibits insulin-stimulated receptor autophosphorylation and reduces insulin-stimulated thymidine incorporation into DNA and receptor down-regulation suggests that the extracellular partof the insulin receptor @-subunitplays arole in the regulation of insulin receptor protein-tyrosine kinase activity.

subunits (Mr135,000 bySDS-PAGE),’ comprising the insulin binding site and located entirely at the extracellular face of the plasma membrane, and two @-subunits (Mr 95,000 by SDS-PAGE) that are protein-tyrosine kinases and span the plasma membrane. Insulin binding to insulin receptor @subunit causes rapid phosphorylation of tyrosine residues on the receptor @-subunit(for a review, see Ref. 1). Although the structure of insulin receptor is characterized (2,3) and therelevance of its protein-tyrosinekinase activity is defined (4-9), less information is available on the relative importance of different domains of the molecule in the regulation of its enzymatic activity (10-12). The following data suggest that the insulin receptor is composed of functionally independent domains: first, a-subunits of chimeric receptors both lacking the cytoplasmic portion of the @-subunit and presentingsubstitutionsinthis domain bind insulin with normal affinity (13, 14); second, in the absence of the asubunit, the cytosolic part of insulin receptor P-subunit, expressed in Chinese hamster ovary cells, is a protein-tyrosine kinase more active than the entire receptor (15); third, the protein-tyrosine kinase of the cytoplasmic domain of insulin receptor can be activated by autophosphorylation independently of its organization within the native receptor oligomer (16); fourth, some agents (sodium orthovanadate, hydrogen peroxide, anti-@-subunitmonoclonal antibodies) activate insulin receptor protein-tyrosine kinase directly acting on @subunits (17-19). Monoclonal antibodies are convenient probes to understand the functional topography of receptor molecules (20-23). In previous reports we demonstrated that protein-tyrosine kinase activity, stimulated in the entire insulin receptor by insulin and insulin mimickers in vitro, can be inhibited by the anti-insulin receptor monoclonal antibody MA-10 (24, 25). Based on this finding we suggested that MA-10 recognizes a district, conserved in human andrat insulin receptor, involved in the regulation of protein-tyrosine kinase activity but different from the insulin binding site (24). In this paper we investigate: (i) whether MA-10 recognizes insulin receptors in intact rat cells, (ii) whether the portion of insulin receptor molecule, recognized by antibody MA-10, The insulin receptor is a transmembraneglycoprotein com- is important to regulate receptor autophosphorylation in inposed of distinct subunits linked by disulfide bonds: two a- tact cells and whether the inhibition of the enzyme by MA-

* This work was supported in part by grants from M.P.I. (to R. DP.) and by Grant 870008204 from Consiglio Nazionale delle Richerche and grantsfrom M.P.I. and NOVAItalia (to R. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore he hereby marked “aduertisernent” in accordance with18U.S.C. Section 1734 solely to indicate this fact. 7To whom correspondence shouldbe addressed I.S.M.I. University of Genova, Viale Benedetto XV, 6, 16132 Genova, Italy.

* The abbreviations used are: SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; DTT, dithiothreitol; NEM, Nethylmaleimide; IgG, immunoglobulin G; WGA, wheat germ agglutinin; AhPC, antibody PC; DME, Dulbecco’s modified essential medium; PBS, phosphate-buffered saline; Hepes, N-2-hydroxyethylpiperazine-N’-2-ethanesulfonicacid; BSA, fraction V fatty acid-free bovine serum albumin; EGTA, [ethylenebis(oxyethylenenitrilo)]tetraacetic acid MEM, minimal essential medium; mAb, monoclonal antibody.

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10 has biological significance, and (iii)whether MA-10 is able to inhibit the protein-tyrosine kinase activity of the isolated P-subunit of insulin receptor. Data presented herein demonstrate that MA-10 inhibits insulin receptor autophosphorylation and thetransduction of two insulin actions in insulin-responsive rat hepatoma cells binding to a region of the receptor molecule whichis different from the insulin binding site and which is localized in the protein backbone of the extracellular portion of insulin receptor P-subunit. EXPERIMENTAL PROCEDURES

Materials-Porcine insulin, '261-A-14-monoiodohuman insulin (226 mCi/mM) and '251-A-14-monoiodoporcine insulin (230 mCi/ mM)were kindly supplied byNovo Industries (Copenhagen, Denmark). [y3'P]ATP (3000Ci/mM), [meth~l-~HIthymidine (20 Ci/ mmol), [35S]methionine(1150 Ci/mmol), and '261-proteinA (81 pCi/ pg) were purchased from Du Pont-New England Nuclear. Hybond-N nylon blotting membranes and Amplify werefrom Amersham (Buckinghamshire, England).ATP, Triton X-100, phenylmethysulfonyl fluoride, bacitracin, sodium orthovanadate, N-acetyl-D-glucosamine, dithiothreitol (DTT), N-ethylmaleimide (NEM), L-methionine, normal mouse immunoglobulin G (IgG), and polyethylene glycol 6000 were purchased fromSigma. Tunicamycin was from Boehringer Mannheim. Wheat germ agglutinin-agarose (WGA) was obtained from Vector Laboratories (Burlingame, CA), histone HF2B was from Cooper Biomedical (Malvern, PA). Protein A-Sepharose CL4B was from Pharmacia (Uppsala, Sweden), GF5 desalting columns were from Pierce. SM-2 Bio-Beads, gelatin, and all materials for SDSPAGE were purchased from Bio-Rad. Prestained molecular weight markers for SDS-PAGE were from Bethesda Research Laboratories. Cell culture media, fetal calf serum, and dialyzed fetal calf serum were from Gibco. Tissue culture flasks and dishes were from Falcon (Oxnard, CA). All other chemicals were from sources cited in Ref. 26. Antibodies-Monoclonal antibody MA-10 is an IgG2b raised in mice against highly purified human placental receptors as previously described (27). MA-10 is devoid of any phosphatase or ATPase activity, it is not able to bind iodinated insulin, although an antiinsulin antiserum did (24), and itdoes not react with IGF I receptors (27). Antibody PC is a polyclonal antibody raised in rabbit against a synthetic peptide corresponding tothe deduced sequence of the carboxyl-terminal 17 amino acids of the human insulin receptor. This antibody immunoprecipitates both human and rat insulin receptors but is unable to immunoprecipitate IGF I receptors.233Anti-insulin receptor antibody ARA (a kind gift of Dr. Lawrence Mandarino) is a human polyclonal antibody that recognizes both human and rat insulin receptors (28). Antibody H 65.6 is a monoclonal IgG2b that recognizes a monomorphic epitope present on HLA class I1DQ molecules. Cells-Insulin-sensitive rat hepatoma cells Fao (kindly provided by Dr. Bernard Rossi, University of Nice, France) and human hepatoma cells Hep G2 (a generous gift of Dr. Ora Rosen, Sloan-Kettering Cancer Center, New York) were grown in DME with 10% fetal calf serum. Insulin and MA-10 Binding-Insulin binding to Fao and Hep G2 cells was measured for 4 h at 4 "C as described in Ref. 6. Scatchard analysis of insulin binding to both human and rat hepatoma cells, measured under these conditions, indicated that Fa0 and Hep G2 cells have approximately 31,000 and 26,000high affinity insulin receptors per cell, respectively. The affinity constants of '"I-insulin for high affinity receptors of Fao and Hep G2 are both about 1 nM. The binding affinities of both human and porcine '"I-insulin to rat (Fao) and human (Hep G2) hepatoma cells are superimposable. To measure MA-10 binding, cells in six-well dishes were incubated with different concentrations of MA-10 or mAb H 65.6 together with 1.5 x IO5 cpm '251-proteinA/well for 4 h at 4 "C. Then cells were washed, solubilized, and counted for radioactivity as described in Ref. 6. In some experiments '251-insulinbinding to Hep G2 and Fao cells was measured at 37 "C. MA-10 Induced Receptor Internalization-Confluent Fao or Hep G2 cells in six-well dishes were incubated with M MA-10 or *A. Pessino, R. Longhi, G. Damiani, and R. Cordera, submitted for publication. A. Pessino and R. Cordera, unpublished data.

