BIOCHEMICAL-CHARACTERIZATION OF MUTANT EGF RECEPTORS EXPRESSED IN THEHEMATOPOIETIC-CELL LINE BAF 3/

The Epidemal Growth Factor (EGF) receptor appears to require a fully a ctive tyrosine kinase domain to transmit mitogenic signals. However, w aved-2 mice carrying a mutation in the alpha-helix C of their EGF-R, w hich abolishes tyrosine kinase activity, only display a mild phenotype and are fully viable. This suggests that the mutant EGF-R signals thr ough heterodimerization with endogenous, kinase active members of the EGF-R family such as ErbB-2 or ErbB-4. We have examined the biochemist ry of EGF-Rs carrying mutations in the alpha-helix C of the human EGF- R (V741G and Y740F), in the ATP binding site (K721R) and at the C-term inus (CT957), by expression in BaF/3 cells which are devoid of EGF-R f amily members. The in vitro kinase activity of the alpha-helix C EGF-R mutants was severely impaired as a result of reduced phosphotransfer activity without appreciable changes in the affinity for either ATP or peptide substrate. Surprisingly, EGF stimulation of cells carrying th e different mutant or wild type EGF-Rs resulted in tyrosine phosphoryl ation of EGF-R proteins; this phosphorylation was abolished in crude p lasma membrane preparations, and appears to be due to activation of a membrane-associated or a cytosolic kinase, Receptor-mediated internali zation of EGF,vas profoundly suppressed in the V741G, K721R and CT957 receptor mutant, and high affinity EGF binding was undetectable in the V741G and K721R receptors, We conclude that specific residues in the C-helix of the EGF-R kinase are essential for full kinase activity; mu tations in this region do not affect ATP binding, but impair the recep tors' phosphotransfer ability. High affinity binding of EGF is not dep endent on tyrosine kinase activity or sequences in the C-terminus.