Although the mechanism is not well understood, binding of different EGF receptor (EGFR) ligands may influence distinct biological responses following activation of the tyrosine kinase domain. Ligand-induced dimerization of the EGFR has been suggested to enhance ligand-binding affinity and to elevate tyrosine kinase activity through transphosphorylation. The extracellular domain of the EGFR is required for both ligand binding and homo- or heterodimerization of EGFRs, but the amino acid residues which govern these interactions are unknown. Using directed mutagenesis strategies, the aims of this proposal are designed to identify these domains. The first objective of this proposal is to identify amino acid residues in the extracellular region of the EGFR involved in ligand- binding or dimerization using a charged-to-alanine scanning mutagenesis strategy and subsequent transfection into CHO cells for structure/function assays.
The second aim i s to examine the functional consequences of inhibiting dimerization on EGFR signaling. Issues of EGFR heterodimerization with HER2 will also be addressed using a similar approach. The third objective is to develop an alternative, novel strategy for generating numerous mutants of the EGFR ectodomain that can be rapidly screened for ligand binding or dimerization. The phenotypic selection system will involve the construction of novel chimeric molecules consisting of the EGFR ectodomain linked to the TGF beta type II receptor cytoplasmic domain (EB-I) or t he HER2 ectodomain linked to the TGP beta type I receptor cytoplasmic domain (NEB-I). Mutant EB-II receptors will be introduced into cells expressing NEB-I and stable clones will be selected based on growth advantage following stimulation with EGF. Structure/function assays will be performed and mutants of interest will be sequenced to determine sites of mutation for further studies.
| Troyer, K L; Luetteke, N C; Saxon, M L et al. (2001) Growth retardation, duodenal lesions, and aberrant ileum architecture in triple null mice lacking EGF, amphiregulin, and TGF-alpha. Gastroenterology 121:68-78 |
| Saxon, M L; Lee, D C (1999) Mutagenesis reveals a role for epidermal growth factor receptor extracellular subdomain IV in ligand binding. J Biol Chem 274:28356-62 |