M normal mouse IgG together with 1.5 X lo5 cpm IZ51-protein A/well for 4 h at 4 "C. Subsequently cells were washed with ice-cold PBS and incubated with prewarmed binding buffer (50 mM Hepes, pH 8,0, 150 mM NaCl, 10 mM CaC12,10 mM MgS04, 10 mM glucose, 1%BSA) for 15 min at 37 "C. Cells werethen moved to 4 "C and either washed with PBS four times (controls) or washed twice for a total of 6 min with 0.5 M sodium acetate buffer, pH 3.5, containing 150 mM NaCl to remove MA-10-'251-proteinA complexes bound to thecell surface. This procedure, modified from Ref. 29, removes insulin or antibody bound to thecell surface (6). The acid-treated cells were then washed two more times with PBS. Cells were solubilized by the addition of 0.1% SDS, and the radioactivity of the lysates was counted. The radioactivity associated with the cells, washed at pH 7.4 in PBS, represented the total cell-associated ligand, while the radioactivity present inthe lysates of acid-washed cells represented the internalized (acid wash-insensitive) MA-10-'"I-protein A. Nonspecific "'I-protein A binding was measured incubating cells with lo-' M normal mouse IgG. Insulin Receptor Down-regulation-Confluent monolayers in sixwell dishes were incubated at 37 "C for 20 h in complete medium with mAb H 65.6 M) or insulin M) in the presence of M mAb H 65.6 or lo-' M MA-10. Followingincubation, cells were placed at 4 "C, surface-bound insulin or immunoglobulins were removed by washing twice for a total of 6 min with 500 mM sodium acetate buffer, pH 3.5, containing 150 mM NaC1, followed by three additional washes with PBS. The residual insulin or MA-10binding activity on the cell surface was then determined by incubation with 1261-insulinor MA10 and "'I-protein A for 4 h at 4 "C, as indicated above. Thymidine Incorporation into DNA-Assayswere performed as described in Ref. 4 except that cells were serum-starved in DME containing 0.5% dialyzed BSA for 30 h, and the incubation with insulin and MA-10 prior to theaddition of [3H]thymidinewas carried out in 0.5% dialyzed BSA instead of 0.5% fetal calf serum. Receptor Phosphorylation in Intact Fa0 Cells-Confluent Fao cells (lo' cells/plate) were incubated for 1 h at 37"C in phosphate-free MEM containing 20mM Hepes buffer, pH 7.4, and 1%dialyzed BSA, following which[32P]orthophosphate(0.25 mCi/ml) was added. After 90 min at 37 "C, either mAb H 65.6 M) (in some experiments, normal mouse IgG M)),insulin M) plus MA-10 (at different M) (in some experiments, concentrations), or mAb H 65.6 normal mouse IgG (10" M)) was added, and the incubations were continued for 10 min. Cells were then rapidly cooled in ice, washed three times with ice-cold PBS containing 5mM EDTA, 5 mM EGTA, and 1 mM sodium orthovanadate and dissolved in 0.5 ml of 50 mM Hepes buffer, pH 7.4, containing 2% Triton X-100, 5 mM EDTA, 5 mM EGTA, 20 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 20 mM NaF, 1 mM phenylmethylsulfonyl fluoride, and 20 pg/ ml each of aprotinin and leupeptin. The cell extracts were clarified by centrifugation at 100,000 X g for 30 min at 4 "C, and the supernatant fluid was applied to 0.3-ml columns of WGA-agarose;columns were washed with 20 volumes of 50 mM Hepes buffer, pH 7.4, containing 0.1% Triton X-100,20 mM sodium pyrophosphate, 20 mM NaF, 1 mM sodium orthovanadate, 5 mM EDTA, and 5 mM EGTA. The insulin receptor was then eluted in the same buffer containing 0.3 M N-acetylglucosamine. The eluate was incubated with the antipeptide polyclonal antibody AbPC or with the polyclonal anti-insulin receptor antibody ARA for 16 h a t 4 "C and the immune complex precipitated by protein A-Sepharose (8).Immunoprecipitated receptors were analyzed by SDS-PAGE and subjected to autoradiography. Gels were then rehydrated in 1 M KOH and heated a t 55 "C for 1 h. This treatment hydrolyzes most of phosphates incorporated into serine and threonine residues of the receptor but leaves phosphotyrosine residues relatively intact (30). Following incubation in KOH, gels were washed in 10% acetic acid, 40% methanol, 1%glycerol for 1 h, neutralized, dried, and subjected again to autoradiography. Both autoradiograms were scanned with an Ultroscan LKB laser densitometer. PSIMethionine Labeling of the Insulin Receptor-Confluent monolayers of Fao or Hep G2 cells in 100-mm dishes were incubated with methionine-free MEM containing 10% dialyzed fetal calf serum for 2 h. [35S]Methionine(200 pCi/ml) was added, and the incubation was continued further for 30 min. The medium waschanged to DME containing 10% fetal calf serum and 10 mM unlabeled L-methionine, and the incubation was continued for up to 8 h. Cells were then solubilized in 50 mM Hepes buffer, pH 7.4, containing 2% Triton X-100,5 mM EDTA, 5 mM EGTA, and 10 pg/ml each of aprotinin, leupeptin, and soybean trypsininhibitor. Cell lysates were centrifuged at 150,000 x g for 30 min at 4 "C. The

