Calcium effects on epidermal growth factor receptor-mediated endocytosis in normal and SV40-transformed human fibroblasts

Lowering of extracellular Ca²⁺ levels will reversibly arrest the growth of human fibroblasts (WI38). Simian virus₄₀(SV₄₀)-transformed WI38 cells do not exhibit this Ca²⁺-dependent arrest. One possibility for this difference in Ca²⁺ requirement is that extracellular or surface membrane-bound Ca²⁺ may be required for growth factor receptor-mediated endocytosis and this Ca²⁺ requirement may differ in normal versus transformed cells. In this study we have evaluated the role of Ca²⁺ in the binding, internalization, and degradation of epidermal growth factor (EGF) in the WI38 and SV₄₀ WI38 cell. The binding of ¹²⁵I]EGF to the cell surface is not significantly altered by lowering of Ca²⁺ to 10^?5-M levels in either the normal or transformed cell. At this Ca²⁺ level, growth of the normal cell is inhibited. The subsequent internalization of EGF is reduced nearly threefold in the normal cell but not in the transformed cell following Ca²⁺ deprivation. Degradation of the EGF-receptor complex is also sensitive to Ca²⁺. A twofold reduction in the rate of release of acid-soluble ¹²⁵I occurs in the normal but not the transformed cell under conditions of lowered medium Ca²⁺. In contrast, 2-chloro-10-3-aminopropyl phenothiazine (CP), an inhibitor of the Ca²⁺-dependent regulator protein calmodulin, causes an inhibition of ¹²⁵I]EGF internalization and degradation in both the normal and transformed WI38 cell, and a marked inhibition of ¹²⁵I]EGF binding to the cell surface receptor of the transformed cell but not the normal cell.