THE JOURNAL OF BIOLOGICAL CHEMISTRV 0 1986 by The American Society of Biological Chemists, Inc

Vol. 261, No. 32, Issue of November 15, pp. 15281-15287,1986 Printed in U.S.A.

Structural Requirements for the Transmembrane Activation of the Insulin Receptor Kinase*

(Received for publication, March 17, 1986)

Marianne Boni-SchnetzlerS, Joshua B. Rubin, and Paul F. Pilchs From the Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02118

Tetrameric insulin holoreceptor (a&J was reduced with dithiothreitol into a@ dimers such that they main- tain up to 50% of insulin binding at tracer ligand concentrations. Scatchard analysis of insulin binding to dimers revealed that they had a reduced affinity for ligand by a factor of 3-6 compared to holoreceptor, whereas the maximum number of high affinity binding sites was not affected. The a@ dimers can be separated from holoreceptor by sucrose density gradient centrif- ugation, and hence, they are not associated by nonco- valent interactions. Insulin-dependent autophospho- rylation of a@ dimers isolated from low ionic strength sucrose density gradients was minimal and was always accompanied by reoxidation of dimers to the tetrameric holoreceptor. The reformed tetramer exhibited a strong insulin-dependent autophosphorylation reac- tion. Reoxidation was prevented by isolating arj3 dimers in sucrose density gradients containing 0.15 M NaC1. Under these conditions, no insulin-dependent auto- phosphorylation was observed. When insulin receptor was first autophosphorylated and then reduced, recep- tor kinase activity, as assayed by histone phosphory- lation, was not affected. Also, the insulin-independent, basal autophosphorylation was maintained after re- duction into a@ dimers. We conclude that a@-aO inter- action is not necessary for the maintenance of basal kinase activity or for insulin-activated kinase activity once autophosphorylation occurs. However, dimer-di- mer interaction appears critical for the insulin-de- pendent activation of the receptor’s intrinsic kinase activity.

The insulin receptor consists of two (Y subunits (Mr 135,000) and two @ subunits (Mr 95,000) which are linked together by disulfide bonds (1, 2). Affinity labeling techniques demon- strated that the insulin binding domain is located on the (Y

subunit (3-5). The transmembrane B subunit contains tyro- sine kinase activity on its cytoplasmic domain that results in rapid receptor autophosphorylation (611). Activation of the kinase towards exogenous substrates is apparently preceded by this insulin-dependent autophosphorylation reaction of the @ subunit (12). The biochemical details of how insulin binding causes transmembrane activation of the receptor’s

* This research was supported by Grants AM 36424 and HL 26895 from the United States Public Health Service. The costs of publica- tion of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertise- ment” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Recipient of a Postdoctoral Fellowship from the Juvenile Dia- bet,es Foundation.

United States Public Health Service. Recipient of a Research Career Development award from the

intrinsic kinase activity are not completely understood. The physiological significance of the receptor kinase activity is also incompletely understood, although it is likely to play an important role in the expression of insulin’s action(s).

Dilution studies with detergent-solubilized, purified recep- tor showed that the initial rate of autophosphorylation is receptor concentration independent (13, 14). The receptor appears to exist as a monomer in detergent solution as deter- mined by gel filtration (15) and ultracentrifugation studies (16, 17). The insulin-induced autophosphorylation reaction can therefore be regarded as an intramolecular reaction. This conclusion is further supported by a recent study, where greater than 95% of the receptors have been incorporated into phospholipid vesicles at a receptor to vesicle ratio of 1:l. These preparations have been shown to retain insulin-induced (3 subunit autophosphorylation (18). Hence, the kinase acti- vation initiated by receptor occupancy with ligand is mediated by interactions within a single receptor molecule.

Recent amino acid sequence data deduced from cDNA cloning allow us to draw conclusions about some aspects of these interactions. It has been predicted that the a subunit containing the insulin binding site is located entirely extra- cellularly and that the @ subunit contains only one membrane spanning region (19,20). It has been shown that the a subunit does in fact behave like a peripheral membrane protein as it can be released from membranes by simultaneous exposure to urea and dithiothreitol (21, 22). With regard to the signal transferring process within the receptor molecule, this leads to the following assumption: since the a subunit is located entirely extracellularly, the interaction of the a subunit with the /3 subunit upon insulin binding must also take place entirely on the extracellular domains of the subunits. This interaction results in enhanced autophosphorylation of the cytoplasmic domain of the /3 subunit and raises the critical question of how the /3 subunit, having only one membrane spanning region, transduces the signal across the phospholipid bilayer.

