RECRUITMENT OF EPIDERMAL GROWTH-FACTOR AND TRANSFERRIN RECEPTORS INTOCOATED PITS IN-VITRO - DIFFERING BIOCHEMICAL REQUIREMENTS

The biochemical requirements for epidermal growth factor (EGF) and tra nsferrin receptor-mediated endocytosis were compared using perforated human A431 cells. Morphological studies showed that horseradish peroxi dase (HRP)-conjugated EGF and gold-labeled anti-transferrin (Tfn) rece ptor antibodies were colocalized during endocytosis in vitro. The sequ estration of both ligands into deeply invaginated coated pits required ATP hydrolysis and cytosolic factors and was inhibited by GTPgammaS, indicating mechanistic similarities. Importantly, several differences in the biochemical requirements for sequestration of EGF and Tfn were also detected. These included differing requirements for soluble AP (c lathrin assembly protein) complexes, differing cytosolic requirements, and differing sensitivities to the tyrosine kinase inhibitor, geniste in. The biochemical differences detected between EGF and Tfn sequestra tion most likely reflect specific requirements for the recruitment of EGF-receptors (R) into coated pits. This assay provides a novel means to identify the molecular bases for these biochemical distinctions and to elucidate the mechanisms involved in ligand-induced recruitment of EGF-R into coated pits.