ARCHIVES

OF BIOCHEMISTRY

AND

BIOPHYSICS

Vol. 290, No. 1, October, pp. 79-85, 1991

The Native cy2p2Tetramer Is the Only Subunit Structure of the Insulin Receptor in Intact Cells and Purified Receptor Preparations’ Esther Schenker2 and Ronald A. Kohanski Department

of Biochemistry,

Mount Sinai School of Medicine,

1 Gustave L. Levy Place, New York, New York 10029

Received May 8, 1991

The native subunit structure of the insulin receptor was reinvestigated by two-dimensional nonreducingjreducing gel electrophoresis. Human insulin receptor expressed in murine fibroblasts was found to be a single oligomer, the CC& heterotetramer. The structure was assessed using receptor metabolically labeled with [35S]methionine, and using receptor autophosphorylation at two levels of purification: the insulin affinity-purified receptor and the more commonly used wheat germ agglutinin-Sepharose-enriched fraction from whole membrane extracts. Lower molecular weight oligomers and free subunits were observed only upon heating the sample prior to electrophoresis. This artifact of sample handling was dependent upon three factors: (i) temperature, (ii) time of heating, and (iii) impurities typically present in partially purified receptor preparations. We conclude that the (Y&~ tetramer is the only insulin receptor subunit structure native in intact cells and subsequently isolated from cell membranes. 0 1991 Academic PWS. IN.

The insulin receptor, a disulfide-linked heterotetramer, is built of two extracellular a-subunits (M, N 135 kDa), which are anchored to the cell membrane by two transmembrane ,&subunits (n/r, = 95 kDa; reviewed in (1,2)). The two subunits are encoded by a single gene, which is translated into the 210-kDa insulin receptor precursor (3, 4). The disulfide bonds, linking (Y- and ,&subunits, are formed post-translationally, followed by enzymatic cleavage of the precursor into (Y- and P-subunits, thus forming the a& heterotetramer (15). The intracellular domain of the P-subunit is autophosphorylated and displays protein tyrosine kinase activity, both of which are stimulated upon insulin binding to the extracellular cy1 Supported by Grant DK 38893 from the National Health. * To whom correspondence should be addressed. 0003-9861/91$3.00 Copyright 0 1991 by Academic Press, All -:-I-&^ ^c_^-_^_l_.^r:^:- ^_.. c-_-

Institutes

of

subunit. The kinase activity is thought to play a crucial role in signal transduction of the insulin receptor. Alterations of either the insulin binding domain or the catalytic domain are often accompanied by altered kinase activity. The nature of the oligomeric structure of the insulin receptor has been controversial. Several groups, using gel electrophoresis, identified multiple oligomeric forms of the insulin receptor, as well as free (Y- and P-subunits (513). These observations are contradictory to the conclusions drawn from investigations with the insulin receptor precursor, the single polypeptide &-monomer, which is processed in the endoplasmatic reticulum and the Golgi into the mature a& heterotetramer (14, 15). There was no evidence obtained which would support the formation of different receptor species during this post-translational processing. Consistent with these latter results, the insulin-affinity-purified insulin receptor was found to retain the heterotetrameric cyzpZstructure, and no other oligomerit forms were detected throughout the entire purification procedure (16). This fact was essential to establish that autophosphorylation of the insulin receptor preceeded insulin stimulation of glucose transport in whole 3T3-Ll adipocytes (17). It seems therefore unlikely that different oligomeric forms of the insulin receptor are present in the membranes of living cells. However, the possibility remains that disulfide bonds linking the subunits to the native heterotetramer are disrupted either during the purification process or while preparing samples for gel electrophoresis. To establish the native oligomeric state of the insulin receptor present in intact cells as well as in solubilized receptor preparations, we analyzed human insulin receptor from transfected mouse NIH 3T3 fibroblasts by twodimensional nonreducing/reducing gel electrophoresis. Using insulin receptor labeled in situ as well as receptor from different stages of the purification protocol, we show that forms other than the native a& heterotetramer clearly result from an artifact of sample preparation. 79