One possible way by which this transmembrane signalling could occur might be by interaction of two (YB dimers within the tetrameric receptor such that following insulin binding to the a subunits, the @ subunits would interact to allow en- hanced autophosphorylation. If an association of two mem- brane spanning domains is required for the transmembrane signalling, their separation should block this process. In order to achieve this separation, the disulfides connecting the ( ~ / 3 dimers to form the tetrameric insulin receptor have to be reduced in such a way that functional dimers are obtained. The disulfide bonds between receptor subunits have been grouped into two classes according to their sensitivity to reductants (23). Class 1 disulfides link two (YB receptor halves together and are susceptible to reduction under mild condi- tions. On the other hand, class 2 disulfides which link (Y to 6 are virtually impossible to reduce without denaturation of the

15281

15282 Transmembrane Activation of the Insulin Receptor Kinase

receptor (21-23). Previously published reduction protocols involved the treatment of the receptor with 20 mM DTT’ (23). With detergent-solubilized receptor, this procedure results in a complete loss of insulin binding. We therefore developed conditions that led to the generation of aB receptor dimers with a minimum of additional structural perturbations. This allowed us to investigate whether aB dimer interactions are required for the insulin-induced transmembrane activation of the kinase (autophosphorylation) or for the maintenance of the activated kinase towards exogenous substrates. The struc- tural requirements for the maintenance of the basal, insulin- independent, kinase activity were also examined.

EXPERIMENTAL PROCEDURES

Preparation of Soluble Insulin Receptor-The insulin receptor was purified from human placenta according to previously published procedures (8, 13). Briefly, placental microsome preparations were solubilized with 1% Triton X-100 in the presence of 1 mM phenyl- methylsulfonyl fluoride for 1 h a t 4 ‘C. The soluble fraction was centrifuged a t 100,OOO X g and subsequently chromatographed over Sephacryl S-400, hydroxylapatite, and DEAE-Trisacryl (8, 13). The insulin-binding fractions were finally equilibrated with 30 mM Hepes (pH 7.6) buffer containing 0.1% Triton X-100 and 0.02% azide. For some experiments this receptor preparation was further purified and concentrated by adsorption to and elution from wheat germ agglu- tinin-agarose (Miles-Yeda) (24). Insulin-binding fractions were iden- tified with a polyethylene glycol precipitation insulin-binding assay (25). Individual receptor preparations were assayed for insulin-induc- ible autophosphorylation, and we only used receptor that showed more than a 4-fold ligand effect for these studies.

Velocity Sedimentation-Velocity sedimentation was performed in a linear 5-25% sucrose density gradient containing 30 mM Hepes (pH 7.6), 0.1% Triton X-100, and 0.02% azide. Receptor preparations (250 pl ) were layered onto 4-ml gradients and centrifuged for 14 h at 170,000 X g at 4 “C in a Beckmann SW 56 rotor. 100-pl fractions were collected from the bottom and analyzed for insulin binding and autophosphorylation.

Reduction of Insulin Receptors into a@ Dimers-Class 1 disulfide bonds were selectively reduced by incubating receptor preparations a t room temperature with 1.25 mM dithiothreitol and 75 mM Tris (pH 8.5) for 30 minor the times indicated. The reactions were stopped by centrifuging the samples for 5 min at 1OOO X g through 3-ml syringes, packed with desalting gel (Bio-Gel B-GDG, Bio-Rad), and equilibrated with 30 mM Hepes (pH 7.6). This results in removal of >98% of the DTT and Tris.

Receptor Cross-linking-Cross-linking was performed essentially according to Pilch and Czech (3). Receptor preparations were incu- bated for 30 min at room temperature with 1251-labeled insulin at the concentrations indicated in the text. The samples were cooled on ice and incubated for 15 min with disuccinimidyl suberate, which had been freshly dissolved in dimethyl sulfoxide to 0.1 M. The reaction was stopped either by the addition of 0.1 M Tris (pH 7.6) or by the addition of Laemmli sample buffer.

Phosphorylation Assay-Phosphorylation assays were performed as described previously (8, 13), with some minor modifications. The reaction mixture contained 15 pCi of [T-~’P]ATP, and the reaction time for autophosphorylation was 6 min. The reaction was stopped by adding 50 mM EDTA.

