[Structure and function of the insulin receptor].
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Animals↗
Biomedical subjects
M Kasuga
Publications and source records attributed to M Kasuga.
[Endoscopic preoperative intralesional injection of OK-432 in early gastric cancer].
The study group consisted of 35 patients with early gastric cancer, 16 of whom were admitted for preoperative immunotherapy. Ten to 40 K.E. of OK-432 was injected intralesionally by endoscope, and then gastrectomy was performed. After the intralesional injection, fever, nausea, vomiting and epigastralgia occurred. In cancer lesion and regional lymph node, histological findings from OK-432 treated group were compared to those of the control group. Lymphoid cell infiltration at cancer lesion was marked in OK-432 treated group, and degenerated cancer cells were found in 3 cases. On the other hand, lymphoid follicles showed a marked statistical increase in OK-432 treated group. Also the cases with marked lymphoid follicle showed increased numbers of peripheral blood lymphocyte. From the results, intralesional injection of OK-432 may confirm the tumor-associated antigenicity and serves as a useful method to potentiate the specific and/or non-specific immunity in regional lymph nodes.
Adult↗
Characterization of the insulin receptor kinase purified from human placental membranes.
The insulin receptor purified from human placenta by sequential affinity chromatography on wheat germ agglutinin- and insulin-Sepharose to near homogeneity retained tyrosine-specific protein kinase activity. This purified insulin receptor kinase specifically catalyzed the incorporation of 32P from [gamma-32P]ATP into not only the beta-subunit of the insulin receptor but also histone H2B, a synthetic peptide which is sequentially similar to the site of tyrosine phosphorylation in pp60src (a gene product of the Rous sarcoma virus) and antibodies to pp60src present in the sera obtained from three rabbits bearing tumors induced by the Rous sarcoma virus. In each case, phosphorylation occurred exclusively on tyrosine residues. Insulin stimulated phosphorylation of these substrates 3- to 5-fold. Kinetic analysis using the synthetic peptide indicated that insulin acted by increasing the Vmax of peptide phosphorylation from about 3.1 to 9.5 nmol X mg-1 of protein X min-1, whereas the value of the Km for the peptide, about 1.5 mM, was not significantly changed. This kinase acted weakly on casein, alpha-S-casein, actin, and a tyrosine-containing peptide analogue of a serine-containing peptide used commonly as a substrate for the cyclic AMP-dependent protein kinases. These data show that the insulin receptor kinase displays specificity toward exogenous substrates similar to the substrate specificity observed for pp60src and the protein kinase activity associated with the receptor for epidermal growth factor. The data suggest that the catalytic sites of these three tyrosine kinases are similar and that insulin activates its receptor kinase by increasing the Vmax.
Animals↗
Biosynthesis and glycosylation of the insulin receptor. Evidence for a single polypeptide precursor of the two major subunits.
