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Genetics of cell-surface receptors for bioactive polypeptides: a variant of mouse BALBc/3T3 fibroblasts possessing
Abstract:
An insulin-nonresponsive variant was isolated from mutagenized mouse BALBc/3T3 fibroblasts. Selection was based on the insulin's mitogenic action upon quiescent cells and subsequent arrest at mitosis by vinblastine sulfate to remove insulin-responsive cells. Among four surviving colonies, one, designated IN-2, exhibited no binding for [123I] insulin at 2 x 10(-10) M and at 4 degrees C. The binding ability, however, recovered substantially at 15 degrees C and increased with higher temperature and at higher ligand concentrations. The binding profiles, Scatchard plot analysis, and the dissociation kinetics indicated that the receptors expressed on IN-2 cells possess lower affinity than the parental 3T3 cells. The IN-2 cells were negative for stimulating effects of insulin on 2-deoxyglucose uptake, thymidine incorporation, and cell growth. The IN-2 cells were also negative for cross-reactivity to antibodies which react with insulin receptors on 3T3 cells and for the susceptibility to a cytotoxic chimeric insulin which was cross-linked to diphtheria toxin fragment A. This negative response of IN-2 cells can be attributed to a deficiency in "high-affinity receptors" for insulin. The insulin bound to the "low-affinity binding sites" of IN-2 cells, however, undergoes internalization and intracellular degradation. Therefore, such processing by itself does not account for insulin's mitogenic action.
Insights
A new mouse cell variant, IN-2, lacks high-affinity insulin receptors, showing reduced insulin binding and no mitogenic response. This suggests high-affinity receptors are crucial for insulin
Area of Science:
- Cell Biology
- Molecular Endocrinology
- Signal Transduction
Background:
- Insulin binding to its receptor initiates crucial cellular processes, including mitogenesis.
- Understanding insulin receptor function is key to metabolic and growth regulation.
- Mutagenesis screens are valuable for identifying critical components of signaling pathways.
Purpose of the Study:
- To isolate and characterize a mouse fibroblast cell line variant unresponsive to insulin's mitogenic effects.
- To investigate the role of insulin receptor affinity in mediating insulin's cellular actions.
- To elucidate the binding and processing characteristics of insulin in nonresponsive cells.
Main Methods:
- Mutagenesis of BALBc/3T3 fibroblasts followed by selection for insulin nonresponsiveness.
- Vinblastine sulfate was used to arrest cells in mitosis, aiding selection.
- Characterization of insulin binding using radiolabeled insulin ([123I] insulin), Scatchard plot analysis, and kinetic studies at varying temperatures and ligand concentrations.
Main Results:
- An insulin-nonresponsive variant (IN-2) was isolated, exhibiting significantly reduced [123I] insulin binding at low temperatures and concentrations.
- IN-2 cells displayed lower-affinity insulin binding compared to parental cells, confirmed by binding profiles and Scatchard analysis.
- IN-2 cells showed no response to insulin regarding 2-deoxyglucose uptake, thymidine incorporation, or cell growth, and lacked cross-reactivity to anti-insulin receptor antibodies or a cytotoxic insulin conjugate.
Conclusions:
- The IN-2 cell line's unresponsiveness is attributed to a deficiency in high-affinity insulin receptors.
- Insulin binding to low-affinity sites on IN-2 cells is followed by internalization and degradation, but this process does not mediate mitogenesis.
- High-affinity insulin receptors are essential for mediating insulin's mitogenic and growth-promoting effects in BALBc/3T3 fibroblasts.