Polyacrylamide Gel Electrophoresis and Autoradiography-Prior to addition of the electrophoresis sample buffer, the samples were in- cubated for 5 min with 20 mM N-ethylmaleimide. The final concen- trations of the electrophoresis sample buffer were 50 mM Tris (pH 6.8), 10% glycerol, and 1% SDS. Samples were subjected to SDS- polyacrylamide gel electrophoresis according to Laemmli (26). The separating gels contained gradients of 3-10% acrylamide. Coomassie Blue-stained and dried gels were exposed to Kodak X-Omat AR film in the presence of a Cronex Lightning Plus enhancer screen. Incor- porations of 32P into protein bands were quantitated by excising the bands and counting their Cerenkov radiation. All experiments were performed on at least three independent occasions and representative data are shown.

The abbreviations used are: D m . 1,4-dithiothreitol; Hepes, 4-(2- hydroxyethy1)-1-piperazineethanesulfonic acid; SDS. sodium dodecyl sulfate; EGF, epidermal growth factor.

Materials-Monocomponent porcine insulin was a gift of the Eli Lilly Company (Dr. R. Chance) and was iodinated to a specific activity of 100-150 pCi/pg using NalZ5I from Amersham Corp. and Enzymo- beads from Bio-Rad. For some experiments, B26-monoiod0-’~~I-in- sulin was used and it was bought from Amersham Corp. [-y-”P]ATP was prepared from [32P]orthophosphate (NEN Corp.) using a Gam- maprep ki t from Promega Biotech. Hepes and dithiothreitol were purchased from Research Organics. Electrophoresis reagents were obtained from Bio-Rad. Disuccinimidyl suberate was purchased from Pierce Chemicals. Unless otherwise noted, other reagents were ob- tained from Sigma.

RESULTS

Reduction of Tetrameric Insulin Receptor to aB Dimers- Studies on the minimal covalent subunit structure of the insulin receptor have shown that treatment of the receptor with 20 mM DTT at pH 7.6 results in selective cleavage of the interhalve disulfides (class 1 disulfides) and in the con- version of most of the tetrameric receptor into the CUB dimer form (23). However, when assaying the insulin binding capa- bility of cup dimers generated under these conditions, the specific binding at tracer hormone concentration was found to be below 5% when compared with an untreated control (Fig. la , A uersus E) . We therefore worked out an alternative reduction protocol which involved treatment of receptors with 75 mM Tris at pH 8.5 and 1.25 mM DTT for 30 min. The pH of this mixture is above the pK of sulfhydryl groups. This procedure has been reported to increase the effectiveness of reduction and to favor simultaneous reoxidation of reduced, deprotonated sulfhydryls. The overall effect is such that only those disulfides that are most accessible to the reductant become reduced with a high efficiency (27, 28). Using this protocol we achieved almost complete reduction of receptor into a@ dimers while maintaining 25-50% of binding at tracer concentrations of insulin (Fig. la, B) . Also, there was no generation of free a and B subunits (Fig. Ib, B) . Separate treatment of receptor with either 1.25 mM DTT (Fig. la, C) or 75 mM Tris (pH 8.5) (Fig. la, D ) had no significant effect upon hormone binding nor did it promote the appearance of a@ dimers.

Characterization of the Reduced Binding to as Dimers- Reduced binding to as dimers at tracer hormone concentra-

I A B C D E A B C D E

FIG. 1. Reduction of insulin receptor to afl dimers. Soluble insulin receptor was treated for 30 min with 20 mM DTT at pH 7.6 (A), with 1.25 mM D n and 75 mM Tris a t pH 8.5 ( B ) , with 1.25 mM D m (C), with 75 mM Tris at pH 8.5 (D), and without addition (E). The reaction was stopped by centrifuging the samples over a syringe column containing desalting gel as described under “Experimental Procedures.” Samples were then subjected to a soluble insulin-binding assay using 2 X 10”’ M insulin as tracer. Results are expressed as percent of the untreated control ( p a n e l a). To determine the structural consequence of reduction, insulin receptors were phosphorylated prior to reduction and samples were analyzed by SDS-polyacrylamide gel electrophoresis in the presence of 50 mM N-ethylmaleimide. Labeled bands were visualized by autoradiography ( p a n e l b ) .