Inc. _^_^ _.^_I

80 EXPERIMENTAL

SCHENKER

AND

PROCEDURES

Materials. Trar?S-label and [32P]orthophosphate were obtained from ICN Radiochemicals; [y3’-P]ATP was synthesized following the method of Walseth and Johnson (18) and purified (19). Bio-Gel P-6DG was from Bio-Rad. Triton X-100, Hepes,3 and ATP (disodium salt) were from Boehringer Mannheim; EDTA (tetrasodium salt) and Mn(CH,CO& were from Sigma; HPLC solvents, SDS, and BCA reagents for protein determination were from Pierce Chemical Co.; chemicals for electrophoresis were from United States Biochemicals. Cell culture reagents were from Gibco; Er?hance was from New England Nuclear. All other chemicals were of reagent grade. Monoclonal antibodies were a gift from Dr. K. Siddle. NIH 3T3 HIR3.5 cells Metabolic labeling of human insulin receptor. were grown in 100.mm-diameter dishes in DMEM, supplemented with 10% FBS. At 80% confluence, cells were incubated for 22 h in methionine free DMEM, containing Tra#S-label (carrier free; 0.5 mCi/dish) and 10% dialyzed FBS. Cells were then washed three times with PBS and lysed in 50 mM Hepes (pH 7.6), 150 mM sodium chloride, 1% Triton X100 (w/v), 100 mM sodium fluoride, 10 mM EDTA, 10 mM pyrophosphate, 2 mM vanadate, 10% glycerol (w/v). Half of the plates were incubated with 2 PM insulin for 10 min prior to the lysis. 35S-labeled insulin receptor was either partially purified over a WGA-Sepharose column or immunoprecipitated directly from whole membrane extracts with the mixture of the anti& and anti-0 antibody (see below). Samples were either heated 10 min at 90°C or kept at room temperature and then analyzed by twodimensional SDS-PAGE. Gels were stained, destained, and soaked for 1 h in En3hance and autoradiograms were taken from the dried gels. Receptor was purified from NIH Purification of the insulin receptor. 3T3 fihroblasts transfected with the cDNA of human insulin receptor (clone HIR3.5, Ref. (20)). The receptor was extracted from membranes with 2% Triton X-100 (w/v) in 50 mM Hepes, 1 mM EDTA, pH 8.5, and partially purified as a WGA-Sepharose eluate (WGASE); pure receptor was obtained by insulin-affinity chromatography as described earlier (16). The reaction was performed Insulin receptor autophosphorylation. at room temperature in 50 mM Hepes, 1 mM EDTA, 0.12% Triton X100 (w/v) at pH 6.9. The reactions were initiated by the addition of carrier free [y3*-P]ATP (-5000 Ci/mmol), and 5 mM Mn(CH&O&. Autophosphorylation was carried out in either the absence of insulin or the presence of 1 PM insulin. Reactions were quenched with either concentrated Laemmli sample buffer or by gel filtration chromatography using a Bio-Gel P-6DG column. Isolation of the insulin receptor subunits and oligomers. Partially purified receptor (WGASE) or pure receptor was autophosphorylated as described above and the reaction quenched by adding concentrated Laemmli sample buffer, giving final concentrations of 2% SDS, 20 mM EDTA, 0.4 M Tris, but no DTT. The reaction mixture was divided into aliquots which then were heated at 90°C for 2-10 min. Control reactions were not heated. The samples were analyzed by two-dimensional nonreducing/reducing gel electrophoresis. The first-dimension separating tube gel (0.3 X 11.5 cm) was 4.2% total a&amide (50:2.5, acrylamide: bis); the second-dimension separating slab gel (0.8 mm thick) was 10% total acrylamide. The receptor subunit oligomers were localized on the dried gels by autoradiography. For quantification, gel segments containing the different subunit oligomers were excised and radioactivity measured by Cerenkov counting in a Beckman LS9000 scintillation counter.

