KINETICS OF BINDING, ENDOCYTOSIS, AND RECYCLING OF EGF RECEPTOR MUTANTS

This report describes analysis of factors which regulate the binding o f EGF to EGF receptor, receptor internalization, and receptor recyclin g. Three different methods were used to inhibit high-affinity EGF bind ing as measured at equilibrium: treatment of cells with an active phor bol ester (PMA), binding of a mAb directed against the EGF receptor (m Ab108), and truncation of most of the cytoplasmic domain of the recept or. These treatments reduced the rate at which low concentrations of E GF bound to cells, but did not affect the rate of EGF dissociation. We conclude that high-affinity EGF binding on living cells results from a difference in the apparent on rate of EGF binding. We then used thes e conditions and cell lines to test for the rate of EGF internalizatio n at different concentrations of EGF We demonstrate that internalizati on of the EGF receptor is stimulated roughly 50-fold at saturating con centrations of EGF, but is stimulated an additional two- to threefold at low concentrations (< 1 nM). Four treatments reduce the rate of int ernalization of low concentrations of EGF to the rate seen at saturati ng EGF concentrations. Phorbol ester treatment and mAb108 binding to " wild type" receptor reduce this rate (and reduce high-affinity binding ). Point mutation at Lys721 (kinase negative EGF receptor) and point m utation at Thr654 (removing a major site of protein kinase C phosphory lation) reduce the internalization rate, without affecting high-affini ty binding. We suggest that while EGF stimulates endocytosis for all r eceptors, high-affinity receptors bind and are internalized more quick ly than low-affinity receptors. Tyrosine kinase activity and the Thr65 4 region appear necessary for this response.