Transmembrane Actiuation of the Insulin Receptor Kinase 15283

tion can either be due to a reduced number of binding sites or to a reduced affinity or both. Therefore, insulin binding to a@ dimers and to the tetrameric receptor form was analyzed by a competition binding assay and transformation to Scat- chard plots. Fig. 2 shows a representative Scatchard plot obtained upon transformation of the binding data. Since the low affinity binding component is a very minor component in these purified receptor preparations, the maximum number of binding sites can be determined by extrapolation of the linear part of the curve to the abscissa. This analysis always revealed a reduced affinity by a factor of 3-6 for the a@ dimer, whereas the maximal number of binding sites was not af- fected. This can be confirmed by chemical cross-linking of '""Ilabeled insulin at saturating concentrations (see inset, Fig. 2). The amount of ligand cross-linked to a@ halves and the proteolytic form a@' after reduction was equal to that linked to the tetrameric holoreceptor form and its proteolyt- ically derived lower molecular weight tetramers. We therefore used insulin concentrations near saturation for all the subse- quent experiments involving kinase activation.

a@ Dimers Are Not Associated by Noncovalent Interac- tions-Following the reduction protocol described above, the migration of the receptor in SDS as apparent a@ dimers does not rule out the possibility that this species remains as a noncovalently associated tetrameric form in Triton X-100. In order to address this question, we subjected reduced or intact receptor to velocity gradient sedimentation in sucrose gra- dients, and we then measured tracer insulin binding in the resultant fractions as shown in Fig. 3. Insulin binding to the reduced form (fractions 19-25) shows a markedly slower sedi- mentation profile compared to the binding profile of the intact receptor (fractions 11-19). Previous studies had shown that the tetrameric receptor from turkey erythrocytes had an s value of 10.2 in similar velocity sedimentation experiments, whereas the dimeric receptor form had an s value of 6.6 under the conditions employed, namely cross-linking of receptor prior to centrifugation (17). The data in Fig. 3 are quite similar to that of the previous study. Moreover, tracer insulin

1 2 3 bound (MI

FIG. 2. Reduction with DTT results in a decreased affinity and does not affect the maximum number of binding sites. Receptor preparations were subjected to the standard reduction pro- tocol (-) or left intact (-), and binding was determined by a competition binding assay using 40 PM of '251-labeled insulin as a tracer and adding increasing concentrations of cold insulin. Results were transformed to a Scatchard plot and the maximum number of binding sites, given at the intersection of the x axis, was determined by linear regression of the linear part of the binding curve. The inset shows reduced ( B ) and nonreduced (A ) receptor to which a near saturating '251-insulin concentration (5 X lo-') was cross-linked (see "Experimental Procedures") prior to electrophoresis and autoradiog- raphy.

I 10-3

JO-

20 -

1 0 -

A FIG. 3. a@ dimers can be separated by velocity centrifuga-

tion. Receptor preparations, reduced with the standard reduction protocol or left intact, were separated on sucrose density gradients exactly as described under "Experimental Procedures." Specific bind- ing of tracer insulin concentrations was determined to individual fractions of gradients loaded with intact receptor (U) and reduced receptor (0- - -0).

binding to the separated a@ dimer comprises 40% of the total binding to intact receptor, a result entirely consistent with the data in Figs. 1 and 2 where the species were not separated. We also note that the separation of the dimer from the tetramer is essentially complete under our conditions (Fig. 3).

In order to demonstrate that the slower sedimenting recep- tor form in Fig. 3 was in fact the a@ dimer, we cross-linked '2sI-labeled insulin to tetrameric receptor and to receptor dimers which had been obtained upon reduction, and we centrifuged them through sucrose gradients. Following frac- tionation, we analyzed receptors by gel electrophoresis fol- lowed by autoradiography. Fig. 4a shows that the tetrameric receptor peaks in fraction 15, whereas the dimeric receptor peaks in fraction 23-24 (Fig. 46). The tetrameric receptor does not trail into fractions where the dimeric receptor peaks, confirming that this separation yields a pure population of dimeric receptors halves. We obtain exactly the same result (data not shown) if we perform the cross-linking analysis after velocity sedimentation, namely complete separation of di- meric receptor from intact receptor. These data also allow us to analyze the question of whether a@ dimers stay together by noncovalent interactions. If this is the case, reduced recep- tors should appear in the fractions of the tetrameric receptor upon gel electrophoresis. We never see a@ dimers in fractions where the tetrameric receptor sediments (Fig. 4B).

From the data of Figs. 3 and 4, we make the following conclusions: 1) after our reduction protocol in Triton X-100, (Y@ dimers are not associated by noncovalent interactions. However, this does not exclude the possibility that interac- tions might transiently occur to some degree, particularly when the receptor concentration is high; 2) separated or not from residual tetramer, a@ dimers appear to retain the ligand binding characteristics shown in Fig. 2, that is, an unchanged Bmnx with a reduced affinity for ligand.

