Proc. Natl. Acad. Sci. USA Vol. 77, No. 12, pp. 7137-7141, December 1980 Biochemistry Electrophoretic resolution of three major insulin receptor structures with unique subunit stoichiometries (crosslinking/plasma membranes/receptor heterogeneity/disulfide-linked subunits/insulin action) JOAN MASSAGUE, PAUL F. PILCH, AND MICHAEL P. CZECH Section of Physiological Chemistry, Division of Biology and Medicine, Brown University, Providence, Rhode Island 02912 Communicated by Herman M. Kalckar, August 29, 1980 ABSTRACT Plasma membrane insulin receptors, affinity erogeneous population of three major insulin receptor structures labeled by covalent crosslinking to receptor-bound "25I-labeled in membranes from all rat and human tissues studied. The insulin, are shown to appear as a heterogeneous population of resolution of these heterogeneous insulin receptor structures three major disulfide-Iinked complexes (Mr 350,000, 320,000, and 290,000) upon electrophoresis in highly porous dodecyl clarifies the apparent diversity of results obtained in different sulfate/polyacrylamide gels in the absence of reductant. This laboratories concerning subunit components of the receptor. pattern is consistent in all rat and human tissues that were an- Our results reveal that the three receptor structures resolved alyzed. Upon reduction of disulfide bonds, each of these re- consist of the Mr 125,000-135,000, Mr 90,000, and Mr ceptor structures is dissociated in two successive steps. Low 45,000-49,000 subunits, identified in various laboratories (1-9), concentrations of dithiothreitol promote a first step of disulfide in unique stoichiometries. bond reduction in which the Mr 350,000 species splits into a Mr 210,000 form and the Mr 290,000 species splits into a Mr 160,000 form. In contrast, both the Mr 210,000 and Mr 160,000 receptor MATERIALS AND METHODS fragments are generated from the native Mr 320,000 species upon partial reduction, indicating an asymmetrical structure. Preparation of Membranes. Adipocytes were isolated from The second step of receptor reduction occurs upon treatment the epididymal fat pads of 150- to 175-g Sprague-Dawley rats of the native disulfide-linked receptor complexes with high by digestion with collagenase (Worthington) (10); plasma concentrations of dithiothreitol. Under these conditions, the Mr membranes were prepared from these cells as described (2). Rat 350,000 receptor yields a Mr 125,000 subunit, denoted as a, and liver plasma membranes were purified by the method of Ne- a Mr 90,000 subunit, denoted as P, whereas the Mr 290,000 re- ville (11) as modified (12). To prepare crude plasma membrane ceptor dissociates into the a subunit and a Mr 49,000 subunit, denoted as , The Mr 320,000 receptor band is found to consist fractions from rat kidney and lung and from human placenta, of a, P, and fi1 subunits upon complete reduction. The partially we minced samples of these tissues and homogenized them in reduced Mr 210,000 receptor fragment is composed of the a 250 mM sucrose/10 mM Tris, pH 7.4/1 mM EDTA with a subunit disulfide-linked to the p subunit, whereas the Mr Potter-Elvehjem homogenizer with a Teflon pestle. The ho- 160,000 species consists of the a subunit disulfide-linked to the mogenate was centrifuged at 10,000 X g for 10 min. The su- PI subunit. Thus, the stoichiometry of the three ubiquitous na- pernatants were centrifuged at 30,000 X g for 30 min, the tive insulin receptor structures of Mr 350,000, 320,000, and pellets were resuspended in the same buffer, and the latter 290,000 are (a)2(A, (a)2(#X#,), and (a)2(#1)2, respectively. centrifugation step was repeated twice. Final pellets were Recent efforts of this (1, 2) and other (3-8) laboratory groups resuspended in 10 mM Tris, pH 7.4/1 mM EDTA. to elucidate the structural features of the insulin receptor in Affinity Crosslinking of Insulin Receptors. The affinity adipocyte and liver plasma membranes have shown that the crosslinking protocol was typically performed by incubating receptor contains at least one subunit in the 125,000-135,000 membranes (1-2 mg of protein per ml of Krebs-Ringer phos- Mr range and is disulfide-linked in the native membrane (2, 4, phate buffer containing 1% bovine serum albumin) with 10 nM 5). Although the presence of additional subunits has also been 125I-labeled insulin (125I-insulin) for 30 min at 23°C. The in- reported (4-9), the data obtained in different laboratories on cubation was ended by addition of excess cold Krebs-Ringer the molecular characteristics are variable and controversial. buffer and centrifugation (30,000 X g, 30 min). The membrane Thus, Jacobs et al. (4) have reported that insulin receptor pu- pellet was resuspended in the same buffer and incubated with rified from liver membranes consists of Mr 45,000 and Mr 0.25 mM disuccinimidyl suberate for 15 min at 0°C. The 135,000 subunits. More