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supernatant fluid was diluted 20-fold into 50 mM Hepes buffer, pH not inhibit '251-insulinbinding to rathepatoma Fao cells even 7.4 containing 0.1% Triton X-100,150mM NaCl, 5 mM EDTA, 5 mM at M concentrationboth at 4 and 37 "C (Table I). MAEGTA, and the protease inhibitors (10Fg/ml each) and subjected to 10 binds to Hep G2, and insulin M) partiallyinhibits chromatography on WGA-agarose as described in Refs. 6 and 24. The MA-10 binding (Fig. 1);MA-10 also binds to Fao cells but, in insulin receptor was eluted with 0.3 M N-acetylglucosamine. RecepM ) does not affect MA-10 binding (Fig. tors were immunoprecipitated withMA-10, AbPC ormAb H 65.6 and this case, insulin 35S-labeled proteinsanalyzed by SDS-PAGE. Gels were stained, 1).It is noteworthy that: (i) MA-10 binding to Fao cells is destained, soaked (30 min,room temperature) in Amplify, and dried. right-shifted about 1 order of magnitude compared to MA-10 In experimentsdesigned to study whetherMA-10 recognizes nongly- binding to Hep G2 cells; and (ii) in the presence of 1O"j M cosylated insulin receptors Fao cells were incubated in methioninefree MEM (+lo% dialyzed fetal calf serum) in the presenceof 20 fig/ TABLEI ml tunicamycin.Tunicamycin (20pg/ml)was maintainedinthe incubation medium of cells during [35S]methioninepulse and during Effect of MA-10 on insulin binding to human and ratcells chase time. The viability of tunicamycin-treated cells was superimHep G2 and Fao cells were incubated for 4 h at 4 "C or for 2 h a t posable on that of control cells at the end of the incubation. Cells 37 "c in the presence of 'Z51-insulin (0.1 nM) and either 100 nM were then solubilized as described above and, to reduce the concen- monoclonal antibody H 65.6 or the indicated concentrationsof MAtration of Triton X-100 from2 to 0.1%, the clarified supernatant was 10. Cells were then washed three times with PBS at 4 "C andsolubichromatographedthrough a Bio-BeadsSM-2column. The eluted lized with 0.1% SDS. Aliquots of the lysate were analyzed for radiomaterial was concentrated by precipitation with 12.5% polyethylene activity. The average of three independent experiments, performed glycol 6000 and 0.2% 7-globulins (26,31).The pellet was resuspended in duplicate, for each cell line are presented. The values for 100% in 50 mM Hepes buffer, pH 7.4, containing 0.1% Triton X-100, 150 insulin binding were 8760 and 8200 cpm/106 Hep G2 and Fao cells, mM NaC1, aprotinin, leupeptin, andsoybean trypsin inhibitor (10 pg/ respectively, for binding experiments performedat 4 "C and3870 and ml each) and samplessubjected to immunoprecipitation. 3690 cpm/106 Hep G2 and Fao cells, respectively, for binding experReduction of Insulin Receptors into a- and @-Subunits-Insulin iments performed a t 37 "C. receptor disulfide bonds were reduced by incubating unlabeled, 35SInsulin binding activity or 32P-labeledWGA-purified receptor preparations at 22 "C with 100 mM DTT and 75 mM Tris (pH 8.5) for 30 min using a modification MA-10 Hep G2 Fao of the method described by Boni-Schnetzler et al. (32). The reaction 4 'C 37 "C 4 "C 37 "C was stopped by adding NEM (to 100 mM final concentration) and transferring tubes on ice. Then reduced receptors (indicatedas "DTT/ nM Tris-reduced") were chromatographed on a 2-ml GF5 desalting col100 100 100 0 100 umn equilibrated with HTGbuffer (50 mM Hepes, 0.05% Triton X81 96 93 0.1 83 100, 0.5% glycerol, 5 mM NEM) and eluted in the samebuffer. This 54 100 99 49 1 procedure results in a near completeremoval of DTT and Tris. Insulin 36 98 100 10 34 receptor @-subunits were immunoprecipitated with the antipeptide 100 28 98 30 98 polyclonal antibody PC, with MA-10, or withmAb H 65.6 as described in other sections. Insulin Receptor and Histone Phosphorylation in Vitro-Insulin 10000 receptors were partially purified from human placenta and rat liver E membranes using WGA-agarose as described (24). Insulin activation Q 0 and phosphorylation of WGA-purified receptors were conducted as v X previously reported (24,26). When WGA-purified receptors were a phosphorylated prior to being subjected todisulfide bonds reduction, thephosphorylationreaction was stopped by theaddition of 10 *Oo0 0 volumes of a 50 mM Hepes buffer, pH 7.4, containing 5 mM EDTA, a 5 mM EGTA, 1 mM unlabeled ATP, 1 mM sodium orthovanadate, 20 t mM NaF, 20 pg/ml aprotinin and leupeptin, and 1 mM phenylmeth.a ylsulfonyl fluoride. To evaluate the effect of MA-10 on autophos* 6000 phorylation and protein-tyrosine kinase activity of isolated insulin receptor @-subunit,aliquots of DTT/Tris-reducedreceptors were incubated with AbPC and the immune complexes, comprising insulin LD receptor @-subunit, precipitated with protein A-Sepharose CL4B. The immunoprecipitates were washed, resuspended in HTG buffer, and 4000 incubatedwith 10-j M MA-10 or M normal mouse IgG (in some experiments 10-7 M mAb H 65.6) for 4 h a t 4 "C. [y-32P]ATP (50mM (final concentration)MnCl2 were then added for 30 min a t 4 "C. The -E3! reaction was stopped by theaddition of SDSsample buffer and samples subjected to SDS-PAGE followed by autoradiography (24, 8 - 2000 26). Protein kinase activity of immunoprecipitated insulin receptor a @-subunit on histone was assayed in the same reaction mixture as 0 above, except that 0.5 mg/ml histone HFPB was added and incubated 4 h at 4 "C before adding [y-3ZP]ATP. The reactionwas stopped by the addition of SDS sample buffer, and histone phosphorylation was analyzed as described in Ref. 24. 10"O 10 -9 10-8 Immunoblotting-WGA-purified rat liver receptors were subjected to SDS-PAGE underreducing conditions. The proteinswere electro[MA-IO] M phoretically transferred to Hybond-N nylon membranes and reacted first with a 1:lOO dilution of antipeptide polyclonal antibody AbPC, FIG. 1. MA-10 binding to human a n d rat cells. Binding of loF6M antibody MA-10, or 1:lOO dilution of nonimmune rabbit serum MA-10-'251-protein A complexes to the two hepatoma cell lines, rat and then with '251-protein A as described in Ref. 33, with modifica- Fao (0)and human Hep G2 ( O ) ,was measured as described under tions according to the filter manufacturer's instructions (Amersham)."Experimental Procedures." Cells in six-well dishes were incubated Filters were washed and subjected to autoradiography. with increasing amounts of MA-10 (from 10"' to 5 X 10-7 M ) and 1.5 X IO5 cpm/well 1251-proteinA in the absence (-) or presence (- - -) of insulin M ) for 4 h at 4 "C. A and A indicatebinding RESULTS of H 65.6-'251-protein A complexes to Fao and Hep G2 cells, respecAntibody MA-10 Binding to Human and Rat Cells-Antitively. Results are expressed as counts/min (cprn) of "'1-protein A body MA-10 inhibits '251-insulinbinding to human hepatoma bound to lo6 cells and represent the average of three independent Hep G2 cells, 50% inhibition occurring at lo-' M , but it does experiments performed in duplicate.

