Characterization of purified insulin receptor subunits.

Three insulin receptor subunits prepared from the purified receptor were isolated and characterized. Peptide mapping of the isolated subunits revealed that the Mr = 125,000 subunit (alpha) is distinct from the Mr = 90,000 subunit (beta) whereas the Mr = 50,000 subunit (beta 1) shows considerable structural homology to beta, indicating that the alpha and beta subunits are components of the intact insulin receptor. From two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the absence and presence of dithiothreitol, the purified insulin receptor was shown to be composed of heterogeneous disulfide-linked complexes of (alpha 2, 2 beta), (alpha 2, beta, beta 1), (alpha 2, 2 beta 1), (alpha 2), (alpha beta), and (alpha beta 1). The largest disulfide-linked complex (alpha 2, 2 beta) appears to be the minimum unit of the intact insulin receptor whereas the other complexes appear to be generated from (alpha 2, 2 beta) by proteolytic degradation and/or reduction. These studies provide conclusive evidence that the alpha 2 beta 2 complex is the basic structural unit of insulin receptor, as previously proposed from affinity cross-linking experiments using crude membranes by Czech's group (Czech, M. P., Massague, J., and Pilch, P. F. (1981) Trends Biochem. Sci. 6, 222-225). The biochemical approach described here should allow us to further elucidate the mechanism of insulin action.

because only specifically labeled proteins can be detected. For example, essential protein components which may lie far from the insulin binding site may not be labeled with "'I-insulin or membrane components which may exist near the insulin binding site but are not involved in insulin action may be artificially labeled. In fact, with the affinity cross-linking method, the p subunit is very poorly labeled with insulin.
Thus, to achieve a stoichiometric measurement of each subunit requires complete purification of the intact receptor.
Previously, we purified the insulin receptor from human placentas to apparent homogeneity. The purified receptor retained full insulin binding activity (8) as well as tyrosinespecific protein kinase activity (9), indicating that our purified receptor retains basic functions of the native insulin receptor. In addition, the purified receptor contained a significant amount of the p subunit which had not been clearly observed by others (10-13), suggesting that this receptor preparation would be useful for studies on the subunit structure of native insulin receptor. In this report, the subunits of the purified receptor are isolated and characterized by various methods including isoelectric focusing, peptide mapping, two-dimensional SDS-PAGE in the absence and presence of dithiothreitol, and amino acid analysis.

EXPERIMENTAL PROCEDURES AND RESULTS'
Subunit Composition of the Purified Receptor-Among nearly 50 preparations of purified insulin receptor, the most typical SDS-PAGE results of the receptor under reducing conditions are shown in Fig. 1. Both Coomassie blue and silver staining of the gel revealed almost the same densitometric patterns for three protein components with M , = 125,000 (a)," 90,000 (p), and 50,000 ( 0 ' ) : as seen in Fig. 1, A2 and BZ. The three components derived from disulfide-linked complexes with -M, = 300,000 were previously shown to be Portions of this paper (including "Materials and Methods," portions of "Results," and Figs. 2 and 4) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Document No. 83M-1340, cite the authors, and include a check or money order for $2.40 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.
135,000 ( a ) , 90,000 (@), and 60,000 from SDS-PAGE under the Molecular weight of each subunit was previously estimated to be conditions described by Weber and Osborn (19). In this report, the values estimated from SDS-PAGE under Laemmli's conditions are used. It should be noted that these molecular weights estimated from SDS-PAGE are tentative, mainly because the subunits are glycoproteins which are known to give ambiguity on molecular weight estimation.
The M , = 50,000 subunit is termed b1 in this report. Although this subunit appears to be similar to the b1 subunit described by Massague et al. (6), there is no direct evidence that they are the same molecule. The terminology (Dl) used in this report was adopted at the suggestion of the editor.

