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Insulin receptor internalization defect in an insulin-resistant mouse melanoma cell line
M J Androlewicz1, D F Brandenburg, D S Straus
1Division of Biomedical Sciences, University of California, Riverside 92521-0121.
Abstract:
Previous studies from this laboratory demonstrated that the PG19 mouse melanoma cell line does not exhibit a biological response to insulin, whereas melanoma x mouse embryo fibroblast hybrids do respond to insulin. To investigate the molecular basis of the insulin resistance of the PG19 melanoma cells, insulin receptors from the insulin-resistant melanoma cells and insulin-sensitive fibroblast x melanoma hybrid cells were analyzed by the technique of photoaffinity labeling using the photoprobe 125I-NAPA-DP-insulin. Photolabeled insulin receptors from the two cell types have identical molecular weights as determined by SDS gel electrophoresis under reducing and nonreducing conditions, indicating that the receptors on the two cell lines are structurally similar. Insulin receptor internalization studies revealed that the hybrid cells internalize receptors to a high degree at 37 degrees C, whereas the melanoma cells internalize receptors to a very low degree or not at all. The correlation between ability to internalize insulin receptors and sensitivity to insulin action in this system suggests that uptake of the insulin-receptor complex may be required for insulin action in these cells. Insulin receptors from the two cell lines autophosphorylate in a similar insulin-dependent manner both in vitro and in intact cells, indicating that insulin receptors on the melanoma and hybrid cells have functional tyrosine protein kinase activity. Therefore, the block in insulin action in the PG19 melanoma cells appears to reside at a step beyond insulin-stimulated receptor autophosphorylation.
Insights
Mouse melanoma cells resist insulin due to impaired insulin receptor internalization, not receptor function. This suggests receptor uptake is crucial for insulin signaling and action in these cells.
Area of Science:
- Cell biology
- Molecular endocrinology
- Cancer research
Background:
- PG19 mouse melanoma cells lack biological response to insulin.
- Melanoma x mouse embryo fibroblast hybrids are insulin-sensitive.
- Investigating molecular basis of insulin resistance in melanoma cells.
Purpose of the Study:
- Analyze insulin receptors from insulin-resistant melanoma and insulin-sensitive hybrid cells.
- Determine the molecular mechanisms underlying insulin resistance in PG19 melanoma cells.
Main Methods:
- Photoaffinity labeling with 125I-NAPA-DP-insulin.
- SDS gel electrophoresis (reducing and nonreducing conditions).
- Insulin receptor internalization studies at 37°C.
- Insulin-stimulated receptor autophosphorylation assays (in vitro and in intact cells).
Main Results:
- Insulin receptors from both cell types have identical molecular weights, indicating structural similarity.
- Hybrid cells exhibit high insulin receptor internalization; melanoma cells show very low internalization.
- Receptors from both cell lines possess functional tyrosine protein kinase activity, demonstrated by similar insulin-dependent autophosphorylation.
- Insulin action blockade in melanoma cells occurs post-receptor autophosphorylation.
Conclusions:
- Insulin receptor internalization is critical for insulin action in this cellular system.
- The primary defect in insulin resistance of PG19 melanoma cells lies in receptor internalization, not receptor structure or kinase activity.
- Findings suggest a novel requirement for insulin receptor uptake in mediating cellular responses to insulin.