The biosynthesis and carbohydrate processing of the insulin receptor were studied in cultured human lymphocytes by means of metabolic and cell surface labeling, immunoprecipitation with anti-receptor autoantibodies, and analysis on sodium dodecyl sulfate-polyacrylamide gels under reducing conditions. In addition to the two major subunits of Mr = 135,000 and Mr = 95,000, two higher molecular weight bands were detected of Mr = 210,000 and Mr = 190,000. The Mr = 210,000 band and the two major subunits were labeled by [3H]mannose, [3H]glucosamine, [3H]galactose, and [3H]fucose, and were bound by immobilized lentil, wheat germ, and ricin I lectins. On the other hand, the Mr = 190,000 band was labeled only by [3H]mannose and [3H]glucosamine and was bound only by lentil lectin. All four components could be labeled with [35S] methionine; however, in contrast with the other three polypeptides, the Mr = 190,000 band was not labeled by cell surface iodination with lactoperoxidase, suggesting that it is not exposed at the outer surface of the plasma membrane. Pulse-chase studies with [3H]mannose showed that the Mr = 190,000 was the earliest labeled component of the receptor; radioactivity in this band reached a maximum 1 h after the pulse, clearly preceded the appearance of the other components, and had a very brief half-life (t1/2 = 2.5 h). The Mr = 210,000, Mr = 135,000, and Mr = 95,000 bands were next in appearance and reached a maximum 6 h in the chase period. Monensin, an ionophore which interferes with maturation of some proteins, blocked both the disappearance of the Mr = 190,000 protein and the appearance of the Mr = 135,000 and Mr = 95,000 subunits. The mannose incorporated in the Mr = 190,000 component was fully sensitive to treatment with endoglycosidase H while that in the Mr = 210,000 band and the two major subunits was only partially sensitive. Tryptic fingerprints of the 125I-labeled Mr = 210,000 band suggested that this component contains peptides of both the Mr = 135,000 and Mr = 95,000 subunits. In conclusion, the Mr = 190,000 component appears to represent the high mannose precursor form of the insulin receptor that undergoes carbohydrate processing and proteolytic cleavage to generate the two major subunits. In addition, the Mr = 210,000 band is probably the fully glycosylated form of the precursor that escapes cleavage and is expressed in the plasma membrane.
Cell Line↗
Purification of an insulin-like growth factor II receptor from rat chondrosarcoma cells.
An insulin-like growth factor II (IGF-II) receptor was purified from rat chondrosarcoma cells by Triton X-100 solubilization of a 100,000 X g membrane preparation and affinity chromatography on a multiplication-stimulating activity (MSA)-Sepharose column. Analysis of the purified receptor by sodium dodecyl sulfate-gel electrophoresis and silver staining showed a major band of Mr = 210,000 (Mr = 250,000 after reduction with dithiothreitol). When 125I-MSA was chemically cross-linked to the purified receptor and analyzed by sodium dodecyl sulfate-gel electrophoresis (with and without dithiothreitol) and autoradiography, the radioactive bands coincided with the Mr = 210,000 and 250,000 bands identified by silver staining. The purified receptor also appeared to contain an Mr = less than 68,000 species identified by silver staining in addition to the Mr = 250,000 binding component. IGF-I, IGF-II, and MSA-II inhibited binding of 125I-MSA to the purified receptor with the same relative potency as for binding to the intact chondrosarcoma cell (IGF-II greater than MSA-II greater than IGF-I), and insulin did not inhibit binding. The association constant (K alpha) for MSA-II binding to the purified receptor was 2 X 10(9) M-1. The purified receptor bound to concanavalin A-Sepharose and wheat germ lectin-Sepharose columns and was eluted with alpha-methyl-D-mannoside and N-acetyl-D-glucosamine, respectively, showing that the receptor is a glycoprotein.
Animals↗
125I-anti-insulin receptor Fab is internalized by cultured human lymphocytes.
When 125I-anti-insulin receptor Fab components are prepared from serum IgG, this univalent material binds to the surface of cultured human lymphocytes and is subsequently internalized by the cell in an identical fashion to the divalent IgG and to 125I-insulin itself.
Antigen-Antibody Complex↗
Characterization of insulin-mediated phosphorylation of the insulin receptor in a cell-free system.