Characterization of
B.   insulin receptor subunits by immunoprecipitation with antiinsulin receptor antibodies (8,9). Two sets of four different receptor preparations were electrophoresed. One set was stained with Coomassie blue and another with silver. The peaks were quantitated by densitometric scanning. The percentages of three subunits contained in the preparations were estimated to be 72.5 f 4.9 ( n ) , 18.5 f 3.7 (@), and 9.1 f 1.4 (/jI) from the Coomassie blue-stained gels or 70.8 f 2.4 (CY), 16.4 f 3.7 (e), and 12.8 f 1.9 from the silver-stained gels.
Peptide Mapping of the Insulin Receptor Subunits-The three insulin receptor subunits were separated by SDS-PAGE under reducing conditions and stained with Coomassie blue. The stained bands which corresponded to CY, @, and subunits were cut out, iodinated, and digested with TPCK-trypsin. The tryptic peptides of each subunit were spotted on cellulosecoated TLC plates and subjected to electrophoresis followed Molecular weight of the complexes was estimated hy SDS-PAGE on a 5% polyacrylamide gel.
' Subunit composition of each complex was determined from the result of second dimension electrophoresis under reducing conditions (Fig. 5 ) .
Subunit structure of each complex was deduced from its molecular weight, subunit composition, and the color factor (see text). by chromatography. Fig. 3 shows the autoradiograms of the resulting peptide maps. A peptide map of bovine serum albumin is also shown as a control.
The tryptic peptide maps of the (Y and @ subunits were quite different. In contrast, most tryptic peptides found in the subunit were also found in the map of the @ subunit. These results indicate that the bI subunit is generated by limited proteolysis of the @ subunit. When different purified receptor preparations were analyzed by SDS-PAGE, the dl subunit sometimes showed slightly different mobility. The peptide map of such an anomalous M, = 50,000 subunit was very similar to that of the HI subunit (data not shown).
Amino Acid Analysis of the Insulin Receptor Subunit.$-Approximately 18% of the original protein was recovered as isolated subunits from the SDS-polyacrylamide gel. The amino acid composition of each subunit was assayed in duplicate after desalting on Sephadex G-25. The results are summarized in Table I. Since the data obtained are based only on 48-h hydrolysis, the values for Thr, Ser, Met, Val, Leu, Ile, and Tyr could be underestimated relative to the rest of amino acids, whereas Gly appears to be overestimated due to its contamination from the buffers. The (r subunit is relatively rich in Cys and Lys, while the @, subunit is poor in Cys. Although the amount of GlcNac estimated by the amino acid analyzer is not quantitative, this may be an indication of glycoprotein, since this sugar is a major component of the carbohydrate moieties which are usually found in glycoproteins. The results show that all three subunits seem to be glycoproteins and that the (r subunit contains more carbohydrate residues than the @ or subunits.
Two-dimensional SDS-PAGE in the Absence and Presence o f Dithiothreitol-The purified receptor was electrophoresed in a 5% gel under nonreducing conditions for analysis in the first dimension. The gel slice was then placed on top of a 7.5% slab gel and the disulfide-linked complexes were analyzed in the second dimension under reducing conditions. The results are summarized in Fig. 5. The five major bands (a,c,d,e, and f) observed under nonreducing conditions were shown to be composed of (Y + @, (Y + PI, (r, (Y + 8, and N + PI, respectively. Since the molecular weights of the two smaller components (e and f) were 195,000 and 150,000, the subunit structure of the complexes was determined to be (N@) and ((Y&), respectively. The color intensity of the (Y and @ subunits measured by densitometric scanning of the complex "e" allowed the calculation of a relative color factor of 0.32 for the @ subunit  against that of the (Y subunit. The subunit composition of the disulfide-linked complex "a" with M, = 320,000 can therefore be deduced to be 2tu and 28 subunits by quantitating the (r and /j subunits in the complex using the color factor. The other complex "c" appears to be composed of 2tu and 28, subunits while "d" appears to be 2n. Complex "b" (2n, @, l(jl)" may also exist in this preparation. However, resolution in this experiment was not good enough to identify the complex.
As summarized in Table 11, two-dimensional SDS-PAGE revealed that the purified receptor is composed of the heterogeneous disulfide-linked complexes: (20, 2@), (2cu, @, @,), and '.)tu, 21j1) as major complexes and (as), (tup), and (nbl) as minor complexes. Since (2tu, 2d) is the largest disulfide-linked complex found in the preparation after SDS treatment, it is likely that it is an intact disulfide-linked complex and that the others are derived from it by either proteolytic degradation and/or reduction. It should be noted that the n-dimer was clearly detected in our purified receptor preparation as seen in Fig. 5A(2). This indicates that the two tu subunits are linked together with disulfide bridges.
Insulin binding activity appeared as a single peak on Sepharose 6B chromatography, which gave a Stokes radius of 79 A for the native purified insulin receptor-Triton X-100 ' ' Notations used: parentheses denote a disulfide-linked complex; subscript (N,) denotes an m-(r dimer; coefficient denotes the number o f subunits in the complex. --0-+ " 0 " t complex (Data not shown). Since all disulfide-linked complexes were affinity-labeled with ""I-insulin (Fig. 423, 3 and 4, the small complexes ( ( u p ) , (nB), and (CUB,) must exist as larger forms.