3 Abbreviations used: Hepes, 4-(2-hydroxyethyl-l-piperazine) ethanesulfonic acid, EDTA, ethylenediaminetetraacetic acid, WGASE, wheatgerm agglutinin-Sepharose eluate; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; DTT, dithiothreitol; DMEM, Dulbeccos’s modified Eagle medium; FBS, fetal bovine serum; PBS, phosphate-buffered saline; BSA, bovine serum albumin; BCA, bicinchoninic acid.

KOHANSKI

Influence of impurities on the analysis of subunit composition. Autophosphorylated pure receptor was applied to a l-ml Bio-Gel P6DG column in 50 mM Hepes, 1 mM EDTA, 0.12% Triton X-100 (w/ v), pH 6.9. The 32P-labeled receptor was recovered in 200 ~1. Concentrated Laemmli sample buffer was added to the pooled fraction and aliquots were mixed with increasing amounts of WGASE. The samples were then heated for 10 min at 90°C and analyzed on a nonreducing twophase gel system (upper gel 4.2% total acrylamide; 7 cm); lower gel 8% total acrylamide; 5 cm). Controls were not heated. An autoradiogram was taken from the dried gel. The gel was cut according to the autoradiogram, 32P quantified in each subunit/oligomer, and the percentage of radioactivity present calculated for each lane. [32P]Phosphopeptides of the P-subunit. Pure receptor was autophosphorylated and boiled for 10 min and subunits were separated by twodimensional nonreducing/reducing gel electrophoresis. Gel segments from the wet, unfixed gels were excised according to the autoradiogram taken of the wet gel. [32P]Phosphopeptides were obtained by tryptic digestion, and were resolved by HPLC, using a Hewlett-Packard 1090 chromatograph and a Spherisorb C8 column as described elsewhere (21). Immunoprecipitation with mono&mu1 anti-insulin receptor antibodies. Monoclonal antibodies were used to precipitate partially purified 35S-labeled receptor and 32P-labeled receptor. Autophosphorylation was performed with partially purified insulin receptor (44 pg total protein), and 32P-labeled receptor was recovered from a Bio-Gel P-6DG column (see above). The 32P-labeled receptor (50 ~1 = 11 pg total protein) was incubated at 4°C for 16 h with 7 pg of the antibodies (see Ref. (26); antibody 8314 against the a-subunit and antibody 18-44 against the P-subunit). Protein A-Sepharose (prehydrated at 2 mg per 50 ~1) was added and incubated for 2 h at 4°C. The reaction mixture was divided, concentrated Laemmli sample buffer was added, and the reaction mixture was heated for 10 min at 9O”C, or kept at room temperature. Subunit separation was achieved by using the two-phase gel system described above. Quantification was done as described above.

RESULTS

Two-dimensional nonreducing/reducing gel electrophoresis was used to separate disulfide-linked insulin receptor oligomers. First, insulin receptor was labeled with [35S]methionine and its oligomeric structure assessed, as present in live cells. Then, receptor at two different stages of our purification protocol (16) was investigated for lower molecular weight oligomers that might arise during extraction and isolation. Autophosphorylation, which conveniently radiolabels the P-subunit, was used for localization. The subunit compositions were inferred from the 2-D coordinates of the 32P-P-subunits, using the [35S]methionine-labeled receptor for comparison (13, 17). Subunit Structure of Metabolically Insulin Receptor

Labeled Human

When assessing the oligomeric structure of the insulin receptor under nonreducing conditions, samples usually were prepared according to the original protocol of Laemmli (22), where samples were boiled 1 to 5 min in a sample buffer containing SDS. We investigated whether heating the receptor in this buffer breaks the disulfide-linked CQ/& heterotetramer into lower molecular weight oligomers, which are not inherent to the live cell. Therefore, we metabolically labeled the human insulin receptor, expressed in NIH 3T3 fibroblasts. In this way any subunit composition existing in live cells can be de-

