EPIDERMAL GROWTH-FACTOR LIGAND-INDEPENDENT, UNREGULATED, CELL-TRANSFORMING POTENTIAL OF A NATURALLY-OCCURRING HUMAN MUTANT EGFRVIII GENE

The type III deletion-mutant gene for the epidermal growth factor rece ptor (EGFRvIII) is frequently expressed in glioblastomas and in breast and non-small cell lung carcinomas. To understand its contribution to the malignant phenotype in humans, we transfected NR6 cells with the mammalian vector pH beta APr-1-neo containing cDNA for either ECFRvIII or wild-type EGFR. Western blot analyses showed that NR6 transfected with wild-type EGFR (NR6W) contained a normal-sized protein (170 kilod altons); cells transfected with EGFRvIII (NR6M) contained a truncated protein (145 kilodaltons). NR6W cells demonstrated a saturation bindin g curve with I-125-labeled EGF (affinity, 1.8 x 10(8); r(2) = 0.96). N R6M cells, however, showed a low but consistent level of I-125-labeled EGF binding (affinity, 4 x 10(7); r(2) = 0.99) compared with NR6, whi ch lacked binding. The population doubling time was shorter for NR6M ( 0.64 days) than for NR6W (1.1 days) and NR6V (2.27 days). Soft agar fo cus formation assay by NR6M was 4- to 5-fold higher than that by NR6W. In nude mice, NR6M (1 x 10(7) cells), without exogenous ligand, forme d tumors within 12 days; no tumors were observed over 90 days in mice receiving identical doses of NR6W, NR6V, or NR6 cells. EGF stimulated autophosphorylation of receptor in NR6W (4- to 9-fold) but caused only slight (1.8- to 1.9-fold) to no enhancement in NR6M. Further, there w as no difference in constitutive tyrosine kinase activity between NR6M and NR6W. Our results clearly indicate that ECFRvIII functions as an oncoprotein, but its intrinsic tyrosine kinase activity may not be res ponsible for its biological function.