Autophosphorylation of Isolated Dimers-Having estab- lished that a@ dimers generated by our standard reduction protocol are true dimers that retain saturable ligand binding, we tested these species to determine if they could undergo insulin-dependent @ subunit autophosphorylation. Control experiments in which various concentrations of sucrose up to 25% had been added to receptor preparations showed that the

Transmembrane Activation of the Insulin Receptor Kinase

10 13 15 16 18 19 20 21 22 24 25 26 27 29 f r a c t i o n number

FIG. 4. aj3 dimers are not associated by noncovalent inter- actions. Soluble insulin receptor was reduced with the standard reduction procedure (panel 6) or left intact (panel a), passed over a desalting column, and subjected to cross-linking with 5 X lo-* M lZ5I- labeled insulin. The receptor preparations were analyzed by velocity sedimentation as described under “Experimental Procedures.” Indi- vidual fractions were electrophoresed through a SDS-polyacrylamide gel, and bands were visualized by autoradiography. The fractions on the left represent the bottom of the gradient whereas the fractions on the right are the top of the gradient.

presence of sucrose does not interfere with the receptor auto- phosphorylation. Therefore, individual gradient fractions could be assayed directly for autophosphorylation in the pres- ence and absence of insulin. Fig. 5 shows autophosphorylation reactions of reduced receptors which had been centrifuged in a sucrose gradient with a low ionic strength. The upper panel of Fig. 5 shows an autoradiogram of gradient fractions which had been subjected to autophosphorylation and separated by polyacrylamide gel electrophoresis. a@ dimers were character- ized by a marginal insulin-induced autophosphorylation re- action which never exceeded a 1.5-fold stimulation of p sub- unit phosphorylation when compared with the /3 subunit phosphorylation in the absence of insulin. However, under these conditions we always observed tetrameric receptors in the dimer fractions and these tetramers exhibited a marked insulin-dependent autophosphorylation reaction. Since tet- rameric receptor forms were observed in the dimer fractions and no dimers were seen in the tetramer fractions, the for- mation of tetramers in the dimer fraction was most likely due to reoxidation of some ap dimers to the tetrameric receptor form, Reoxidation however requires the association of two dimers and might be abolished by preventing the formation of noncovalent interactions between dimers. Dimers were

f r a c t i o n n u m b e r

FIG. 5. Autophosphorylation of gradient fractions isolated from low ionic strength sucrose density gradients. Receptor preparations were reduced with the standard reduction protocol and separated on a 5-25% sucrose density gradient containing 30 mM Hepes (pH 7.6), 0.1% Triton X-100, and 0.02% azide. Individual fractions were subjected to autophosphorylation in the presence (+) or in the absence (-) of 3.5 X lo-’ M insulin (ins). Samples were separated on a nonreducing SDS-polyacrylamide gel, and bands were visualized by autoradiography (upper part of Fig. 5). To quantify the 32P incorporation, bands were excised and Cerenkov radiation was determined. The graph shows autophosphorylation of tetrameric re- ceptor in the presence of insulin (-) and in the absence of insulin ( 0 - - -O), autophosphorylation of ab dimers in the presence of insulin (M) and in the absence of insulin (0- - -0).

therefore isolated by centrifugation through a media with a higher ionic strength (0.15 M NaCI). During the subsequent kinase assay, the salt concentration is 75 mM. In a series of separate control experiments using both intact and reduced receptor, we determined that this level of NaCl does not interfere with ligand binding as determined by Scatchard analysis or with receptor autophosphorylation (data not shown). Thus the intact receptor shows a 5-fold insulin effect after sedimentation in the NaC1-containing gradient (Figs. 6A and 7). When reduced receptor was separated in the salt- containing gradient, no receptor reoxidation was observed (Fig. 6B). Furthermore, a@ dimers showed no insulin-induced autophosphorylation (Fig. 7). These data strongly support the notion that CY@ dimer-dimer interaction is required for the activation of the kinase. The level of 32P incorporation into dimers roughly corresponded to the level of the insulin-inde- pendent, basal kinase activity of the tetrameric receptor (Fig. 7, compare the basal kinase of the tetramer, fraction 15, with that of the dimer in fraction 21 or 23). The basal activity of the dimer was, however, slightly higher than that of the tetramer due to the stimulatory effects of the DTT (see Ref. 13 and Fig. 8).

