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Inhibition of epidermal growth factor receptor biosynthesis caused by the src oncogene product, pp60v-src
W J Wasilenko1, M Nori, N Testerman
1Department of Microbiology and Cancer Center, University of Virginia Health Sciences Center, Charlottesville 22908.
Abstract:
We have previously shown that an intracellular mechanism down regulates epidermal growth factor (EGF) receptor levels in rodent fibroblasts transformed by the src oncogene (W. J. Wasilenko, L. K. Shawver, and M. J. Weber, J. Cell. Physiol. 131:450-457, 1987). We now report that this down regulation is due to an inhibition of EGF receptor biosynthesis. With Rat-1 (R1) cells infected with a temperature-sensitive src mutant, we found that 125I-labeled EGF binding to cells began to decrease soon after the activation of pp60v-src by shift down to the permissive temperature for transformation. This effect of src on EGF receptors was reversible. Pulse-chase studies with [35S]methionine-labeled cells revealed that the tyrosine protein kinase activity of pp60v-src had little if any effect on EGF receptor degradation rate. By contrast, the expression of pp60v-src caused a large reduction in the apparent rate of EGF receptor biosynthesis. Northern (RNA) blot analysis demonstrated that pp60v-src also caused marked reductions in the steady-state level of EGF receptor mRNA. These data indicate that one way the expression of the src oncogene can affect the machinery of growth control is by affecting the expression of specific genes for growth factor receptors.
Insights
The src oncogene inhibits epidermal growth factor (EGF) receptor biosynthesis, not degradation, by reducing EGF receptor mRNA levels. This src-induced downregulation of EGF receptors impacts cell growth control.
Area of Science:
- Molecular Biology
- Oncology
- Cellular Biology
Background:
- Previous studies demonstrated that the src oncogene downregulates epidermal growth factor (EGF) receptor levels in rodent fibroblasts.
- The underlying intracellular mechanism for this downregulation remained to be elucidated.
Purpose of the Study:
- To investigate the mechanism by which the src oncogene downregulates EGF receptor levels.
- To determine if the downregulation is due to altered receptor biosynthesis or degradation.
Main Methods:
- Utilized temperature-sensitive src mutant in Rat-1 (R1) cells to study EGF receptor regulation.
- Employed 125I-labeled EGF binding assays to measure cell surface receptor levels.
- Conducted pulse-chase studies with [35S]methionine to assess EGF receptor biosynthesis and degradation rates.
- Performed Northern (RNA) blot analysis to examine EGF receptor mRNA levels.
Main Results:
- Activation of pp60v-src led to a rapid and reversible decrease in 125I-labeled EGF binding.
- pp60v-src tyrosine protein kinase activity minimally affected EGF receptor degradation rates.
- Expression of pp60v-src significantly reduced the apparent rate of EGF receptor biosynthesis.
- Northern blot analysis revealed a marked reduction in EGF receptor mRNA steady-state levels upon src expression.
Conclusions:
- The src oncogene downregulates EGF receptor levels primarily by inhibiting EGF receptor biosynthesis.
- This inhibition occurs at the level of gene expression, leading to reduced EGF receptor mRNA.
- Altering the expression of growth factor receptor genes is a mechanism by which the src oncogene influences cell growth control.