OLIGOMERIC

STRUCTURE

tected, including those that would not exhibit kinase activity. Such oligomers might not be detected when labeling the receptor through autophosphorylation. After continuously labeling the cells with Tran35S-label for 22 h in methionine-free medium, half of the plates were incubated with 2 PM insulin for 10 min. One single receptor species, the c-u& heterotetramer, was isolated with receptor immunoprecipitated by the mixture of the anti-a- and anti-&subunit antibody (Fig. 1A) or partially purified by WGA-Sepharose chromatography (Fig. 2A). Insulin binding to intact cells did not influence the native subunit composition of the insulin receptor. Different oligomers, assigned oligomer I and II, including free psubunits were observed only after heating the receptor in Laemmli sample buffer (Figs. 1B and 2B). The lower molecular weight oligomers observed were noticeably less abundant after heating insulin receptor purified by immunoprecipitation than using partially purified receptor (cf. below impurities and Table III). Subunit

Structure

of the Insulin

Receptor after Heating

Partially purified insulin receptor (WGASE), the most commonly used preparation, and pure receptor were examined next. Insulin receptor present at each stage of purification was autophosphorylated and each reaction

non-reducing

ho

aP-

t

t

t

I

II

INSULIN

81

RECEPTOR non-reducina

ho

DTT)

(I-W B-

aB-

tt T

t I

II

t T

FIG. 2. Subunit structure of partially purified human insulin receptor. Metabolically labeled insulin receptor (A and B) as well as unlabeled receptor (C and D) were partially purified and analyzed for their oligomerit structure by two-dimensional nonreducing/reducing gel electrophoresis. The unlabeled, partially purified receptor was autophosphorylated for 35 min with carrier-free [y-a*P]ATP, 5 mM Mn(CH,CO&, and 1 pM insulin. The reaction was stopped by adding Laemmli sample buffer. Samples of each preparation were either analyzed directly by SDS-PAGE (A and C) or heated for 10 min at 90°C (B and D). Arrows indicate the receptor tetramer (T) and oligomers I and II as separated in the first dimension, and the migration of cy- and p-subunits in the second dimension.

DTT)

aP-

T

OF THE

FIG. 1. Subunit structure of metabolically labeled human insulin receptor. NIH 3T3 fibroblasts, expressing human insulin receptor, were continuously labeled with Tran3’S-label for 22 h, the receptor was extracted and immunoprecipitated with the combination of anti-a- and anti-/3-subunit antibodies (see Experimental Procedures). Samples were analyzed by two-dimensional nonreducing/reducing gel electrophoresis. Sample A was not heated, B was heated for 10 min at 90°C in Laemmli sample buffer prior to gel electrophoresis. Arrows indicate the receptor tetramer (T), and oligomers I and II as separated in the first dimension, and the migration of 01-and P-subunits in the second dimension.

quenched with Laemmli sample buffer. One-half of each mixture was heated 10 min at 9O”C, while the other half was kept at room temperature (25°C). Figures 2B and 2D illustrate the results obtained from autophosphorylated WGASE without and with heating, respectively. Receptor in the WGASE and the purified receptor each appeared as a unique molecular weight species, the cra& heterotetramer, when not heated prior to electrophoresis (Figs. 2A and 2B). After heating, however, the amount of radioactivity present in the a.& tetramer decreased while two other, lower molecular weight species of the insulin receptor appeared (Figs. 2C and 2D). They were assigned oligomer I (see above), and free P-subunit, according to a molecular weight standard, run on a nonreducing twophase SDS-PAGE (see below). The total radioactivity present in the &$ tetramer resolved without prior heating was distributed quantitatively in the three receptor species, resolved after heating. Thus, the origin of the lower molecular weight oligomers was the aePz heterotetramer and the appearance of these resulted from sample heating. Time-Dependence

of Heterotetramer

Breakdown

We then investigated the time of heating necessary to break down the a~~/$tetramer into lower molecular weight oligomers and free subunits. Autophosphorylated receptor was prepared as above and samples were heated for increasing periods at 90°C. A constant decrease of cr.& tetramer was observed, while the amounts of oligomer I and

82

SCHENKER TABLE

Insulin (1 PM)

TABLE Resolved

by SDS-PAGE

Percent distribution (subunit structure)

Heated at 90°C (min)

Percent distribution (subunit structure)