Insulin stimulates phosphorylation of both alpha- and beta- subunits of its own receptor in a cell-free system. A solubilized lectin-purified preparation of insulin receptors from rat liver membranes was preincubated with or without insulin at 4 degrees C and labeled for 10 min with Mn[gamma- 32P]ATP; the receptor subunits were isolated by specific immunoprecipitation with anti-receptor antibodies, followed by gel electrophoresis in sodium dodecyl sulfate. In gels run under reduced conditions, two bands (Mr = 135,000 and 95,000) were selectively labeled. These correspond exactly to the position of the alpha- and beta-subunits of the insulin receptor. Labeling of the Mr = 95,000 band was approximately 5-fold that of the Mr = 135,000 band. No labeled bands were detected when identical samples were immunoprecipitated in control serum. Phosphorylation of the receptor subunits required the presence of the divalent cation Mn2+ or Co2+; other cations such as Mg2+, Cr3+, Ca2+, and Zn2+ were ineffective. [gamma- 32P]ATP served as the 32P donor, whereas [gamma- 32P]GTP was ineffective. Phosphorylation of both subunits was stimulated 4-6-fold after a 60-min exposure to 10(-7) M pork insulin. Insulin-stimulated phosphorylation was half-maximal after 5 min of incubation with 10(-7) M insulin or after 18 h with 3 X 10(-10) M hormone. The enhanced phosphorylation was specific for insulin and its analogs; guinea pig insulin was about 2% as potent as pork insulin, whereas epidermal growth factor, adrenocorticotropic hormone, and glucagon, as well as cAMP, were ineffective. The rapidity and specificity of this reaction, as well as the presence of all necessary components in the plasma membrane, suggest that insulin-mediated receptor phosphorylation is one of the earliest biochemical steps following insulin binding.
Adenosine Triphosphate↗
Conservative surgery for regional lymphadenectomy in the treatment of early gastric carcinoma.
The relationship between lymph node metastases and postoperative prognosis in 209 patients with early gastric cancer was studied. As to the postoperative prognosis in relation to the extent of lymph nodes dissection, no significant difference was observed among the age-corrected 5-year survival rates following three surgical procedures in patients with early gastric cancer. Age-corrected 5-year survival rates were 0.92 +/- 0.44 R1-resection, 0.95 +/- 0.44 in R2-resection, and 1.00 +/- 0.06 in R3-resection, respectively. In addition, in 71 patients including 33 with early gastric cancer and 38 patients with advanced but relative early gastric carcinoma, the relationship between the immunostatus and postoperative prognosis was investigated. Postoperative age-corrected 5-year survival rate (0.904 +/- 1.153) of the optimal responders with good immunostatus was significantly higher than that (0.582 +/- 1.153) of the suboptimal responders with impaired immunostatus (P less than 0.01). Thus, conservative surgery for regional lymphadenectomy may be an effective procedure for cure of early gastric carcinoma, particularly in cases of a carcinoma limited to the mucosal area of the stomach.
Adult↗
The structure of the insulin receptor: studies using external and internal labeling techniques.
Using both biosynthetic and external labeling techniques, we have demonstrated that the insulin receptor consists of two major subunits with a molecular weight of approximately 135,000 and 95,000, respectively. These two major subunits are glycoproteins. Our data and observations from other laboratories have led to the suggestion that the insulin receptor is a heterodimer of the 134,000 and 95,000 subunits and, in fact, has an immunoglobulin-like structure with heavy and light chains held together by disulfide bonds. Biosynthetic studies showing the incorporation of all four labeled monosaccharides (fucose, mannose, galactose and glucosamine) into the two major subunits of the insulin receptor suggested that both subunits were likely to contain carbohydrate chains of the complex, N-linked type. External labeling techniques demonstrated that a portion of the protein and carbohydrate moiety of both subunits was exposed at the external cell surface. Further, labeling of the external oriented carbohydrates revealed remarkable differences in the nonreducing termini of the carbohydrate chains of both major subunits. Indeed, in the Mr 134,000 subunit, there appeared to be more exposed galactosyl or N-acetylgalactosaminyl (or both) residues, whereas the Mr 95,000 subunit seemed to have a higher degree of sialylation.
Chemical Phenomena↗
Tyrosine-specific protein kinase activity is associated with the purified insulin receptor.