Effect of Various DTT Concentrations on the Basal Kinase Activity-The autophosphorylation experiments with the gra- dient fractions revealed that the basal, insulin-independent kinase is slightly stimulated by reduction and does not seem

Transmembrane Activation of the Insulin Receptor Kinase 15285

FIG. 6. a/3 dimers isolated in the presence of 0.15 M NaCl do not show insulin-dependent kinase activation. Receptor prepa- rations were reduced (panel R ) or left intact (panel A ) and fractionated over a 5-25% sucrose density gradient containing 30 mM Hepes (pH 7.6), 0.1% Triton X-100, 0.02% azide, and 150 mM NaCI. Individual fractions were subjected to autophosphorylation in the presence (+) or absence (-) of 3.5 X 10" M insulin. Samples were analyzed by SDS-polyacrylamide gel electrophoresis as described in the legend to Fig. 4.

I r a c l i m number ..

Flc. 7. Quantitation of the autophosphorylation of gradient fractions isolated in the presence of 0.15 M NaCl. Quantitation of the autophosphorylation experiment shown in Fig. 5 was performed by excising the bands and counting their Cerenkov radiation: Auto- phosphorylation of tetrameric receptor in the presence (-) and in the absence (0- - - 0 ) of insulin and autophosphorylation of a@ dimers in the presence (M) and in the absence (0- - - 0 ) of insulin.

to require dimer interactions. To examine this in further detail, the effect of various concentrations of DTT a t pH 7.6 on the receptor was analyzed (Fig. 8). At low concentrations of DTT, when the reduction is not complete, an increase of the basal kinase activity is observed on the tetrameric, as well as on the dimeric receptor structures. At higher concentra- tions where the dimeric structure is the predominant form, the basal kinase was also slightly enhanced compared to the untreated tetramer. This shows that even if reduction condi- tions (20 mM DTT a t pH 7.6) are used, which result in a complete inactivation of the binding function, the basal, in- sulin-independent autophosphorylation is not compromised, and if anything, is slightly stimulated although to a far less degree than by insulin. We interpret this to mean that our reduction protocol leaves the basal kinase domain essentially in its native state.

(, 1 2 5 2 5 5 70 20 4 0 mu OllhmIh~es101

Flc. 8. The insulin-independent basal kinase activity is not diminished upon treatment with various D T T concentrations. Insulin receptor was treated with various concentrations of DTT for 30 min at pH 7.6. DTT was removed by passing the samples over desalting columns, and the resulting receptor preparations were sub- jected to insulin-independent autophosphorylation. The reactions were stopped by adding 40 mM EDTA final concentration. The samples were treated with 50 mM N-ethylmaleimide prior SDS- polyacrylamide gel electrophoresis. '"P incorporation into tetrameric receptor (-) and into no dimers (o"-o) was determined as previously described.

Effect of Reduction on the Actiuated Kinase-hsulin-de- pendent autophosphorylation of the /3 subunit results in an enhanced ligand-independent kinase activity toward exoge- nous substrates (12). To see whether the activated kinase is affected by reduction, we first determined in a control exper- iment that our receptor preparation showed a 4-fold enhance- ment of ligand-dependent autophosphorylation. We then re- duced the autophosphorylated receptor, after 8 min in the presence of insulin and ATP, either with 1.25 mM at pH 8.5 or with 20 mM DTT at pH 7.6. To remove the insulin, we precipitated the receptor preparations with polyethylene gly- col and resuspended them in an insulin-free buffer. Insulin- independent exogenous kinase activity was measured by add- ing histones as substrate for various times. The same general results were obtained for both reduction procedures. Fig. 9 shows a representative time course of histone phosphorylation where 20 mM DTT at pH 7.6 has been used for reduction. There was never an impairment of the exogenous kinase activity. The rate of histone phosphorylation was even slightly higher in the case of the reduced receptor despite a 15-20% loss of ["PI-label in dimers as compared to the tetramers after reduction. We do not know why this loss of label occurs, but in any case, these data suggest that dimer-dimer interac- tions are not necessary for the maintenance of the kinase activity once autophosphorylation takes place. This finding has recently been independently made by Pike et al. (29).

DISCUSSION

The insulin receptor belongs to a group of receptors which have some functional and structural features in common. This group includes receptors for epidermal growth factor, platelet- derived growth factor and insulin-like growth factor-1. These all undergo ligand-induced stimulation of intrinsic tyrosine- specific kinase activity (for review see Ref. 30). Their basic structure as represented by the EGF receptor is composed of an extracellular domain with the binding site, a single mem- brane spanning region, and a cytoplasmic domain containing the tyrosine kinase activity (31). It is an open question of how ligand binding to the extracellular domain results in a trans- membrane activation of the kinase while having only one membrane spanning region within the molecule. In contrast to the EGF receptor, the insulin receptor appears to have

