Preferential degradation of the beta subunit of purified insulin receptor. Effect on insulin binding and protein kinase activities of the receptor.

Collagenase preparations (a mixture of enzymes including collagenase, clostripain, and a casein-degrading protease) degraded the beta subunit (Mr = 95,000) of the purified insulin receptor into fragments of Mr less than 15,000, without degrading the alpha subunit. The resulting beta-digested insulin receptor preparations were found to bind insulin as well as control insulin receptor, as assessed by either cross-linking of 125I-insulin to the digested receptor or by separating insulin bound to receptor from free insulin by high performance liquid chromatography. Moreover, the beta-digested insulin receptor preparations were still precipitated by a monoclonal antibody directed against the insulin-binding site. In contrast, the beta-digested insulin receptor lacked protein kinase activity since it no longer phosphorylated either itself, or an exogenous substrate, calf thymus histone. These results support the identification of the beta subunit of the insulin receptor as a protein kinase.

. Thus, the harsh conditions required for separating the two subunits would by unlikely to yield molecules that retained either binding or kinase activity. In this report, we demonstrate that treatment of purified receptor with a mixture of proteases degrades the @ subunit without affecting the CY subunit. In the present study, therefore, protease treatment was used to assess the contributions of the p subunit to the insulin binding and protein kinase activities of the insulin receptor. We found that degradation of the @ subunit eliminated the protein kinase activity of the receptor, but did not affect its insulin binding activity.

MATERIALS AND METHODS
Protease Digestions of the Insulin Receptor-Insulin receptors were purified from IM-9 cells cultured in the presence of [35S]methionine (New England Nuclear) (12) via the use of monoclonal anti-insulin receptor antibody and wheat germ agglutinin affinity columns (Miles Laboratories, Inc., Elkhart, IN), as described previously (8), except that the insulin receptor was eluted from the antibody column with 1.5 M MgCI,, 120 mM borate, and 0.1% Triton X-100, pH 6.5 (13). The insulin receptor was also isolated from human placenta particles via the same procedure. For the 35S-labeled receptor, the receptor peak was identified by measuring the radioactivity in the fractions; for the placental receptor, the peak was identified by its insulin binding activity as measured by the polyethylene glycol precipitation method described below. Bovine serum albumin, 0.2 mg/ml, was added to the purified receptors and they were then stored at 4 "C.
The isolated placental receptor was labeled in some experiments by incubation with 6 PM [y-32P]ATP (Amersham Corp.) (8), or by cross-linking to '251-insulin by addition of 0.5 mM disuccinimidyl suberate (Pierce Chemical Co.) (14). The '251-insulin-receptor complex was then repurified on the wheat germ agglutinin affinity column.
The proteases tested were: Type I collagenase (CLS) ( The digestions were performed by incubating the indicated concentrations of protease, and either 35S-labeled IM-9 receptor (800 to 1200 cpm), or placental receptor (20 to 40 fmol of insulin binding activity), for 1 h a t 37 "C in 30 pl of buffer containing 50 mM Hepes', pH 7.6, 150 mM NaCI, 0.1% Triton X-100, and 2 mM CaC12. After it was determined that bovine serum albumin had no effect on the digestions, 0.2 mg/ml of bovine serum albumin was added to reduce nonspecific losses. For the experiments analyzing the structure of the digested receptor, the preparations were made 1% in SDS, and 5% in P-mercaptoethanol, heated for 1 min at 100 "C, and electrophoresed on a 7.5% SDS-polyacrylamide gel (12). The gels were stained and destained. The gels containing '"Ior 32P-labeled receptor were dried and autoradiographed with the use of Du Pont Cronex Lightning-Plus intensifying screens, whereas gels containing [3sS]methioninelabeled receptor were treated with Enhance (New England Nuclear), dried and autoradiographed.
Immunoprecipitation of the Receptor-@-Digested receptor preparations were incubated with either control mouse IgC, or monoclonal anti-insulin receptor antibody for 14 h at 4 "C. The antibodies were precipitated by the addition of Staphylococcus aureus (New England Enzyme Center, Boston, MA), coated with rabbit antibodies to mouse immunoglobulin (N. L. Cappel Laboratories, Inc., Cochranville, PA), washed three times, solubilized, and analyzed by SDS-gel electrophoresis (12).