Highly purified human placental insulin receptors were obtained by sequential affinity chromatography on wheat germ agglutinin and insulin-agarose. The preparation had an insulin binding capacity of 4,700 pmol/mg of protein approaching theoretical purity. The purified receptor revealed three major bands of Mr 135,000, 95,000, and 52,000 in NaDodSO4/polyacrylamide gel electrophoresis after reduction by dithiothreitol. All three bands were immunoprecipitated by anti-insulin-receptor antibodies. When this preparation was incubated with [gamma-32P]ATP in the presence of MnCl2 (2 mM) and analyzed in NaDodSO4/acrylamide gel electrophoresis, only the Mr 95,000 band was labeled. Preincubation with several concentrations of insulin increased the 32P incorporation into this peptide in dose-dependent fashion, whereas insulin-like growth factors were approximately equal to 2% as potent and epidermal growth factor had little or no effect, consistent with their known affinities for the insulin receptor. Insulin stimulation of phosphorylation of the Mr 95,000 subunit of the receptor was observed also in immunoprecipitates of this highly purified insulin receptor by anti-insulin-receptor antibodies. Phosphoamino acid determination revealed only phosphotyrosine in both the basal and insulin-stimulated states. These data suggest that a tyrosine-specific protein kinase activity is closely associated with insulin receptor, and this may be important in the signal transmission required for insulin action.
Female↗
Antireceptor antibodies as probes of insulinlike growth factor receptor structure.
Insulin receptors and Type I insulinlike growth factor (IGF) receptors have a similar structure with a major binding subunit of Mr approximately 130,000 linked by disulfide bonds to other membrane proteins to form a Mr greater than 300,000 complex. Both insulin and Type I IGF receptors also interact with both insulin and IGF, although with different binding affinities. We used a panel of human and rabbit sera containing antibodies to insulin receptors to determine whether these sera also interact with Type I IGF receptors. Immunoglobulins from five of five human sera inhibited binding of 125I-insulin and 125I-IGF-I to insulin receptors and Type I IGF receptors in human placenta and human lymphocytes. The rank order of reactivity with both receptors was the same; two sera, however, appeared to be selectively less reactive with the Type I IGF receptor, especially in placenta. Sera from five of seven patients and from a rabbit immunized with purified insulin receptor effectively immunoprecipitated both placental insulin receptors and Type I IGF receptors. Of the remaining sera, one had only a low titer against the insulin receptor and did not immunoprecipitate the IGF receptor, whereas the second serum effectively immunoprecipitated cross-linked and surface-iodinated insulin receptors, but had negligible reactivity against the Type I IGF receptor. These results suggest that most antisera to the insulin receptor also contain antibodies to Type I IGF receptors. Whether both specificities are inherent in the same or different antibody molecules remains to be determined. These data support the hypothesis that the insulin and IGF-I receptors are separate but related molecules, although there remains a small possibility that both receptors are domains on the same protein.
Animals↗
Receptors for insulin-like growth factors and growth effects of multiplication-stimulating activity (rat insulin-like growth factor II) in rat embryo fibroblasts.
Levels of multiplication-stimulating activity (MSA) in fetal rat serum are high (2-4 micrograms/ml), suggesting that MSA may have a role in fetal growth. We now demonstrate that fibroblasts derived from rat embryos (REFs) have specific MSA receptors and respond to MSA with increased DNA synthesis. Two types of insulin-like growth factor (IGF) receptors were demonstrated by competitive binding of radioiodinated MSA, IGF-I, and IGF-II and by chemical cross-linking of [125I]iodo-MSA and [125I]iodo-IGF-I to REFs. One type of receptor (mol wt, 260,000 under reducing conditions) did not interact with insulin, and another type of receptor (mol wt, 130,000, under reducing conditions) was recognized by insulin. Scatchard analysis of [125I]iodo-MSA binding data was consistent with one class of noninteracting binding sites. A biological response of MSA, increased DNA synthesis, was demonstrated with autoradiography in REFs. During a 16-hr incubation, DNA synthesis was stimulated by normal rat serum, and platelet-poor plasma plus platelet-derived growth factor (PDGF), but not by serum from hypophysectomized (hypox) rats or hypophysectomized (hypox) platelet-poor plasma plus PDGF. However, when MSA was added to hypox serum or to hypox platelet-poor plasma plus PDGF, DNA synthesis was stimulated to the level achieved by normal rat serum. By contrast, during a longer cell multiplication experiment, REFs grew equally well in normal or hypox rat serum, raising the possibility that REFs may produce a MSA-like factor.
