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

Abstract

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 SV40WI38 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.