Insulin Binding Assays-@-Digested and control receptor preparations were incubated with 100 p~ '2511-insulin for 100 min at 24 "C. Receptor-bound insulin was assessed by three methods. One method was to precipitate the receptor with 9.5% polyethylene glycol in the presence of 1 mg/ml of human y globulin as described previously (15). The second technique was to add 0.5 mM disuccinimidyl suberate (14), to covalently link the hormone to the receptor, incubate for 15 min at 0 "C, and electrophorese the samples as described above. The 135,000 band was identified by autoradiography and excised, and the radioactivity in this region was determined in a y counter. The third technique used to quantitate the receptor-bound insulin was to separate the free insulin by high performance liquid chromatography. Chromatography was carried out on a Waters Model 720 microprocessor-controlled instrument with a Waters Intelligent Sample Processor Model 710K, and a single Waters 1-125 column with a guard column (Waters Associates, Milford, MA). The column was equilibrated in 20 mM sodium phosphate, pH 7.4, 0.15 M NaCI, and 0.1% bovine serum albumin at 25 "C, and eluted at a flow rate of 2 ml/ min. Fractions (0.7 ml) were collected via a Cilson fraction collector, and radioactivity was measured in a y counter.
Protein Kinase Assays-Either @-digested or control receptor was incubated for 60 min at 24 "C with 2 mM MnC12, 100 nM insulin, and 10 pg of histone (HFZB, Worthington); then 6 p~ [y-32P]ATP was added and the reaction was allowed to proceed for 1 h at 24 "C, and then the reaction was stopped by the addition of 1% SDS and 5% mercaptoethanol. The samples were electrophoresed, as described above, the 95,000 band and the histone band were excised, and the radioactivity in these bands was determined in a scintillation counter. To avoid the loss of a proteolytic fragment of the receptor, the collagenase-treated receptor preparations were used without further purification. However, several results indicated that the collagenase remaining in the receptor preparation did not affect the results. First, the Coomassie-stained gel of the phosphorylation reaction showed that the histone and albumin bands were not degraded. Second, inhibition of the remaining collagenase with ECTA prior to the phosphorylation reaction did not affect the results. Third, collagenase-treated receptor, which was first freed of collagenase by binding to and eluting from a monoclonal antireceptor column, gave the same results as receptor preparations which were not fractionated.

RESULTS
Protease Digestion of the /3 Subunit-Metabolically "Slabeled purified insulin receptor from IM-9 cells was incubated for 1 h at 37 "C with various concentrations of a crude collagenase preparation used to prepare isolated rat adipocytes (Type I, Worthington). This enzyme preparation contains collagenase, clostripain, and a casein-degrading protease. The receptor was then examined by SDS-polyacrylamide gel electrophoresis in the presence of reducing agents; the /3 subunit ( M , = 95,000) was found to be degraded into fragments of M, < 15,000 (observed at the dye front of the gel), in a dose-dependent manner (Fig. 1, upper). A detectable effect was observed with 0.3 pg/ml, and a maximal effect was found with 12 pg/ml of the collagenase preparation. In contrast, the a subunit ( M , = 135,000) was not affected by concentrations of the collagenase preparation up to 30 pg/ml. The degradation of the / 3 subunit was not blocked by 1 mM N-ethylmaleimide (an inhibitor of clostripain), but was blocked by 3 mM ethylenediamine tetraacetic acid (an inhibitor of collagenase and the casein-degrading protease) (Fig. 1,  . Using another lot of the same type of collagenase preparation gave similar results. More purified preparations of collagenase, CLSPA, CLOSA, and Form 111, were then tried. All were found to degrade the p subunit of the receptor without affecting the a subunit. However, the potency of these purified preparations was inversely related to their purity; CLSPA and Form 111 collagenase, the least and most pure, required 10 and 100 pg/ml, respectively, to completely degrade the /3 subunit. With these purified preparations of collagenase, a very small amount of a fragment of the , B subunit (M, = 45,000) was observed (Figs. 2 and 3).
The increased amounts of purified collagenase required to degrade the receptor suggested that the collagenase itself was not responsible for the degradation. To test this hypothesis, the purified collagenase (Form 111) was compared with a preparation enriched for the casein-degrading protease present in collagenase preparations, but low in collagenase. The latter preparation was found to preferentially degrade the / 3 subunit at a concentration of 12.5 pg/ml, a value 10 times lower than that of purified collagenase (Fig. 1, lower).