recently, these workers have obtained crosslinking reaction was quenched by addition of excess ice- data suggesting that the insulin receptor structure consists of cold 10 mM Tris, pH 7.4/1 mM EDTA. Finally, the crosslinked two Mr 135,000 and two Mr 45,000 subunits, all disulfide-linked material was pelleted at 30,000 X g for 30 min and resuspended (5). However, Yip and coworkers (7) claim that Mr 90,000 and in 10 mM Tris, pH 7.4/1 mM EDTA. Mr 130,000 species are the only components specifically labeled Dodecyl Sulfate/Polyacrylamide Gel Electrophoresis and when liver plasma membrane insulin receptors are subjected Autoradiography of Gels. Crosslinked membranes in 10 mM to photoaffinity labeling and to electrophoresis under disso- Tris, pH 7.4/1 mM EDTA were subjected to electrophoresis ciating conditions. Lang et al. (9) have also visualized putative according to Laemmli (13). Prior to electrophoresis, samples receptor subunits of Mr 90,000 and lower. Taken together, these were boiled for 1 min in Laemmli's sample buffer in the pres- data have presented a puzzling picture of the insulin receptor ence of the concentration of dithiothreitol indicated in each subunit composition. experiment. A second electrophoresis of individual receptor We provide here direct evidence for the presence of a het- bands resolved after a first electrophoresis was performed by slicing the first-dimension gel bands into 2-mm sections (Figs. The publication costs of this article were defrayed in part by page 2 and 4). The radioactivity in the gel slices was determined in charge payment. This article must therefore be hereby marked "ad- a gamma counter. The second electrophoresis was carried out vertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. Abbreviation: l25I-insulin, l25I-labeled insulin. 7137 Downloaded by guest on September 25, 2021 7138 Biochemistry: Massague et al. Proc. Natl. Acad. Sci. USA 77 (1980) by grinding the gel slices in a stainless steel mortar that had been Mr Mr Ur chilled at -30'C, soaking them in 100 mM Tris, pH 6.9/20% 350,000 320,000 290,000 (vol/vol) glycerol/2% NaDodSO4 and dithiothreitol as indicated for 30 min, and electrophoresing them in the presence of the 125,000 same buffer. The gel slabs were stained in 0.05% Coomassie -Am-. 90,000 -c4m. N blue, dissolved in 25% 2-propanol/7% acetic acid (vol/vol) and destained in 5% 2-propanol/7% acetic acid (vol/vol). The gels were dried and subjected to autoradiography (14). To estimate 1 }- 49,000--C D Mr values, we subjected the following standards to electro- phoresis in the presence of 50 mM dithiothreitol: myosin (Mr 43,000- 200,000), (3-galactosidase (Mr 116,000), phosphorylase b (Mr 94,000), bovine serum albumin (Mr 68,000), ovalbumin (Mr 45,000), and carbonic anhydrase (Mr 31,000). All autoradiographic results depicted were verified by on different membrane FIG. 2. Electrophoretic identification of affinity-labeled insulin multiple experiments performed receptor subunits. Affinity-labeled adipocyte membranes were preparations. At least 30, 3, 9, and 5 experiments confirmed the subjected to dodecyl sulfate/polyacrylamide gel electrophoresis in results of Figs. 1, 2, 3, and 4, respectively. the presence of 5 mM N-ethylmaleimide' and in the absence of di- Reagents. Porcine insulin was a gift of R. Chance (Eli Lilly). thiothreitol. The three specifically labeled bands (Mr 350,000, Mr 125I-Insulin was prepared by the lactoperoxidase method (En- 320,000, and Mr 290,000) resolved in this first electrophoresis were zymobeads, Bio-Rad). Disuccinimidyl suberate was prepared selectively isolated and individually subjected to electrophoresis in in this laboratory (1). the presence of 50 mM dithiothreitol in 8.5-13% polyacrylamide gradient gels (acrylamide/bisacrylamide, 100:2.6). The autoradiogram RESULTS of a representative gel is shown. The presence of a native, unreduced, disulfide-linked complex second electrophoresis with complete reduction. All receptor of insulin receptor (Mr - 300,000) that minimallypenetrated forms under these conditions yielded a strongly labeled band standard dodecyl sulfate/polyacrylamide gels has been reported at Mr 125,000 that represents a subunit we denote as a and a (2). Fig. 1 shows that more detailed resolution of the affinity- Mr 43,000 species which we conclude represents a proteolytic labeled disulfide-linked complexes of insulin receptor was ob- fragment of this a subunit. This conclusion is based on peptide tained when highly porous gels (5% acrylamide; acrylamide/ mapping experiments showing that homologous peptides were bisacrylamide, 100:1) were used. In the absence of dithiothre- generated from the Mr 125,000 and Mr 43,000 species upon itol, 125I-insulin-linked insulin receptors appeared as three major proteolysis (not illustrated). The Mr 43,000 species therefore distinct species with Mr 350,000,320,000, and 290,000.