5

g

-

s -

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insulin, MA-10 binding to Hep G2 cells is similar to MA-10 A B C binding to Fao cells in the absence or in the presence of 7 K O H l insulin (Fig. 1). Superimposable results were obtained performing MA-10 binding experiments at 37 "C instead of 4 "C 1 2 3 1 2 3 Mr 1 2 3 4 Mr - KDa KDa (data not shown). When Fao and Hep G2 cells are probed with MA-10 in indirect immunofluorescence experiments, in 200 200 the absence of insulin, both Fao and Hep G2 cells exhibit 116 membrane-associated fluorescence, while coincubation with 11692 92 - 010"' M insulin reduces staining of Hep G2cells butnot 66 staining of Fao cells (data not shown). When insulin receptors 66 were down-regulated in both Fao and Hep G2 cells,l2'1-insulin 45 45 and MA-lO-"'I-protein A binding were reduced to the same extent in both Fao and Hep G2 cells, thus suggesting that + + INSULIN - + + + + + + MA-10 binds to insulin receptor in these cell lines (Table11). - - + - - + - + + + MA-10 Effect of Antibody MA-10 on Insulin Receptor Phosphorylation, Thymidine Incorporation into DNA, Insulin Receptor FIG. 2. Insulin receptor phosphorylation in intact cells. Panel A, Fao cells (2 X lo'), incubated with [:"P]orthophosphate for Internalization, and Down-regulation-MA-10 antibody decreases insulin-stimulated protein tyrosine kinase activityof 90 min, were treated with either normal IgG, insulin, or MA-10 for WGA-purified rat insulin receptors withoutaffecting insulin 10 min as described under"Experimental Procedures."After cell solubilizationinsulinreceptors were purified, immunoprecipitated binding (24). To understand whether this phenomenon occurs with AbPC, and subjected to SDS-PAGE (7.5% polyacrylamide) as also when insulin receptors are located in the cell membrane described. An autoradiogram of the drygel, exposed for 16 ha t -50 "C and todefine the mechanism by which MA-10 reduces insulin with an intensifying screen, is shown. The band seen on this autoreceptor autophosphorylation, the effect of this antibody on radiogram is the :'2P-labeled insulin receptor &subunit purified from cells treated with lo-' M normal IgG (lane I ) , insu!in M) plus insulin receptor phosphorylation in intact cells was studied. normal IgG (lane 2), and insulin M ) plus 10" M MA-10 (lane In Faocells, labeled with ["Plorthophosphate, insulin 3 ) . Positions of molecular mass markers are indicated in kDa on the M, for 10 min at 37 "c)stimulates 32Pincorporation into the left. Panel R, afterthe exposureshown in panel A, the gel was insulin receptor @-subunit 9-fold compared to control cells rehydrated in 1 M KOH a t 55 'C for 1 h, then washed in 10% acetic incubated with both normal mouse IgG (Fig. 2, A and B) or acid,40% methanol, 1% glycerol for 1 h,neutralized, dried,and with the irrelevant monoclonal IgG2B H 65.6 (Fig. 2C). MA- subjected toautoradiography (exposure 36 h a t -70 "C with an 10 reduces insulin-stimulated '*P incorporation into insulin intensifying screen).Lunes are as in Panel A. Panel C,Fao cells (2 X receptor @-subunit aindose-dependent manner,half-maximal 10'). labeled with ["P]orthophosphate for 90 min, were treated with insulin M) plus lo-' M mAb H 65.6 (lane I ) , or withinsulin effect beinga t IO-" M (Fig. 2). *'P incorporated into the insulin(lo-' M ) plus MA-10: M (lane 2), 5 X M (lane 3). and M receptor @-subunitwas partially resistant to alkalinehydrol- (lane 4 ) as descrihed under "Experimental Procedures." Insulin recepysis, and MA-10 reduces the incorporation of both alkalitors were purified, immunoprecipitated with ARA, and analyzed by sensitive and -resistant'"P into the insulin receptorsuggest- SDS-PAGE (7.5% polyacrylamide) as described above. An autoradiing that the contentof phosphotyrosine as well as phospho- ogram of the dry gel, exposed for 12 h a t -70 "C with an intensifying serine residues is reduced in cells incubated with MA-10 (Fig. screen, is shown. Positions of molecular mass markers (in kDa) are indicated on the left. 2, A and R). It is noteworthythat MA-10 M ) does not affect basal (not insulin-activated) insulin receptor autophosphorylation(datanotshown)asalreadydemonstrated in In rat hepatoma Fao cells insulin (lo-" M ) stimulates thymidine incorporation into DNA 2.5-fold and MA-10 (10" M) partially purified receptors (Ref. 24 and Fig. 8). In order toverify whether the inhibitionof insulin receptor inhibits insulin action by 75% (Table 111). MA-10 inhibition autophosphorylation produced by MA-10 is relevant in terms of insulin-stimulated thymidine incorporation into DNA is of insulin action, the effect of MA-10 on insulin-stimulated dose-dependent with half-maximal effect at lo-" M (data not thymidine incorporation into DNA of Fao cells was studied. shown). On the contraryMA-10 does not affect basal thymidine incorporation intoDNA of Fao cells (Table 111). The functional relevance of MA-10 inhibition of insulin TABLE I1 receptor phosphorylation was investigated also measuring the Effrct of insulin rpceptor down-regulation on insulin and MA-IO binding t o human and rat cells effect of theantibodyon insulin-induced receptor downHep G2 or Fao cells, in six-well dishes, were incubated in complete regulation. Fao cells were exposed to lo-' M insulin in the medium in the absence or in the presence of M insulinfor 20 h M mAb H 65.6 (control cells) or lo-' MA-10, presence of a t X "C. Surface-bound ligands were then removed by washing cells for 20 h in complete medium, following which the residual at pH 3.5 as described under "Experimental Procedures" and '?'Ibinding activity was measured. The insulin binding activity insulin (150.000 cpm/well) or MA-lO-"'I-protein A complexes (10" on Fao cells exposed to insulin was reduced by about 40%, M MA-10 + l..i X lo:' cpm/well '"1-protein A) binding was measured while that of cells incubated with insulin plus MA-10 was for 4 h a t 4 "C. The results are the average of three independent experiments performed in duplicate and are expressed as percent of reduced by IO%, 10" M MA-10 reduces the insulin-induced the insulin or MA-10-protein A binding observed in control cultures. receptor experiments down-regulation by 75%. Control The values for 1004 insulin binding activity were 8860 and 8210 showed that MA-10 does not down-regulate insulin receptor cpm/l0" Hep G2 and Fao cells, respectively. The values for 100% in Fao cells per se but, at the same concentration, inhibits MA-10-protein A binding activity were 9046 and 7860 cpm/lOR Hep insulineffect on receptordown-regulation (Table 111).As G 2 and Fao cells, respectively. ~previously reported (6), MA-10 induces insulin-independent Insulin binding MA-10 binding internalization of human insulin receptors. However MA-10 activity Cultures fails to produce the sameeffect in rat cells: in fact the amount Fao Hep G2 Fao Hep G 2 of internalized MA-10-12'I-protein A complex is less than 10% % % of the total antibody bound to Fao cells after 15 min a t 37 "C, Control 100 100 100 100 thus suggesting that the epitopeof insulin receptor molecule, 67 62 65 64 Down-regulated to which MA-10 binds in rat cells, is not involved in antibody~~

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Regulation of Insulin Receptor Kinase bya Monoclonal Antibody TABLE I11 Effect of MA-IO on thymidine incorporation into DNA and on insulin receptor down-regulation Fao cells, in 24-well dishes, were incubated in DME containing 0.5% dialyzed USA for 30 h a t 37 "C. The irrelevant monoclonal IgG H 65.6 (lo-' M), monoclonal antibody MA-10 (IO-' M), insulin M ) plus mAb H 65.6 (IO-' M ) or plusMA-IO (lo-' M ) were then added for 16 hprior to a 3-h pulse with 0.5 PCi of [3H]thymidine/well. Cells were washed three times with ice-cold PBS and solubilized in 0.03% SDS. Trichloroacetic acid (final concentration 10%)was then added to the extractsf x 1 h at 4 "C, and the precipitates were collected by filtration onto Whatman No. 3MM filters, washed with 20 ml of icecold 10% trichloroaceticacid, and assayedforradioactivity. For insulin receptor down-regulation, cells in six-well dishes were incubated in complete medium in the presence of mAbH 65.6 M ) or insulin (10"' M ) plus mAb H 65.6 (lo-' M) or MA-10 M ) for 20 h at 37 'C. Surface-hound ligands werethen removed by washing cells at pH 3.5 as detailed under "Experimental Procedures," and '"Iinsulin binding activitywas measured for 4 h a t 4 "C. The results are the average of four independent experiments performed in quadruplicate (thymidine incorporation) or duplicate (insulin receptor downregulation) and are expressed as percent of thymidine incorporation or insulin binding observed in control cultures exposed to mAb H 65.6. The value for 100% thymidine incorporation was 5360 cpm/106 cells. 100% insulin binding activity was 8210 cprn/l0' cells. A. Thymidine incorporation into DNA