15286 Transmembrane Activation of the Insulin Receptor Kinase

I , , 0.5 1 2 4 6 8 minuter

FIG. 9. The insulin-activated exogenous kinase activity does not require aB dimer interactions. The insulin receptor kinase was activated by subjecting the receptor to autophosphoryla- tion. The reaction was stopped with 50 mM EDTA, and the insulin, ATP, and cations were removed by precipitation with polyethylene glycol. The receptors were resuspended in 30 mM Hepes (pH 7.6) containing 0.1% Triton X-100 and 0.02% azide and either left intact or reduced with 20 mM DTT for 30 min. Histone H2b was added at a final concentration of 0.5 mg/ml, and phosphorylation was initiated by adding ATP and cations to a final concentration of 50 p~ ATP, 10 mM MgCIz, and 2 mM MnCIz. The reaction was stopped by adding Laemmli sample buffer, and samples were separated in a 7.5-15% polyacrylamide gradient gel and autoradiographed. Histone bands were excised and their 3zP incorporation was determined by Cerenkov counting. U, histone phosphorylation by tetrameric receptor; M, histone phosphorylation by dimeric receptor. To examine completeness of reduction, samples were removed after DTT exposure and analyzed by gel electrophoresis: Lane A on the inset shows the untreated receptor and lane B shows the a0 dimer obtained upon reduction.

duplicated each of its structural domains and each receptor molecule therefore contains two associated transmembrane subcomponents which are linked by disulfide bonds. However, the EGF receptor has been found to be associated by nonco- valent interactions: 80% of the EGF receptors appear in a dimer state upon detergent solubilization of membranes (32). I t is possible that the interaction of two membrane spanning basic receptor units is required to transduce the signal of the ligand across the membrane. This may be a general require- ment for receptors possessing intrinsic tyrosine kinase activ- ity.

This report describes an approach to obtain functional insulin receptor halves in order to examine whether they form a minimal functional unit or whether their interaction is necessary for the transmembrane activation of the receptor kinase and for the maintenance of the activated kinase. In- sulin receptor halves ((YO dimers), which are obtained upon virtually complete reduction of class 1 disulfides with 1.25 mM DTT at pH 8.5, maintain up to 50% of tracer insulin binding, whereas previously used reduction procedures which involve treatment of receptors with 20 mM DTT at pH 7.6, result in a complete loss of binding for detergent-solubilized receptor. The effect on binding is most likely due to reduction of disulfides in the binding domain rather than as a conse- quence of denaturation: DTT is highly specific for sulfhydryl groups due to its extremely negative redox potential. Indeed the (Y subunit contains a region which is enriched in cysteines and is postulated to form a ligand-binding domain (19, 20). The EGF receptor has a similar sulfhydryl-rich region (30). Scatchard analysis and chemical cross-linking of '2sII-insulin revealed that the reduced binding at tracer hormone concen-

tration seen for receptor halves is due to a reduction in affinity for insulin and that the total number of binding sites is not affected. Scatchard plots of insulin receptor samples tend to be curvilinear and it has been postulated that a receptor preparation consists of high affinity binding and of low affin- ity binding receptors. They differ in their affinity by a factor of 25 (33). a@ dimers obtained upon reduction of interhalve disulfides show a reduction in high affinity binding by a factor of 3-6, which is not dramatic when compared to the difference in affinity between high and low affinity binding receptors. We interpret this to mean that our reduction conditions lead to a relatively minor loss in ligand affinity for the high affinity insulin-binding site. To test ligand-inducible autophosphory- lation we therefore always used near saturating insulin con- centrations, where the affinity loss is inconsequential with respect to receptor occupancy.

During all the experiments involving reduction with DTT, it is of critical importance to either remove the DTT by gel filtration or to inactivate it with N-ethylmaleimide prior SDS gel electrophoresis. Omission of this step results in further reduction of the receptor in SDS and does not allow the deduction of the actual receptor structure in Triton X-100 solution. Data from such protocols where DTT was not re- moved or inactivated prior SDS-polyacrylamide gel electro- phoresis are therefore not easily interpretable. Thus, the result of a recent publication (34) claiming that the CUB recep- tor form is more active as a kinase than is the tetrameric receptor form is problematic, because the DTT was not re- moved prior to the addition of SDS.