%P*

Oligomer I

P

0

100

0 9.5 * 1.3 24.2 + 6.8 48.4 f 1.2 0 53.8 f 4.5

2 5

80.8 i 2.0 58.4 + 5.5

10

31.0 f 2.1

0 9.6 f 0.7 17.4 f 3.4 20.6 f 2.2

-

0 10

100 29.6 f 4.8

0 16.6 + 0.7

Note. The distribution of insulin receptor oligomers and free subunits was established by two-dimensional nonreducing/reducing SDS-PAGE. The insulin receptor in the WGASE was first autophosphorylated with carrier-free [y3*-P]ATP, 5 mM Mn(CHsC02)2, at room temperature for 60 min; 1 pM insulin was present or absent as indicated. The autophosphorylation reaction was quenched and then divided into aliquots before heating at 90°C or kept at room temperature (25’C, “10 min”). 25 pg of protein was used per assay. The three subunit structures were located by autoradiography (cf. Fig. 2D). Further details are given in the text. These data are averages of triplicate samples.

free P-subunit increased accordingly (Table I). Again, the pure receptor preparation appeared to be less heat-labile than the partially purified receptor (Fig. 2, and below). However, the breakdown into smaller oligomers was observed at each time of heating for both receptor preparations. No significant differences in the amounts of oligomer I or free P-subunit were observed whether or not the autophosphorylation reaction was carried out in the presence or in the absence of 1 PM insulin (Table I). Therefore, the binding of insulin, while inducing a conformational change (25), does not alter the heat-lability of the disulfide bonds. Based upon these results, we autophosphorylated the receptor in all further experiments in the presence of 1 PM insulin to enhance radiolabeling of the P-subunit. Temperature-Dependence

of Heterotetramer

Breakdown

The temperature-dependent heat lability of the disulfide bonds was examined with partially purified receptor, autophosphorylated in the presence of 1 yM insulin. These samples were heated at different temperatures for 10 min and lower molecular weight species quantified (Table II). Heating of the insulin receptor at temperatures from 50 to 90°C always generated lower molecular weight species, with higher temperatures giving more breakdown of the native structure. Again, without heating only the o$$ receptor tetramer was detected. and Heterotetramer

II

Temperature of Sample Heating and Subunit Structures

Structures

by SDS-PAGE

+ + + +

Impurities

KOHANSKI

I

Sample Heating Time and Subunit Resolved

AND

Breakdown

The different amounts of oligomers generated by the two receptor preparations after heating suggested that

Temperature (“C, 10 min)

25 50 70

100 89.3 + 6.0 69.5 + 5.2 31.0 + 1.4

90

Oligomer I

P

0

0 0 15.8 f 3.9 45.0 f 1.0

10.7 + 6.0 14.7 + 1.3 24.0 F 1.4

Note. Partially purified receptor (WGASE) was autophosphorylated in the presence of insulin for 30 min under the same conditions as described in Table I. The samples were heated in Laemmli sample buffer for 10 min at the indicated temperature.

impurities coeluting with the receptor from the WGASepharose column contribute to reduction of the disulfide bonds (see Fig. 2C versus 2D). To illustrate this effect, pure receptor was autophosphorylated, freed from [y3’P]ATP by gel filtration, and mixed with concentrated Laemmli sample buffer. Then, unlabeled WGASE was added in increasing amounts to the sample mixture. These samples were then heated for 10 min at 90°C. The unheated control samples were without or with 50 pug WGASE. The separation of the subunit structures was achieved this time on a nonreducing two-phase gel system, separating higher molecular weight forms on the upper phase and retaining the free P-subunit on the denser lower phase. Autoradiography and subsequent quantitation of the excised gel segments revealed increasing free P-subunit with increasing amounts of WGASE added to the pure receptor (Table III). TABLE

III

Sample Impurities and Subunit Structures Resolved by SDS-PAGE

WGASE added (PiiT) 0

50 0

5 25 50

Heated at 90°C (min) 0 0 10 10 10 10

Percent distribution (subunit structure) dz 100 100 49.9 f 1.8 34.7 f 2.0 31.1 + 0.5 21.7 f 1.4