B. Insulin binding activity %

%

mAb H 65.6 (lo" M ) mAb H 65.6 (lo" M ) insulin (10"' M) MA-IO (IO-? M) MA-IO (IO" M ) + ind i n (lo-* M)

+

100 mAb 268 102 140

H 65.6 (IO" M ) mAb H 65.6 (lo" M ) + insulin ( 1 P M) MA-IO (IO"' M) MA-10 (lo-' M ) + insulin M)

100 60 101 90

mediated receptor internalization. Antibody MA-10 Immunoreacts with Human and Rat Insulin Receptors-As shown in Fig. 3, MA-10 immunoprecipitates both human and rat insulin receptors. At lo-* M concentration, MA-10 immunoprecipitates rat insulin receptors 1015 times less than human insulin receptors. This difference in efficiency is predicted by the difference in MA-10 binding activity to rat and human insulin receptors in intact cells. On the contrary, a t saturating antibody concentration (2 x M), MA-10 immunoprecipitates both human and rat receptors to the same extent.["S]Methionine pulse-chase experiments also show that MA-10 recognizes both human (not shown) and rat insulinreceptor precursor (molecular mass 210 kDa) (Fig. 3C and data not shown). Next, the possibility that MA-10 immunoprecipitates isolated insulin receptor @-subunits was investigated. Interchain disulfide bonds of insulin receptors, WGA-purified from human or rat hepatomacells, were reduced using a modification of the DTT/Tris method reported by Boni-Schnetzler et al. (32). As depicted inFig. 4 this procedure yields isolatedinsulin receptor @-subunits that are immunoprecipitated by an antipeptide antibody directed against the carboxyl-terminal 17 amino acids of the insulin receptor (AbPC).As shown in Fig. 4 antibody MA-10 immunoprecipitates reduced insulin receptor @-subunitsof insulin receptors WGA-purified from ['"SS] methionine-labeled Fao cells. In experimentsperformed using reduced receptors WGA-purified from human Hep G2 cells, MA-10 immunoprecipitates both a- and @-subunits(Fig. 4). Reduction of class 2 disulfide bonds is not 100% complete: in factdiscreteamounts of a/@ dimersarepresentandare immunoprecipitated by MA-10. Thesmallamount of rat insulin receptor a-subunit evident in the MA-10 immunoprecipitates of reduced rat receptors (Fig. 4) probably depends onthe coprecipitation of a-subunits linked to@-subunits through hydrogen bonds or other noncovalent bonds. How-

-

Mr KDa

C

0

A

-

1 2

Mr KDa

1

2

3

Mr KDa

20011692-

200-

200

1 2

-

m,-

66-

116

-

45 -

92

-

11692-

66-

66-

FIG. 3. Immunoprecipitation of humanand rat insulin receptors with MA-10. Panel A, WGA-purified human placenta (lane I ) and rat liver (lane 2) receptors (40 fmol of insulin binding activity) were incubated with insulin M), phosphorylated, immunoprecipitated with M) MA-10, and subjected toSDS-PAGE (7.5% polyacrylamide) under reducing conditions as described under "Experimental Procedures." The autoradiogram was exposed 3 days a t -70 "C with an intensifying screen. Panel R, Hep G2 cells (1 X 10') were labeledwith [:''SS]methionine for 30min, washed, and exposed to medium containing 10 mM unlabeled methionine as described under "Experimental Procedures." After 8 h cells were solubilized and insulin receptors were WGA-purified and immunoprecipitated using the nonrelevant mAb H 65.6 M), MA-10 M), or AbPC (1:lOO dilution) as indicated in the figure. The immunoprecipitated proteinswere subjected to SDS-PAGE (6% polyacrylamide), the gel was stained, destained,soaked 30 min a t room temperature in Amplify, dried, and exposed 24 h at -70 "C. Panel C, Fao cells (1 x 10') were labeled with ["'SS]methioninefor 30 min, washed,and chased for 4h inthe presence of 10 mM methionine.Cells were there solubilized, insulin receptor WGA-purified, and immunoprecipitated M ) as indicated in the with mAb H 65.6 M ) , MA-IO (2 X figure. The immunoprecipitated receptors were subjected to SDSPAGE (6% polyacrylamide). Positions of molecular mass markers, in kDa, are indicated on the left of each panel.

ever the ratioof a to @ subunits immunoprecipitatedfrom rat DTT-reduced receptors by MA-10 and AbPC (as measured by densitometer quantitationof autoradiograms) was similar (0.21 uersus 0.23), while the ratio of a- to @-subunitsimmunoprecipitated from human DTT-reduced receptors by MA10 was -3-fold higher (0.70) (Fig. 4). In order toexclude the possibility that MA-10 immunoprecipitates rat insulin receptor @-subunitsby cross-reacting with the small amountof noncompletely reduced a-subunits, also present in our DTT-reduced preparations, we performed immunoblottingexperimentsprobing WGA-purified rat liver receptorswith MA-10 or with the anti-insulin receptor @subunit antipeptide antibody AbPC. As demonstrated in Fig. 5, both MA-10 and AbPC reactwith a major band of approximately M, 95,000 that corresponds to insulin receptor @subunits. No immunoreactivity is evident at the molecular weight corresponding to insulin receptor a-subunits (Fig. 5). Finally MA-10 immunoprecipitates invitro phosphorylated insulin receptor @-subunits purified from both human placenta and ratliver (data not shown). Antibody MA-10 Immunoprecipitates Nonglycosylated Insulin Receptors-Fao cells were incubated with tunicamycin to inhibit protein glycosylation and labeled with ["S]methionine. Cells were solubilized and the clarified supernatant,