Animals↗
[Induction of diabetes in miniature swine and the effects of insulin on the cells of diabetic animals].
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Adipose Tissue↗
Intratumor chemoimmunotherapy with mitomycin C and BCG in C3H/He mice transplanted with MH134.
Experiments were performed to explore the influence of local chemoimmunotherapy by intratumoral administration of mitomycin C (MMC) and/or BCG on the survival rate, lymph-node metastasis, growth pattern of rechallenge tumor, and host immune function, using a host-tumor system consisting of C3H/He mice and syngeneic tumor MH134. Combined intratumoral regimens of MMC plus BCG yielded a significantly higher survival rate than those achieved with BCG or MMC alone. Furthermore, the incidence of metastatic involvement of regional lymph nodes was also remarkably reduced in the combined regimen group. The group given the combined MMC-BCG regimen also showed a marked suppression of the growth of rechallenge tumor after surgical removal of the primary tumor and showed a greater induction of tumor-specific immunity than that seen in the group given intratumoral BCG injection alone. Both the delayed-type hypersensitivity, as measured by the footpad swelling assay, and the antibody response in spleen lymphocytes, as estimated by the plaque-forming cell assay, were found to be substantially depressed following a systemic injection of MMC, whereas the intratumoral administration of MMC had little or no effect on these parameters.
Animals↗
[Structure and function of insulin receptor].
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Animals↗
Effect of a protein-bound polysaccharide preparation, PS-K, on dimethyl hydrazine induction of intestinal tumors in rats.
The effect of a protein-bound polysaccharide preparation, PS-K, on the induction of intestinal tumors by dimethyl hydrazine (DMH) was assessed in Wistar rats. One hundred and fifty-one rats were randomly divided into two groups. Seventy-two rats were treated with DMH alone and 79 rats were treated with DMH and PS-K. All animals were subjected to a sequential autopsy and all lesions within the gastro-intestinal tract were examined macroscopically and histologically. Tumor incidence in the DMH plus PS-K-treated group was significantly lower than that in the group treated with DMH alone. The most interesting histological finding was marked lymphoid infiltration in and around the tumors of the rats in the PS-K-treated group. The number of circulating lymphocytes dropped below the control range in the 25th and 35th weeks for the group treated with DMH alone, but the drop in the PS-K-treated group was smaller. Delayed-type hypersensitivity reaction to purified protein derivative was well maintained in the PS-K-treated rats. The most interesting findings in these experiments were differences in the serum blocking activities and serum immunosuppressive substance in these two groups; they were markedly reduced in the PS-K-treated rats. The present results may be explained in terms of competitive action of PS-K against the immunosuppressive factor produced by a tumor-bearing host.
Animals↗
Insulin receptor phosphorylation in intact adipocytes and in a cell-free system.
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Adipose Tissue↗
Insulin stimulation of phosphorylation of the beta subunit of the insulin receptor. Formation of both phosphoserine and phosphotyrosine.
Rat hepatoma cells were labeled with [32P]orthophosphate and the insulin receptor subunits were identified by immunoprecipitation and sodium dodecyl sulfate-acrylamide gel electrophoresis. In the basal state, only the Mr = 95,000 (beta) subunit of the insulin receptor was phosphorylated. The covalent labeling with 32P of this subunit was stimulated about 3-fold by insulin (10(-6) M). This stimulation was due to an increase in the content of phosphoserine, the appearance of phosphotyrosine, and a possible increase in phosphothreonine as well. These results suggest phosphorylation of the insulin receptor at multiple sites is an early event in insulin action.
Amino Acids↗