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Antibody-induced down-regulation of a mutated insulin receptor lacking an intact cytoplasmic domain
Abstract:
Insulin receptor down-regulation was studied in various Chinese hamster ovary (CHO) cell lines expressing transfected human insulin receptor cDNAs. In addition to a cell line expressing the normal receptor (CHO.T line), three lines expressing mutated receptors were studied: the CHO.T-t line, which expresses a receptor with a degraded cytoplasmic domain due to the removal of the C-terminal 112 amino acids, and the CHO.YF1 and CHO.YF3 lines, in which important autophosphorylation sites of the receptor kinase (tyrosines-1162 and -1163) have been replaced by phenylalanine. A monoclonal anti-receptor antibody, but not insulin itself, was found to down-regulate cell surface receptor levels in all four cell lines by 60-80% after 18-h treatment at 37 degrees C. Down-regulation of the CHO.T and CHO.T-t receptors occurred at similar antibody concentrations and with a similar time course, although the maximum level of CHO.T-t down-regulation (60%) was generally lower than the level of CHO.T down-regulation (80%). Pulse-chase labeling of these two cell types with [35S]methionine revealed that antibody treatment of both CHO.T and CHO.T-t cells resulted in a similar increase in the rate of degradation of mature receptor subunits. These results indicate that antibody-induced down-regulation of the insulin receptor in these cells can occur in the absence of various autophosphorylation sites of the receptor and that the mechanism of antibody-induced down-regulation is different from that for insulin.
Insights
Antibody treatment down-regulates insulin receptors in Chinese hamster ovary cells, even when key receptor parts are missing or altered. This antibody-induced insulin receptor down-regulation differs from insulin
Area of Science:
- Molecular biology
- Cell biology
- Biochemistry
Background:
- The insulin receptor plays a crucial role in glucose metabolism.
- Understanding insulin receptor regulation is key to metabolic disease research.
- Down-regulation mechanisms, particularly those involving antibodies, are not fully understood.
Purpose of the Study:
- To investigate insulin receptor down-regulation in Chinese hamster ovary (CHO) cell lines.
- To compare the effects of antibody-induced versus insulin-induced receptor down-regulation.
- To examine the role of specific receptor domains and autophosphorylation sites in antibody-mediated down-regulation.
Main Methods:
- Utilized Chinese hamster ovary (CHO) cell lines expressing normal and mutated human insulin receptors.
- Employed a monoclonal anti-receptor antibody and insulin to induce down-regulation.
- Quantified cell surface receptor levels after treatment.
- Performed pulse-chase labeling with [35S]methionine to assess receptor degradation rates.
Main Results:
- A monoclonal anti-receptor antibody, but not insulin, significantly down-regulated cell surface insulin receptors (60-80%) in all tested CHO cell lines.
- Antibody-induced down-regulation occurred similarly in cells with normal receptors and those with a degraded cytoplasmic domain.
- Antibody treatment increased the degradation rate of mature receptor subunits in both normal and mutated receptor-expressing cells.
- Down-regulation was observed even when key autophosphorylation sites (tyrosines-1162 and -1163) were mutated.
Conclusions:
- Antibody-induced insulin receptor down-regulation can occur independently of the receptor's cytoplasmic domain and autophosphorylation sites.
- The mechanism of antibody-induced insulin receptor down-regulation differs fundamentally from that of insulin itself.
- These findings provide insights into the distinct pathways regulating insulin receptor levels.