To test whether the collagenase preparations would degrade the / 3 subunit of the insulin receptor of other tissues, the insulin receptor of human placenta was also isolated and studied. When the placental receptor was labeled by first cross-linking '"I-insulin to the molecule, the labeled a subunit was not degraded (Fig. 2). In contrast, when the /3 subunit of the receptor was labeled by phosphorylation, it was found to be degraded (Fig. 2).
Immunoprecipitation of p-Digested Receptor-The P-digested receptor was tested for its ability to be precipitated by a monoclonal antibody directed against the insulin-binding site of the receptor (12). The antibody was found to precipitate the a subunit of &digested receptor, as well as controls of undigested receptor (Fig. 3).
Effect of Protease Digestion on Insulin Binding-Placental insulin receptor preparations were first incubated with 20 pgj ml of the crude collagenase preparation, and then incubated with ""I-insulin. To separate bound insulin from free insulin, the preparation was precipitated with polyethylene glycol (12). Using this technique, binding of insulin to &digested receptor was decreased by 50%. However, when a control of "'I-insulin cross-linked to the receptor was examined, the precipitation of the "'I-insulin-receptor complex was also decreased 40% after protease digestion. These results suggested that polyethylene glycol precipitation was not accurately reflecting the binding activity of the receptor, but instead was affected by the decreased molecular weight of the insulin receptor after digestion.
Two other methods for assessing the insulin-binding capability of the 8-digested receptor were then tried. Digested receptor was incubated with "'I-insulin, the bound insulin was cross-linked to the receptor with disuccinimidyl suberate (14), and the amount of insulin linked to the receptor was determined by SDS-gel electrophoresis. Protease digestion did not significantly decrease the amount of "'I-insulin bound to the receptor when binding was assessed by this technique (Fig. 4).
The ability of P-digested receptor to bind insulin was also measured by high performance liquid chromatography. Trial experiments demonstrated the feasibility of separating "'Iinsulin bound to receptor from free "'I-insulin by chromatography on a Waters 1-125 column. The amount of '"I-insulin in the receptor peak was proportional to the amount of receptor incubated with the "'I-insulin and was completely displaced by 1 p~ unlabeled insulin. When @-digested and control receptors were compared, the amount of "'I-insulin in the receptor peak was found to be the same for treated (35 k 5%, n = 3) and control receptor (30 & 4%, n = 3) (Fig. 5).
Effect of Protease Digestion on the Kinase Activity of the Receptor-&Digested and control receptor preparations were incubated with ['"'PIATP and then the reaction mixtures were analyzed by SDS-gel electrophoresis. The crude collagenase preparation at concentrations of 3 pgjml and 20 pg/ml decreased the extent of phosphorylation of the @ subunit by 50 and 90%, respectively (Fig. 4). Since this decrease in phosphorylation could occur as a result from either a decrease in kinase concentration or from a decrease in the substrate for the kinase, an exogenous substrate, calf thymus histone (16), was included in the reaction. Protease digestion of the receptor decreased the phosphorylation of this substrate to the same extent as the phosphorylation of the p subunit (Fig. 4).
More limited protease digestion was found to result in the formation of a M, = 45,000 fragment that retained a small amount of protein kinase activity. This activity was not stimulated by insulin to either phosphorylate itself or histone (Fig. 6). is that the @ subunit of the receptor is not exposed in the intact cell to the proteases, and hence is not degraded.

K
Prior studies of Jacobs et al. (11) have shown that the insulin receptor subunits are held together via strong nonco-K valent interactions. Thus, even after reduction of the inter-chain disulfide bonds of the receptor, the subunits were still held together. These authors found, however, that the subunits could be separated after boiling the receptor in SDS and " mercaptoethanol. When we attempted to isolate the subunits I by the same procedure, we found that a monoclonal antibody insulin-binding site (Fig. 3). The protease treatment was therefore used to assess the contributions of the @ subunit to

ORIGIN-
the insulin binding and protein kinase activities of the receptor.