Regulation of Insulin Receptor Kinase by a Monoclonal Antibody

8632

C

B

A

KDa 200 -

sy -I

200

200 116 -

97.4-

. 1

a

-

116 -

92 66 -

66 -

200 116

68 -

-

43

66 -

-

L

J

FIG. 5. Immunoblotting of rat insulin receptor. WGA-purified rat liver receptors were subjected to SDS-PAGE under reducing DTT/tris + + + + conditions followedby immunoblotting with MA-10,polyclonal antipeptide antibody AbPC (1:lOO final dilution), or nonimmune (NO FIG. 4. Immunoprecipitation of reduced human and rat in- rabbit serum as described under “Experimental Procedures.’’ The sulin receptors. Panel A, Fao cells (2 X 10’) were labeled with [3sS] autoradiogram was exposed for 18 h at -70 “C. The positions of the methionine for 30 min, washed, and exposed to medium containing molecular mass markers are indicated in kDa on the left. 10 mM unlabeled methionine as described under “Experimental Procedures.” Insulin receptors were WGA-purified and subjected to the disulfide bond reduction procedure detailed under “Experimental Procedures.” The neutralized and alkylated receptors were immunoprecipitated using the polyclonal antibody AbPC (1:lOO) or a preimmune rahbit serum (as indicated in the figure) and the immunoprecipitated material subjected to SDS-PAGE (6% polyacrylamide) unKDa der nonreducing conditions. The autoradiogram was exposed for 36 h at -70 “C. The autoradiogram of AbPC immunoprecipitate was scanned densitometrically. The absorbance of the -130 kDa band 200 was 825 arbitrary units, the absorbance of the -95 kDa band was 3587 arbitrary units (ratio 130:95 kDa = 0.23). Panel B,Fao cells (2 X 10’) werelabeled with [”S]methionine, solubilized, and insulin receptors, WGA-purified, were reduced by DTT/Tris as described 116 under “Experimental Procedures.’’ Receptors were then immunoprecipitated with mAb H 65.6 (10” M) or MA-IO (IO-’ M). The immu92 noprecipitates were subjected to SDS-PAGE (6% polyacrylamide). The autoradiogram was exposed for 24 h a t -70 “C, and the lane corresponding to MA-10 immunoprecipitate was scanned densito66 metrically. The absorbance of the -130 kDa band was 218 arbitrary units, the absorbance of the -95 kDa band was 1038 arbitrary units (ratio 130:95kDa = 0.21). Panel C, Hep G2 cells (2 X 10’)were TUNICAMYCIN labeled with [“S]methionine, solubilized, and WGA-purified insulin receptors reduced by the DTT/Tris method. Insulin receptors were then immunoprecipitated with mAb H 65.6 (IO-’ M) or with MA-IO FIG. 6. Immunoprecipitation of nonglycosylatedinsulin (lo” M). The immunoprecipitates were subjected to SDS-PAGE (5% receptors with MA-10. Fao cells (2 X IO’) were incubated in polyacrylamide). The autoradiogram was exposed for 24 h a t -70 “C, methionine-free MEM (+IO% dialyzed fetal calf serum) in the presandthe lane corresponding to MA-IO immunoprecipitate was ence of20 pg/ml tunicamycin, labeled with [“S]methionine for 30 subjected to densitometric scanning. The absorbance of the -130 min, washed, and exposed to DME containing 10 mM methionine for kDa band was 2205 arbitrary units, the absorbance of the -95 kDa 8 h, as described under “Experimental Procedures,” in the presence band was 3150arbitrary units (ratio13095 kDa = 0.7).The positions of tunicamycin (20 pglml). Cellswere solubilized, Triton X-I00 of molecular mass markers, in kDa, are shown on the left of each concentration in the clarified supernatant was loweredby chromatogpanel. raphy on a SM-2 Bio-Beads column, and the “S-labeled proteins concentrated by polyethylene glycol precipitation as described under concentrated by polyethylene glycol precipitation, was im- “Experimental Procedures.” Insulin receptors were then immunopreM) or with AbPC (1:lOO final dilution), cipitatedwith MA-IO (2 X munoprecipitated. MA-10 immunoprecipitated35S-labeled as indicated in the figure, and analyzed by SDS-PAGE (6% polyacrylproteins with M, molecular mass values ranging from 90 t o amide) under reducing conditions. In order to localize the exact 110 kDa correspondingto nonglycosylated insulinreceptor 8- position of mature (glycosylated)insulin receptor a- and &subunits, and a-subunits (Fig.6) (34, 35). an aliquot of insulin receptors immunoprecipitated by MA-10, as An indentical pattern of immunoprecipitation is produced shown in Fig. 4C, was run in parallel (lost lane on the right). The by the antipeptide polyclonal antibody AbPC (Fig. 6). As autoradiogram was exposed 24 h at -70 “C, molecular mass markers internal controlan aliquot of fully glycosylated insulinrecep- are shown on the left of the figure. tor,immunoprecipitatedby MA-10 from[3sS]methionineexact munoprecipitated with AbPC. Autophosphorylation and hislabeled Fao cells,was run inparallelshowingthe position of glycosylated insulinreceptor a- and p-subunits. tone kinaseactivity of immunoprecipitatedmaterial was Effect of Antibody MA-10 on Autophosphorylation and Ki- measured in the presence of normal mouse IgG (1O“j M) or nase Actiuityof Isolated Insulin Receptor p-Subunits-WGA- antibody MA-10 (10” M). As shown inFig. 7, MA-10 inhibits purified human placental insulin receptors were reduced by both insulin receptor beta subunit autophosphorylation and DTT/Tris treatment and isolated @-subunits purified by im- phosphorylation of histone HFZB. Similar results were ob-

I

+

I

+

~

-

-

-

-

Regulation of Insulin Receptor Kinase by a Monoclonal Antibody

4

1 2 3 -

-.-,..

5

6

8633

insulin-stimulated) autophosphorylation of insulin receptor @-subunitswhile it does not decrease, a t any concentration, basal autophosphorylation of the nativereceptor (Fig. 8). DISCUSSION