DISCUSSION
We have previously reported that our purified insulin receptor retains the basic functions of the native insulin receptor such as a curvilinear Scatchard plot and autophosphorylation of the @ subunit (8,9). In addition, a specific activity of 28 pg of insulin bound per mg of protein for our purified receptor is the highest value so far reported. These results indicate that the analysis of our purified receptor should be useful for directly determining the subunit structure of native insulin receptor.
In the purified receptor, the B subunit was clearly detected after Coomassie blue or silver staining of SDS-polyacrylamide gels. However, densitometric scanning of stained gels showed that the amount of the @ subunit was only about one-fourth of the n subunit. Densitometric scanning of a complex which contained only the N and p subunits allowed the calculation of a relative color factor for the staining of @ subunit relative to a. This factor was only 0.32 which could explain why the amount of the B subunit detected on stained gels always appeared less than that of the CY subunit in the purified receptor. In addition, limited proteolysis could lead to a fur- The M, = 50,000 subunit (PI) was often detected in our purified receptor, although the amount varied from preparation to preparation. The peptide mapping experiments of each subunit revealed that the b1 subunit is related to the 8 subunit.

Characterization of
It seems likely that the /I1 subunit is derived by proteolytic degradation of the @ subunit. Since the molecular weight of PI is fairly consistent in different purified receptor preparations, the p subunit may have a region resistant to proteolytic digestion while the rest of its sequence is sensitive to proteases. This result supports the previous reports by Massaque et al. (7) using affinity cross-linking techniques, where they suggested that the M, = 49,000 subunit is generated as a result of limited proteolytic cleavage of the /3 subunit by lysosomal protease.
The a subunit was found in all disulfide-linked complexes detected in the purified receptor by SDS-PAGE under nonreducing conditions, suggesting that a is selectively concen-trated by purifying insulin receptor with insulin-Sepharose affinity chromatography. In addition, affinity cross-linking of the purified receptor with '2sII-insulin resulted in labeling of all disulfide-linked complexes including (cup) and (abI). These results indicate that the a subunit is necessary for insulin binding as previously predicted (21) and that both ((up)* and ( ( Y @~)~ seem to have insulin binding activity.
The present study has revealed that insulin receptor purified with insulin-Sepharose affinity chromatography is not 320,000 has the subunit structure of (2a, 2@) which appears to be a minimum unit of the intact insulin receptor whereas the other complexes can be derived from it by proteolytic degradation and/or reduction as described in Fig. 6. The presence of ap was clearly shown in the purified preparations Hempel for performing the amino acid analysis and Song Choi for as had previously been observed in crude preparations (4), suggesting that the two a subunits are linked together with disulfide bridges. These results provide conclusive evidence that (a2, 28) is the basic structural unit of insulin receptor, which is consistent with the models proposed by Czech et al. (22,23) and Jacobs et al. (1,24). Recently, Yip et al. (25) and Baron and Sonksen (26) have reported the presence of another component with M, = 40,000 or 65,000, respectively, which does not seem to be related to the p subunit. The PI subunit described in this paper is obviously different from these two components, which may be associated with the receptor in the membrane but cannot be purified by affinity chromatography.
Since more PI subunit has been observed in receptor preparations purified from placental membranes stored at -20 "C for 4-6 months than from freshly prepared membranes: it is likely that during storage, the p subunit becomes more accessible to proteases and/or is more easily degraded by proteases present in the crude membrane preparations during purification. It is also possible, however, that heterogeneous receptors exist on intact cell membranes and are all purified by our methods. It is not clear at this moment whether (az, 2p) is a biologically active molecule. The active form could be (az, 20,) or even a higher molecular weight form such as a dimer (az, 2/32. In a previous paper, we reported that our purified receptor has the highest specific activity so far obtained (8). The value, 28 pg of insulin bound per mg of protein, indicates that 1.4 mol of insulin binds to 1 mol of insulin receptor. This value is not conclusive since the present study revealed the presence of heterogeneity in terms of subunit structure in the receptor preparations. If one of the components such as (a2, 2p) represents native receptor and shows a curvilinear Scatchard ' Y. Fujita-Yamaguchi, unpublished.