Abstract
We have recently characterized a mutant insulin receptor (HIR delta 978) in which the insulin receptor beta-subunit was truncated at amino acid residue 978. Compared with parental Rat1 cells, the cells expressing the truncated receptor exhibited enhanced sensitivity to insulin's biologic actions. All of these effects are now extended to transcriptional events, since we now show enhanced sensitivity to insulin stimulation of c-fos mRNA expression. These effects were insulin-specific, since insulin-like growth factor-1 stimulation of glucose incorporation into glycogen, alpha-aminoisobutyric acid uptake, and thymidine incorporation into DNA were normal. In addition, the truncated receptor exhibited enhanced sensitivity only in vivo, but not in vitro, since the kinase activity of wheat germ agglutinin-purified receptor preparations was comparable between HIR delta 978 and parental Rat1 insulin receptors. Parental rat endogenous insulin-like growth factor-1 receptors and transfected human insulin receptors form hybrid receptors as well as homologous tetrameric receptors. The normal heterotetrameric receptors possess kinase activity in vivo leading to tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and its association with the p85 regulatory subunit of phosphatidyl inositol 3-kinase. Interestingly, preincubation with human-specific anti-insulin receptor antibody abolished the increased insulin sensitivity in glucose incorporation into glycogen in HIR delta 978 cells. Furthermore, microinjection of anti-IRS-1 antibody into HIR delta 978 cells inhibited insulin stimulation of DNA synthesis. In summary: 1) truncated receptors on the cell surface confer enhanced insulin sensitivity in vivo; 2) the normal heterotetrameric receptors are functionally active and couple to IRS-1 efficiently; and 3) IRS-1 is an important molecule transmitting insulin's biological signals in HIR delta 978 cells.
Highlights
We have recently characterized a mutant insulin receptor (HIR.1978) in which the insulin receptor l3-subunit was truncated at amino acid residue 978
Based on our earlier studies, we suggested that the enhanced insulin action in HIR~978 cells was directly mediated by the endogenous rodent insulin receptors, rather than the HIR~978 receptors, and that the mutant receptors increased the coupling efficiency between endogenous insulin receptors and downstream signaling molecules
Induction ofc-fos mRNA-We have previously reported that the ability of insulin to stimulate glucose incorporation into glycogen, AlB uptake, thymidine incorporation, and S6 kinase activity is enhanced in HIR.i978 cells compared to parental Rat l cells [9]
Summary
Cell Lines and Materials-The generation of stable clonal cell lines expressing wild type insulin receptors (HIRe) and truncated mutant insulin receptors (HIR~978), in which the insulin receptor J3-subunit was truncated at amino acid residue 978 according to the numbering system by Ullrich et al [1], and cell cultures have been previously described [9, 10]. I The abbreviations used are IRS-I, insulin receptor substrate-I; IGF, insulin-like growth factor; GST, glutathione S-transferase; AlB, a-[methyl-3HJaminoisobutyric acid; BrdU, bromodeoxyuridine; DMEM, Dulbecco's modified Eagle's medium; PCR, polymerase chain reaction; FCS, fetal calf serum; PAGE, polyacrylamide gel electrophoresis; WGA, wheat germ agarose. Thymidine Incorporation-Confluent cell monolayers were incubated in DMEM containing 0.05% fetal calf serum (FCS) for 24 h, various concentrations of IGF-1 were added. Immunofluorescent staining of the injected cells has indicated that about 75% of the cells were successfully microinjected [17, 18]
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