P-

We previously reported that anti-insulin receptor monoclonal antibody MA-10 affects both insulin binding and insulin-stimulated receptor protein-tyrosine kinase activity in partially purified human insulinreceptors, but that it inhibits this enzymatic activity without inhibitinginsulin binding in partially purified rat insulin receptors (24). The aim of this studywas to investigate the mechanism by which MA-10 inhibits insulin receptor protein-tyrosine kinase HF2BBactivity. First the MA-10 binding site to rat insulin receptor was characterized. Data presented herein indicate that antibody MA-10 binds to rat hepatoma Fao cells. By both MAlO-'*'I-protein A complex binding and indirect immunofluorescence MA-10-specific binding to rat as well as to human cells was demonstrated. The fact that MA-10 does not reduce insulin receptor bindingand, conversely, insulindoes not FIG. 7. Effect of MA-IO on the protein tyrosine kinase ac- inhibit MA-10 binding to Faocells suggests that, in ratcells, tivity of isolated insulin receptor &subunit. WGA-purified hu- MA-10 does not recognize the insulin binding siteof insulin man placenta insulin receptors were reduced by 100 mM DTT,75 mM Tris and immunoprecipitated by AbPC (lanes 1, 2, 4, and 5) or by receptor molecule. In human hepatoma HepG2 cells, MA-10 since it inhibitsinsulin preimmune rabbit serum (lanes 3 and 6).Insulin receptors, reduced interacts with the insulin binding site and immunoprecipitated, were incubated for 4 h a t 4 "C with normal binding as insulin (on a molar basis) (27), and furthermore M) (lanes 1 and 4 ) , lo-' M MA-10 (lanes 2 and 5) in saturating concentrations of insulin reduce MA-10 binding. mouse IgG the absence ( l a n e s 1-3) or presence (lanes 4-6) of histone HFLB. In the presence of M insulin, MA-10 binding to Hep G2 Then phosphorylation was conducted as detailed under "Experimencells is reduced to the same amount found in Fao cells both t a l Procedures," and samples were analyzed by SDS-PAGE (6 and in the presence of insulin. The demonstra15% polyacrylamide in the right and left panels, respectively) under in the absence and nonreducing conditions. Autoradiograms were exposed for 30 h a t tion that insulin receptor down-regulation is associated with -70 "C with an intensifying screen. Arrows mark the position of a reduction of both '2'I-insulin and MA-lO-'*'I-protein A insulin receptor &subunit and histone. complex binding to Faocells suggests that MA-10 binds to an. extracellular portionof insulin receptor in ratcells. MA-10 also immunoprecipitates both phosphorylated and native, '?3-labeled rat insulin receptors. This finding is only 1 2 3 4 5 apparently in contrastwith data reportedby Forsayeth et al. MrKDa (27). These authors reported that MA-10 does not immuno200 precipitate purified rat insulinreceptors. Infact using a --mmrelatively low antibody concentration(lo-' M), the amountof immunoprecipitated rat receptors is 10-15-fold less than the amount of immunoprecipitated human insulinreceptors. Optimizing immunoprecipitation conditions and exposing gels for a convenient period of time (up to 3 days a t -70 "C),it was possible to detect discrete immunoprecipitated material a t a molecular weight exactly corresponding to insulinreceptor @-subunitusing WGA-purified rat liver insulin receptors. 45 It is noteworthy that MA-10 interactions (binding and immunoprecipitation) with the native rat insulinreceptor a t submaximal antibody concentrations occur with a 10 times lower efficiency compared to MA-10 interactions with native FIG. 8. Effect of MA-IO on basal insulin receptor autophoswere in- human insulin receptors. Using saturating antibody concenphorylation. WGA-purified placentainsulinreceptors M), MA-10 immunoprecipitates approxi(2 X M normal mouse IgG trations cubated for 16 h at 4 "C in the presence of (lane I ) , 1O"j M MA-10 (lane 2). 10" M MA-10 (lane 3), lo-' M MA- mately the same amount of insulin receptors from [3sSS]me10 (lane 4 ) , or lo-' M MA-10 (lane 5).The phosphorylation reaction thionine-labeled rat and human hepatoma cells. was then conducted in the presence of 50 p~ [y-"PIATP and 5 mM MA-10 immunoprecipitatesboth a- and@-subunits in MnClz as described under "Experimental Procedures." Samples were only analyzed by SDS-PAGE (7.5% polyacrylamide), the autoradiogram DTT-reduced human receptor preparations but reacts with @-subunits of reduced rat receptors in both immunoprewas exposed 16 h a t -70 "Cwith an intensifying screen. Positions of cipitation and immunoblotting experiments. This fact indimolecular mass markers (in kDa) areindicated on the left. cates that MA-10 recognizes human and rat insulin receptor tained using @-subunits purified from rat liver receptors as @-subunits. While species specificity of the insulin binding source of kinase. In some experiments the nonrelevant mono- site of the receptor is well documented (37-39), data presented clonal antibodyH 65.6, used as negative control, did not affect here give further evidence to our previous observation (24) or his- that a site involved in insulinreceptor protein-tyrosine kinase either insulin receptor @-subunit autophosphorylation tone kinase activity. As expected, insulin does not stimulate activity regulation, recognized by MA-10, is conserved autophosphorylation of reduced (?-subunits (data not shown). through rat and humanspecies. Finally it is noteworthy that MA-10 reduces basal (not Data discussed above suggestthat MA-10 binds toa region

-

8634

Regulation of Insulin Receptor Kinase

of insulin receptor molecule common to both human and rat cells and that this region is not the receptor insulin binding site. The insulin receptor region recognized by MA-10 belongs tothe protein backbone of the molecule because MA-10 immunoprecipitates not completely glycosylated receptors from tunicamycin-treated rat hepatoma Fao cells. Experiments were carried out to explore the functional relevance of the insulin receptor region bound by MA-10 in intact rat cells. MA-10 inhibits insulin-stimulated receptor autophosphorylation in intact rat cells without affecting insulin receptor binding. In intact cells a 10-min exposure to insulin leads to the insulin receptor autophosphorylation in both tyrosine and serine residues (40). Recent evidence suggests that after tyrosine autophosphorylation insulin receptors become substrates for a putative serine kinase (1) and that only insulin receptors phosphorylated in tyrosine residues can undergo to serine phosphorylation (6). Our data, demonstrating that MA-10 similarly reduces 32Pincorporation into bothtyrosine and serine residues of insulin receptor, @-subunitsare concordant with this last finding. The inhibition of insulin-stimulated receptor phosphorylation has biological significance: at the same concentrations, MA-10 reduces to a similar extent insulin-stimulated receptor autophosphorylation, thymidine incorporation into DNA, and insulin-induced receptor down-regulation in rat hepatoma Fao cells. Recent data, obtained in cells expressing human insulin receptor partially (13) or completely (4-9) devoid of protein-tyrosine kinase activity by site-directed mutagenesis, demonstrate that insulin receptor enzymatic activity is essential for cell transduction of insulin receptor signal, thus confirming previous indirect data obtained by injection of antiinsulin receptor monoclonal antibodies into cells (36). Our present findings further substantiate this evidence utilizing nonengineered receptors and avoiding any cell manipulation. Interacting with its binding site insulin induces internalization of its receptors (41). In humancells MA-10 binding to insulin receptor induces receptor internalization (6). In this paper we demonstrate that MA-10 binding to rat insulin receptors is not followed by insulin receptor internalization. This finding suggests that the interaction of the same ligand (MA-10) with different sites of the insulin receptor molecule can result in different biological effects. MA-10 also inhibits autophosphorylation and protein-tyrosine kinase activity of the isolated insulin receptor @-subunit.Thislast finding indicates that the protein-tyrosine kinase activity of isolated insulin receptor @-subunitcan be modulated independently of a-subunit, as suggested by Herrera et al. (16) and that MA-10 binding to isolated @-subunit reduces the basal activity of the enzyme. Recently it hasbeen suggested that the a-subunit of the insulin receptor inhibits protein-tyrosine kinase activity, and, consequently, the physiological responses mediated by the enzyme and the insulin relieves this inhibition (15). The demonstration thatthe digestion of insulin receptor a-subunit by trypsin leads to the activation of insulin receptor autophosphorylation in intact cells agrees with this hypothesis (42, 43). Data presented in this report give more support to this hypothesis: MA-10 does not reduce kinase activity of the @-subunitin the native receptor, that is under the inhibitory effect of a-subunit, but has an inhibitory effect when the @-subunit (protein-tyrosine kinase) is isolated. These findings suggest that MA-10 can substitute the a-subunitfor the inhibition of insulin receptor @-subunitprotein-tyrosine kinase activity. In conclusion, MA-10 antibody recognizes the extracellular portion of the insulin receptor @-subunitin rat cells or tissues. Data presented above indicate that (i) the extracellular part

