STRUCTURE-FUNCTION STUDIES OF LIGAND-INDUCED EPIDERMAL GROWTH-FACTOR RECEPTOR DIMERIZATION

We present a novel 96-well assay which we have applied to a structure- function study of epidermal growth factor receptor dimerization. The b asis of the assay lies in the increased probability of EGFRs being cap tured as dimers by a bivalent antibody when they are immobilized in th e presence of a cognate ligand. Once immobilized, the antibody acts as a tether, retaining the receptor in its dimeric state with a resultan t 5-7-fold increase in binding of a radiolabeled ligand probe. When th e assay was applied to members of the EGF ligand family, murine EGF, t ransforming growth factor alpha, and heparin-binding EGF-like growth f actor were comparable with human EGF (EC50 = 2nM); betacellulin, which has a broader receptor specificity, was slightly less effective. In c ontrast, amphiregulin (AR(1-84)), which has a truncated C-tail and lac ks a conserved leucine residue, was ineffective unless used at >1 mu M . We further probed the involvement of the C-tail and the conserved le ucine residue in receptor dimerization by comparing the activities of two genetically modified EGFs (the chimera mEGF/TGF alpha(44-50) and t he EGF point mutant L47A) and a C-terminally extended form of AR (AR(1 -90)) with those of two other unrelated EGF mutants (I23T and L15A). T he potency of these ligands was in the order EGF > I23T > mEGF/TGF alp ha(44-50) > L47A = L15A much greater than AR(1-90) > AR(1-84). Althoug h AR was much worse than predicted from its affinity, this defect coul d be partially rectified by co-localization of the immobilizing antibo dy with heparin. Thus, it seems likely that AR cannot dimerize the EGF R unless other accessory molecules are present to stabilize its functi onal association with the EGFR.