Turnover of epidermal growth factor binding sites in mouse mammary epithelial cells

An in vitro method of studying epidermal growth factor (EGF) receptors in mouse mammary epithelial cells in serum-free collagen gel culture has been developed. Binding of EGF averaged 108 +/- 19 fmol/mg DNA in cells isolated from freshly dissociated virgin mammary tissue. Initial binding values were maintained in cells cultured in the presence of 0.1 ng/ml EGF, but decreased in either 0 ng/ml or 10 ng/ml EGF. Addition of either chloroquine (100 microM) or ammonium chloride (10 mM) to the culture medium increased receptor levels 10-fold. Cycloheximide (0.1 microgram/ml), ouabain (3 mM), and actinomycin D (5 x 10(-2) micrograms/ml) each decreased receptor levels, in some cases by as much as 80%. Both methylamine (10 mM) and dinitrophenol (0.1 mM) had no significant effect. These studies suggest that the net level of EGF receptors in these target cells is the result of an equilibrium between synthesis and degradation. The difference between the effects of the compounds tested on either receptor degradation or synthesis in comparison to cell growth, may be indicative that receptor degradation is not linked to cell proliferation.