Oligomer I

P

0 0 17.2 f 0.1 23.9 -+ 1.7 20.9 +- 4.6 16.8 f 0.1

0 0 33.0 k 1.7 41.4 +- 0.2 48.0 2 4.2 61.5 + 1.4

Note. Pure insulin receptor was autophosphorylated for 30 min, the reaction was stopped by applying the mixture to a Bio-Gel P-6DG column, and fractions containing the labeled receptor were collected. Laemmli sample buffer and increasing amounts of unlabeled protein from wheat-germ agglutinin&Sepharose eluate (WGASE) were then added as indicated. The mixtures were heated at 90°C or kept at room temperature (0 min negative controls) for 10 min and oligomers separated by SDS-PAGE (see Experimental Procedures).

OLIGOMERIC 200

STRUCTURE

A

150

100

z 0

50

0

0

0

0

40

80

min

FIG. 3. HPLC elution profiles of [32P]phosphopeptides. Pure insulin receptor was autophosphorylated for 30 min in the presence of carrier free [y-32P]ATP, 5 mM Mn(CH,CO,),, and 1 pM insulin. The quenched reaction mixture was boiled for 10 min and the receptor oligomers resolved by two-dimensional nonreducing/reducing gel electrophoresis. Chromatograms represent autophosphorylation sites in the P-subunit of the ai& tetramer (A), the oligomer I (B), and the free P-subunit (C).

Results obtained using different preparations of WGASE, but at identical protein concentrations, gave highly reproducible results (heating at 90°C for 10 min at 25 pugtotal protein; Tables I, II, and III). The results presented in Table III show clearly that impurities alone, present in the WGASE, are not responsible for the generation of lower molecular weight receptor oligomers. Breaking of the disulfide bonds occurred upon heating. Phosphopeptide

OF THE

INSULIN

83

RECEPTOR

patterns compared. Figure 3 is a composite of these patterns showing identical phosphorylation sites for each /3subunit previously isolated as the aa& tetramer, oligomer I, and free P-subunit by two-dimensional gel electrophoresis. Also, the autophosphorylation itself does not yield differential instability of specific subunits of oligomers upon sample heating. Immunoprecipitation with Monoclonal Anti-Insulin Receptor Antibodies Immunoprecipitations were carried out with monoclonal anti-insulin receptor antibodies directed either against the a-subunit (antibody 83-14) or the extracellular portion of the P-subunit (antibody 18-44; Ref. (26)). Every possibly occurring insulin receptor oligomer would therefore be precipitable by one or the other of these two antibodies. Preautophosphorylated partially purified insulin receptor was incubated with both antibodies. Native c& tetramer was identified on a nonreducing two-phase gel (Fig. 4) when the samples were not heated. The supernatant, or nonimmunoprecipitated fraction, also contained exclusively a& tetramer. The oligomer I and free P-subunit were only detected after heating either the immune complex, or the supernatant, prior to the nonreducing electrophoresis. With either antibody used in this study, heating was not required to liberate the insulin receptor for analysis by SDS-PAGE. Stability of the Insulin Receptor in Live Cells In contrast to our results, a recent report (13) showed different oligomeric structures of the insulin receptor

no

A heat

12345

B 10min

9o’C

12345

Mapping

If different insulin receptor oligomers were naturally occurring, they might also function differently. Such a functional difference between various receptor forms could be reflected in changes of the phosphopeptide pattern of the P-subunit. Alternatively, variations in the psubunit autophosphorylation may correlate with the heat-sensitive forms observed. To investigate the latter hypothesis, we used the pure receptor preparation, circumventing any possible interference of the impurities present in partially purified receptor with the autophosphorylation pattern. Pure receptor was autophosphorylated and boiled in sample buffer (for details see Experimental Procedures) and the 32P-P-subunits, corresponding to the (Y& tetramer, the oligomer I, and the free P-subunit, were excised from the wet gel and digested with trypsin. The phosphopeptides were separated by HPLC and the elution

