Fig. 1.

Homologous binding inhibition of ¹²⁵I-EGF to EGFR mutants. Mouse 2.2 cells (3T3 devoid of endogenous EGFR) stably expressing EGFR-P (♦), dimerization mutants [D279A/H280A (□), Del:242–259 (×), and Y251A/R285S (•)], and intramolecular tether mutants [D563A/H566A/K585A (■) and Del:560–590 (▴)] were seeded on 24-well plates and grown to confluence. Cells were treated with a fixed concentration of (3.1 ng/ml) ¹²⁵I-EGF and an increasing concentration of unlabeled EGF for 1 h at room temperature. After washing with PBS the remaining bound radioactivity was detected by using a liquid scintillation counter LS 6500 (Beckman). (Inset) Expression levels of EGFR mutants in 2.2 3T3 cells. Stably transfected cells harboring various extracellular domain mutations in a background of EGFR-P with a myc tag in the C terminus of the molecule are shown. Monoclonal anti-EGFR antibody (mAb108) was used for immunoprecipitation followed by detection with anti-Myc antibodies. The mutants are EGFR-P (a), Del:242–249 (b), Y251/R285S (c), D279/H280 (d), D563A/H566/A/K585A (e), and Del:560–590 (f).