The mouse waved-2 phenotype results from a point mutation in the EGF receptor tyrosine kinase

Mice harboring the waved-1 (wa-1) and waved-2 (wa-2) mutations exhibit skin and eye abnormalities that are strikingly similar to those of TGF-alpha-deficient mice, and wa-1 and TGF-alpha were recently shown to be allelic. Because the wa-2 mutation was mapped previously to the vicinity of the EGF/TGF-alpha receptor (EGFR) gene on mouse chromosome 11, we hypothesized that the wa-2 phenotype might result from a defect in either the expression or activity of EGFR, or both. In the present report, we show that EGFR mRNA and protein of normal size are expressed in wa-2 liver and skin at levels that are comparable to those in the corresponding normal tissues, and that the ability of wa-2 EGFR to bind ligand is unaltered. However, ligand-dependent autophosphorylation of wa-2 EGFR is diminished 5- to 10-fold in vitro, and the ability of wa-2 EGFR to phosphorylate an exogenous substrate is reduced by > 90% compared with that of the control receptor. EGF-induced tyrosine phosphorylation, including that of EGFR itself, is also diminished in skin, particularly at lower dose of exogenous EGF. To establish the nature of the wa-2 mutation, we determined the nucleotide sequence of the coding region of normal and wa-2 murine EGFR cDNAs. A comparison of these sequences revealed a single-nucleotide transversion resulting in the substitution of a glycine for a conserved valine residue near the amino terminus of the tyrosine kinase domain. The importance of this mutation was confirmed by showing that its introduction into an otherwise normal EGFR markedly reduced the receptor's tyrosine kinase activity in transfected Chinese hamster ovary cells. Finally, in situ hybridization analysis demonstrated expression of EGFR predominantly in the outer root sheath of active hair follicles in neonatal mice. As we previously localized TGF-alpha mRNA to the inner root sheath, this pattern of EGFR expression is consistent with the effect of the wa-2 mutation on hair structure, and together with our previous characterization of TGF-alpha-deficient mice, reveals a critical role for signaling by this ligand/receptor system in skin.