-

interface

--P

FIG. 4. Immunoprecipitations with two monoclonal anti-insulin receptor antibodies. Partially purified insulin receptor was autophosphorylated as described in the legend to Fig. 2, and purified over a BioGel P-6 DG column. The “‘P-receptor was incubated first with the antibody for 16 h at 4°C and then with protein A-Sepharose for 2 h at 4°C. The immunoprecipitate was washed and Laemmli sample buffer was added. Samples in A were not heated prior to the two-phase nonreducing SDS-PAGE (for details see Experimental Procedures). Samples in B were heated for 10 min at 90°C. Lane 1 (A and B), partially purified insulin receptor not immunoprecipitated. Lanes 2 and 3 (A and B), immunoprecipitates of the antibody 83-14 (anti-a) and 18-44 (anti-p), respectively. Lanes 4 and 5 (A and B) are the nonprecipitated fractions from each immunoprecipitation.

84

SCHENKER

AND

when iodinated in intact cells. The iodination was achieved in the presence of lactoperoxidase and glucose oxidase. We therefore investigated: (i) the influence of lactoperoxidase and glucose oxidase on the receptor’s oligomeric structure, (ii) the amount of insulin receptor tetramer not adsorbed to the WGA-Sepharose, and (iii) the presence of oligomeric forms from intact cells which do not adsorb to WGA-Sepharose. After incubation of intact cells with lactoperoxidase and glucose oxidase, the insulin receptor was solubilized with Triton X-100, adsorbed to a WGA-Sepharose column, washed, and eluted with N-acetylglucosamine. The WGA-Sepharose flowthrough and the eluate (partially purified insulin receptor) were analyzed as above for the presence of the receptor (Y.& tetramer and possible smaller molecular weight forms. None of the samples was heated prior to the gel electrophoresis and only the azPz tetramer was resolved (Fig. 5A). Only 2% of the receptor tetramer was detected in the material which did not adsorb to the lectin column (Fig. 5B). And no smaller oligomeric receptor forms were detected which might not have the ability to adsorb as effectively to the lectin column as the insulin receptor tetramer. DISCUSSION The molecular weight of the insulin receptor was first deduced by nonreducing SDS-PAGE (23, 24). After covalently crosslinking a photoreactive ‘251-insulin derivative to its receptor a protein reported to be 310 kDa was resolved under nonreducing conditions. However, the protein resolved under reducing conditions appeared to have a molecular weight between 125 and 135 kDa. This suggested that the native insulin receptor was a disulfidelinked oligomer. The observation that the insulin receptor phosphorylated itself on the intracellular domain was accompanied by demonstration of different high molecular weight oligomers, also phosphorylated on the P-subunit (5, 6). This observation of different receptor oligomers was then confirmed by several groups using the partially purified insulin receptor (5-13). The biosynthetic pathway of the insulin receptor, from Golgi to plasma membrane (14, 15), is consistent with oligomers having a 1:l stoichiometry of a$ subunits. Because both subunits are processed from a single precursor, other stoichiometries would not be expected. Furthermore, the a2pZ heterotetramer is the only enzymatically active receptor oligomer purified from 3T3-Ll adipocyte membranes (16). Because others have reported numerous oligomers (5-13), however, we investigated possible sources that might cause cleavage of the insulin receptor’s disulfide-bonds: (i) the method of sample preparation, and (ii) the state of purity. A recent report (13) confirmed the existence of different oligomeric forms of the insulin receptor, metabolically labeled with [35S]methionine. We therefore examined by in uiuo labeling the insulin receptor with [35S]methionine

KOHANSKI non -reducing

(no

DTT)

FIG. 5. Insulin receptor oligomers in intact cells. NIH 3T3 cells overexpressing transfected human insulin receptor were incubated with lactoperoxidase and glucose oxidase as used for surface iodinations (for details see Experimental Procedures). The insulin receptor was solubilized in 1.2% Triton X-100 and partially purified over a WGA-Sepharose column. Flowthrough (B) and eluate (A) were autophosphorylated and analyzed by two-dimensional nonreducing/reducing gel electrophoresis; samples were not heated. The a& heterotetramer (arrows) is the only form resolved from the WGA eluate. Only 2% of the total receptor was not adsorbed to the WGA-Sepharose and was present as the a&r heterotetramer (B).