Since after reduction we always found about 5% of the receptor remaining in the tetrameric form, we separated these from the dimers by velocity centrifugation. Essentially, base- line separation between the two forms was obtained and hence this procedure resulted in the isolation of pure dimeric recep- tors. These were used to assay insulin-inducible autophospho- rylation under conditions where we could be confident that any insulin-induced kinase activity would not be a conse- quence of the a@ dimer serving as a substrate for the residual tetramer. Furthermore, this type of analysis is a potent tool to examine whether reduced receptors remain associated by strong and specific noncovalent interactions. Such interac- tions have been observed between receptors for EGF, which sediment as dimers and as monomers on sucrose density gradients (32). Insulin receptor halves were never observed in gradient fractions of the tetrameric form which suggests that possible noncovalent interactions of a@ dimers are weaker than those between EGF receptors. Some degree of dimer association is indicated by the finding that dimeric receptor obtained from a low ionic strength sucrose gradient reoxidizes to the tetrameric form. This reaction must be preceded by dimer association unless a collision-type mechanism is in- volved. Importantly, the reoxidized receptors are highly in- sulin-sensitive, which is a further indication that the reduc- tion protocol (DTT at pH 8.5) does not result in major structural perturbations of the insulin-binding domain.

In contrast to the reoxidized receptor form, there was a reduced insulin responsiveness on dimers (Fig. 5). One might explain the loss of an insulin response by a selective increase in the basal activity of the dimers. This explanation however, can be ruled out because the small increase in the basal kinase of the a@ dimers following reduction (1.5-2-fold, Figs. 5, 7, and 8) cannot account for the loss of a 5-6-fold ligand de- pendent activation (Figs. 5 and 7). The residual insulin- dependent autophosphorylation reaction in fractions from low ionic strength gradients, as well as the reoxidation reaction, was abolished when dimers were centrifuged through a sucrose

Transmembrane Activation of the Insulin Receptor Kinase 15287

density gradient containing 0.15 M NaC1. At this concentra- tion and at 75 mM NaCl present in the actual kinase assay, the salt interferes neither with the autophosphorylation re- action nor with insulin binding to the reduced and to the tetrameric receptor. We think it highly unlikely, but we cannot completely exclude the possibility that NaCl selec- tively affects only the dimeric structure such that kinase activation is blocked. The finding that there are no reoxidized receptors when dimers were isolated in a high ionic strength solution indicates that NaCl interferes with receptor associ- ation. Furthermore, this concentration of NaCl, which is close to physiological concentration, has been used to prevent the aggregation of the src kinase and has no effect on the auto- phosphorylation reaction of pp60"" (35). Taken together, these data strongly support that the association of dimers is of critical importance for the insulin-dependent activation of the receptor's kinase activity.

A recent report showed that the noncovalent EGF receptor dimer is the only form with EGF stimulability and it has been postulated that EGF stimulates the kinase activity by shifting the association equilibrium towards the monomer form (32). The opposite mechanism, that the EGF binding induces di- merization which results in kinase activation has been pos- tulated by Yarden et al. (37). Although there is controversy over the mechanism of the EGF receptor kinase activation, both reports show that a dimeric EGF receptor structure seems to be involved in the regulation of kinase activation. In the case of the insulin receptor only the tetrameric receptor structure, which is analogous to the EGF receptor dimer, shows ligand dependent kinase activation. Separation of the two receptor halves does not result in an activation of the kinase comparable to the insulin-induced activation. The basal kinase of the tetrameric receptor and the dimeric form isolated in high ionic strength sucrose gradients are not sig- nificantly different. In a dose-response experiment with DTT a t pH 7.6, an increase of basal kinase was observed at low DTT concentrations. Since this increase is observed on the tetrameric form as well (see also, Ref. 13), it is not likely that the separation of the receptor halves accounts for the in- creased basal kinase activity on the cy/? dimers. Rather, there are direct effects of DTT on the kinase function and it has been postulated that the insulin receptor kinase requires a reduced sulfhydryl group near the active center (13). This is a common feature of many kinases including the epidermal growth factor receptor kinase (36). The maintenance of the basal kinase activity, even a t high DTT concentrations, in- dicates that DTT treatment does not affect the structural integrity of the kinase domain. This is further supported by the finding that the insulin-stimulated kinase activity is not diminished upon reduction of activated tetrameric receptor. This finding also implies that dimer interactions are not critical for the maintenance of the activated kinase, unlike insulin-induced autophosphorylation which is greatly dimin- ished on dimers isolated from low ionic strength gradients and completely abolished when noncovalent interactions are prevented.

In conclusion, the data presented in this report strongly suggest that the transfer of the insulin-binding signal to the kinase domain requires the association of two intact receptor halves. We believe this is an attractive mechanism by which insulin could induce a biologically important transmembrane signal in intact cells.

Acknowledgment-We wish to thank Dr. A. Traish for his helpful comments.

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