EPIDERMAL GROWTH-FACTOR STIMULATION OF DNA-SYNTHESIS IS POTENTIATED BY COMPOUNDS THAT INHIBIT ITS CLUSTERING IN COATED PITS

Inhibitors of receptor-mediated endocytosis were used to investigate the mechanism and function of epidermal growth factor [EGF] uptake by cultured cells. When rhodamine-labeled EGF is bound to cell surface receptors on confluent monolayers of BALB/c 3T3 cells, it rapidly collects in cell surface clusters and is internalized. The clustering of occupied receptors requires Ca2+ and is inhibited by primary alkylamines: both of these properties are shared by the enzyme transglutaminase (R-glutaminyl-peptide:amine .gamma.-glutamyl-yltransferase, EC 2.3.2.13). In Chinese hamster ovary cell extracts, methylamine inhibits 25-50% of the transglutaminase activity with a Ki [inhibition constant] of 0.2 mM, and it inhibits the remaining transglutaminase activity with a Ki of 20 mM. Clustering is almost completely inhibited by 10 mM methylamine. The polypeptide antibiotic bacitracin inhibits clustering of rhodamine-labeled EGF or .alpha.2-macroglobulin at 0.7 mM, and it inhibits approximately 40% of the transglutaminase activity in Chinese hamster ovary cells with a Ki of 0.03 mM. Fluorescent ligands bound to cell surface receptors in the presence of bacitracin form clusters within 30 min after bacitracin is removed from the culture medium. A transglutaminase-like enzyme may be required for the clustering and subsequent internalization of occupied receptors. The effects of 10 mM methylamine and 0.7 mM bacitracin on EGF stimulation of DNA synthesis were examined. The stimulation of DNA synthesis by EGF was increased 2- to 7-fold in the presence of methylamine or bacitracin. Alone, methylamine or bacitracin increased DNA synthesis 1.1- to 3-fold. The stimulation of DNA synthesis resulting from the simultaneous presence of the hormone and the clustering inhibitor was always greater than the sum of the stimulations produced by the hormone and the clustering inhibitors alone. The potentiation of EGF activity by clustering inhibitors suggests that the hormone acts at the cell surface. Rapid internalization of occupied receptors via coated pits may be a mechanism to limit the response to hormones.