whether the different oligomers were intrinsic forms or only due to sample heating. Using our standard sample preparation technique we could not detect any other oligomeric state than the CZ~&heterotetramer by two-dimensional nonreducing/reducing gel electrophoresis (Fig. 1A). Oligomers of molecular weights lower than the &I2 tetramer were observed exclusively after heating the receptor in Laemmli sample buffer (Fig. 1B). Therefore, oligomers other than the CQ& tetramer are not present in live cells and represent artifacts of sample handling. The insulin receptor subunit structure was then analyzed from two different stages of our purification protocol: i.e., partially purified (WGASE) and pure receptor. Again, we could not detect any oligomeric state other than the a2p2 heterotetramer by two-dimensional SDS-PAGE (Fig. 2C) or using two-phase nonreducing SDS-PAGE (Fig. 4A). Therefore extraction and purification procedures were not responsible for disulfide-bond cleavage. Heating the sample in SDS prior to electrophoresis was commonly employed to solubilize a protein for analysis by reducing SDS-PAGE. Employing the sample heating when assessing the native oligomeric structure of the insulin receptor always yielded the lower molecular weight oligomers and free P-subunit when analyzed under nonreducing conditions. We observed this with the native [35S]me-

OLIGOMERIC

STRUCTURE

thionine-labeled insulin receptor (Fig. 1B) as well as with WGASE (Figs. 2B and 2D) and/or pure insulin receptor. Qualitatively, loss of the disulfide-bond structure was not dependent on purity of the receptor preparation, and was caused exclusively by the heating. However, impurities such as those present in WGASE increased the observed heat-induced disulfide-bond cleavage (Table III). A precise molecular weight for each cleavage product was not established, as high molecular weight standards migrate anomalously under nonreducing conditions. Based on the interpretation of LeMarchand-Brustel et al. (13), comparing intensities of labeling of each oligomer, we presume oligomer I to be the a&trimer and oligomer II the crz-dimer. The mapping of the phosphopeptides obtained by tryptic digestion of the @-subunits corresponding to the CY& tetramer, the @-half receptor, and the free P-subunit confirmed that all these P-subunits must originate from the intact azPa tetramer. Using two different monoclonal antibodies (26), one against the a-subunit, the other against the extracellular domain of the P-subunit, we confirmed the above results. With either antibody exclusively the 01& tetramer was detected in the immunoaffinity-isolated fraction (Fig. 4A). In both cases the supernatant contained only the CY& subunit structure. Oligomer I and free P-subunit were separated on nonreducing SDS-PAGE only after heating of either the immune complex or the supernatant (Fig. 4B). Heating to liberate the receptor was not required using either antibody, but only resulted in artifactual generation of receptor oligomers and free subunits. Different oligomeric forms were reported also after labeling the insulin receptor in intact cells with 1251(13). We therefore explored the possibility of disulfide bond cleavage due to the surface iodination protocol. Again the only receptor form separated after incubation of cells with lactoperoxidase and glucose oxidase was the (m& tetramer (Figs. 5A and B). We have shown that heating of the insulin receptor prior to gel electrophoresis is not necessary for quantification (see Table l), but sample heating is in fact deleterious to accurate analysis of its oligomeric structure. The breakdown of the CY~&subunit structure was dependent upon temperature and time of heating (Tables I and II). Equally important is our finding that impurities favor this breakdown. Our results stress the fact that purification of the insulin receptor is important, but correct sample handling is crucial when assessing the oligomeric structure. This becomes even more critical when investigating the proper processing and expression of insulin receptor mutants transfected into cells as well as when

OF THE

INSULIN

85

RECEPTOR

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The native alpha 2 beta 2 tetramer is the only subunit structure of the insulin receptor in intact cells and purified receptor preparations.

The native subunit structure of the insulin receptor was reinvestigated by two-dimensional nonreducing/reducing gel electrophoresis. Human insulin rec...
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