by a Monoclonal Antibody

of the insulin receptor &subunit recognized by MA-10 is more conserved, through human and rat species, than the insulin binding domain, and (ii) the extracellular part of the insulin receptor @-subunit is involved in the regulation of insulin receptor phosphorylation and in the transduction of insulin biological actions. Acknowledgments-We wish to thankMike Czech for many helpful discussions and for critical reading of the manuscript. We are also grateful to Ora Rosen for the permission to read her manuscript prior to publication and for Hep G2 cells, to Bernard Rossi for Fao cells, and toLawrence Mandarin0 for ARA. R. G . thanks Paola Briata for suggestions and discussion. We are indebted to Maria Rosa Dagnino for typing the manuscript. REFERENCES 1. Rosen, 0. M. (1987) Science 2 3 7 , 1452-1458 2. Ullrich, A., Bell, J. R., Chen, E. Y., Herrera, R., Petruzzelli, L. M., Dull, T. J., Gray, A., Coussens, L., Liao, Y. C., Tsubokawa, M., Mason, A., Seeburg, P. H., Gmnfeld, C., Rosen, 0. M., and Ramachandran, J. (1985) Nature 313, 756-761 3. Ebina, Y., Ellis, L., Jarnagin, K., Edery, M., Graf, L., Clauser, E., Ou, J., Masiarz, F., Kan, Y. W., Goldfine, I. D., Roth, R. A., and Rutter, W. J. (1985) Cell 40, 747-758 4. Chou, C. K., Dull, T. J., Russell, D. S., Gherzi, R., Lebwohl, D., Ullrich, A., and Rosen, 0. M. (1987) J. Biol. Chem. 262,18421847 5. Ebina, Y., Araki, E., Taira, M., Shimada, F., Mori, M., Craik, C. S., Siddle, K., Pierce, S. B., Roth, R.A., and Rutter, W. J. (1987) Proc. Nutl. Acud. Sci. U. S. A. 84, 704-708 6. Russell, D. S., Gherzi, R., Johnson, E.L., Chou, C.-K., and Rosen, 0. M. (1987) J. Biol. Chem. 262, 11833-11840 7.Mc Clain, D. A., Maegawa, H., Lee, J., Dull, T. J., Ullrich, A., and Olefsky, J. M. (1987) J. Biol. Chem. 262,14663-14671 8. Gherzi, R., Russell, D. S., Taylor, S. I., and Rosen, 0. M. (1987) J. Biol. Chem. 262,16900-16905 9. Hari, J., and Roth, R. A. (1987) J. Bid. Chem. 262,15341-15344 10. Herrera, R., and Rosen, 0. M. (1986)J. Biol. Chem. 261,1198011985 11. Goren, H. J., White, M. F., and Kahn, C. R. (1987) Biochemistry 26,2374-2382 12. Tornqvist, H. E., Gunsalus, J. R., Nemenoff, R. A., Frackelton, A. R., Pierce, M. W., and Avruch, J. (1988) J . Biol. Chem. 263, 350-359 13. Ellis, L., Clauser, E., Morgan, D. O., Edery, M., Roth, R. A., and Rutter. W. J. (1986) Cell 45. 721-732 14. Ellis, L.,‘Morgan, D. O., Koshland, D. R., Jr., Clauser, E., Moe, G. R., Bollag, G., Roth, R. A., and Rutter, W. J. (1986) Proc. Nutl. Acad. Sci. U. S. A. 83, 8137-8141 5. Ellis, L., Morgan, D. O., Clauser, E., Roth, R. A., and Rutter, W. J. (1987) Mol. Endocrinol. 1, 15-24 6. Herrera, R., Lebwohl, D., Garcia de Herreros, A., Kallen, R. G., and Rosen, 0. M. (1988) J.Biol. Chem. 263,5560-5568 7. Tamura, S., Brown, T. A., Whipple, J. H., Fujita-Yamaguchi, Y., Dubler, R. E., Cheng, K., and Larner, J. (1984) J. Biol. Chem. 259.6650-6658 18. Haves,’GI R., and Lockwood, D. H. (1987) Proc. Nutl. Acud. Sci. U. A. 84,8115-8119 19. O’Brien. R. M.. Soos. M. A.. and Siddle. K. (1987) EMBO J. 6 , 4003-4010 ’ 20. Smith, M. M., Merlie, J . P., and Lawrence, J. C. (1987) Proc. Natl. Acad. Sei. U. S. A . 84,6601-6605 21. Ross, A. F., Rapuano, M., Schmidt, J. H., and Prives, J. M. (1987) J . Biol. Chem. 262, 14640-14647 22. Yarden, Y., and Schlessinger, J. (1987) Biochemistry 26, 14341442 23. Morgan, D. O., and Roth, R. A. (1986) Biochemistry 2 5 , 13641371 24. Cordera, R., Andraghetti, G., Gherzi, R., Adezati, L., Montemurro, A,, Lauro, R., Goldfine, I. D., and De Pirro, R. (1987) Endocrinology 121, 2007-2010 25. Gherzi, R., Caratti, C., Andraghetti, G., Sesti, G., Montemurro, A,, Bertolini, S., and Cordera, R. (1988) Biochem. Biophys.Res. Commun. 152, 1474-1480 26. Cordera, R., Gherzi, R., De Pirro, R., Rossetti, L., Freidenberg, G. R., Andraghetti, G., Lauro, R., and Adezati, L. (1985) ~~

s.

Regulation of Insulin Receptor Kinase by a Monoclonal Antibody Biochem. Biophys. Res. Commun. 132,991-1000

27. Forsayeth, J. R., Monternurro, A,, Maddux, B. A., De Pirro, R., and Goldfine, I. D. (1987)J.B i d . Chem. 262,4134-4140

28. Mandarino, L., Tsalikian, E., Bartold, S., Marsh, H., Carney, A. Buerklin, E., Tutwiler, G., Haymond, M., Handwerger, B., and Rizza, R. (1984) J. Clin. Endocrinol. Metab. 69, 658-664 29. Haigler, H. T., Maxfield, F. R., Willingham, M. C., and Pastan, I. (1980) Proc. Natl. Acad. Sci. U. S. A . 75.3317-3321 30. Hunter, T., and Sefton, B.M. (1980) Proc.Natl.Acad.Sci. U. S. A . 7 7 , 1311-1315 31. Cutatrecasas,.P. (1972) Proc. Natl. Acad. Sci. U. S. A . 69, 318322 32. Boni-Schnetzler, M., Rubin, J. B., and Pilch, P. (1986) J. Biol. Chem. 261,15281-15287 33. Burnette, W. H. (1981) Anal. Biochem. 112, 195-203 34. Ronnett, G. V., Knuston, V. P., Kohanski, R. A., Simpson, T. L., and Lane, M. D. (1984) J.Biol. Chem. 259,4566-4575 35. Herzberg, V. L., Grigorescu, F., Edge, A. B. S., Spiro, R. G., and Kahn, C. R. (1985) Biochem. Biophys. Res. Commun.129,789796

8635

36. Morgan, D. O., andRoth, R. A. (1987) Proc.Natl.Acad.Sci.

U. 8. A. 84,41-45 Y., Maddux, B. A,, and Goldfine, I. D.(1982) Proc. Natl. Acad. Sci. U. S. A . 79,73127316 Soos, M. A., Siddle, K., Baron, M. D., Heward, J. M., Luzio, J. P., Bellatin, J., and Lennox, E. (1986) Biochem. J. 235, 199208 De Pirro, R., Rossetti, L., Montemurro, A., Lauro, R., Gammeltoft, S., Maddux, B. A., and Goldfine, I. D. (1985) J. Clin. Endocrinol. Metab. 6 1,986-989 Pang, D. T., Sharma, B.R., Shafer, J. A., White, M. F., and Kahn, C. R. (1985) J. Biol. Chem. 260, 7131-7136 Carpentier, J. L., Gorden, P., Freychet, P., Le Cam, A., and Orci, L. (1979) J. Clin. Inuest. 63, 1249-1261 Leef, J. W., and Larner, J. (1987) J. Biol. Chem. 262, 1483714842 Shoelson, S. E., White, M. F., and Kahn, C. R. (1988) J. Biol. Chern. 263,4852-4860

37. Roth, R. A., Cassell, D. J., Wong,K.

38. 39. 40. 41. 42. 43.