When the &digested receptor was first tested for insulin binding activity by the standard method of precipitation by was observed. However, since '2sII-insulin cross-linked to the receptor was also not precipitated as well after collagenase digestion, this effect most likely occurs as a result of the decrease in precipitability of the smaller digested insulin ceptor by SDS-gel electrophoresis indicated that the IM-9 lymphocyte insulin receptor had a M, of 450,000, which was decreased to 300,000 by collagenase digestion:? These results suggest that polyethylene glycol precipitation may underestimate the insulin binding capacity of isolated receptor preparations where the @ subunit has been partially degraded. This  (17) have demonstrated that the @ subunit of the insulin receptor is especially sensitive to proteolysis. The present data are consistent with these results since crude collagenase preparations were found to preferentially degrade the @ subunit of the insulin receptor. This degradation could have been caused by the collagenase itself, or by a contaminating protease present in collagenase preparations (18). This latter hypothesis is supported by two results: 1) more purified collagenase preparations had weaker potencies than less purified preparations in degrading the @ subunit, and 2) a fraction of the collagenase preparation, which was low in collagenase activity but high in other proteases, also preferentially degraded the @ subunit at concentrations less than that of purified collagenase (Fig. 1). This contaminating protease may be the casein-degrading protease present in collagenase preparations (19), since the activity was not inhibited by the sulfhydryl-modifying reagent, Nethylmaleimide, but was inhibited by chelating agents (Fig.  1).
Thus, these results suggest that predominantly the a subunit interacts with insulin. However, some contribution of the remaining @ subunit fragments to the binding of insulin cannot be excluded by the present studies.
In contrast, when the B-digested receptor was tested for kinase activity with or without an exogenous substrate, the phosphorylating activity was greatly decreased (Fig. 4). More limited digestion of the @ subunit resulted in the formation of a fragment of M, = 45,000 that retained a small amount of kinase activity (Fig. 6). These results, taken together with prior studies demonstrating that the @ subunit of the receptor contains an ATP-binding site @-lo), indicate that the / 3 subunit of the receptor has protein kinase activity.
Thus, the insulin receptor may be viewed as being composed of two distinct domains with separate functions. One domain is composed of the a subunit and binds insulin; the second domain is composed of the @ subunit and is a tyrosine-specific Insulin Receptor Structure and Function protein kinase. These two domains must communicate, however, since binding of insulin to the LY subunit increases the kinase activity of the @ subunit (5-7,10,16,25-28). Moreover, we have preliminary data indicating that binding of adenosine triphosphate to the receptor (presumably to the ATP-binding site of the @ chain) affects the binding of insulin to the 01 chain.4 These effects could be mediated via conformational changes since Pilch and Czech (29) have presented data indicating that binding of insulin to the receptor induces a conformational change in the molecule.
Recent data indicate that the two subunits of the insulin receptor may actually be derived from a single precursor polypeptide of M , = 200,000 (30-32). We have also observed a protein with this molecular weight in our purified receptor preparations (12). This protein was also observed to be digested by collagenase, supporting the identification of this molecule as a precursor of the @ subunit.
The structure of the precursor polypeptide of the insulin receptor therefore appears to be similar to the receptor for epidermal growth factor, another hormone receptor which is also a tyrosine-specific kinase (33, 34). This receptor is composed of a single polypeptide of M , = 170,000 (33-37), that both binds epidermal growth factor and has kinase activity (33,34). These two activities of the molecule appear to be on distinct domains since they have different heat stabilities (38). Moreover, the phosphorylation site and/or kinase activity of the epidermal growth factor receptor is also more sensitive to proteolysis than the epidermal growth factorbinding site (39, 40).
Thus, a general feature of hormone receptors which are also tyrosine kinases may be to contain a domain that determines binding specificity, and a protease-sensitive domain that has kinase activity. Recent data suggest that the receptors for nerve growth factor, M , = 200,000 (41), and platelet-derived growth factor, M , = 185,000 (42,43), may also be members of this group of receptors. More detailed studies of these receptors will be required to determine 1) the relationship between the various tyrosine kinases of these different receptors, 2) the relationship between these receptor tyrosine kinases and the tyrosine kinases encoded by various viral genes, and their cellular counterparts (44), and 3) whether the tyrosine kinase domain and the hormone-binding domain are synthesized as separate polypeptides and joined together (45), or synthesized as a single precursor polypeptide.