Ligand‐induced association of epidermal growth factor receptor to the cytoskeleton of A431 cells

Recently, we have obtained evidence in favor of a structural interaction between the epidermal growth factor (EGF) receptor and the Triton X‐100‐insoluble cytoskeleton of epidermoid carcinoma A431 cells. Here we present a further analysis of the properties of EGF receptors attached to the cytoskeleton. Steady‐state EGF binding studies, analyzed according to the Scatchard method, showed that A431 cells contain two classes of EGF‐binding sites: a high‐affinity site with an apparent dissociation constant (K_D) of 0.7 nM (7.5 × 10⁴ sites per cell) and a low‐affinity site with a K_D of 8.5 nM (1.9 × 10⁶ sites per cell). Non‐equilibrium binding studies revealed the existence of two kinetically distinguishable sites: a fast‐dissociating site, with a dissociation rate constant (k₋₁) of 1.1. × 10⁻³s⁻¹ (1.0–1.3 × 10⁶ sites per cell) and a slow‐dissociating site, with a k₋₁ of 3.5 × 10⁻⁵s⁻¹ (0.6–0.7 × 10⁶ sites per cell). The cytoskeleton of A431 cells was isolated by Triton X‐100 extraction. Scatchard analysis revealed that ∼5% of the original number of receptors were associated with the cytoskeleton predominantly via high‐affinity sites (K_D = 1.5 nM). This class of receptors is further characterized by the presence of a fast‐dissociating component (k₋₁ = 2.0 × 10⁻³s⁻¹) and a slow‐dissociating component (k₋₁ = 9.1 × 10⁻⁵s⁻¹). The distribution between fast and slow sites of the cytoskeleton was similar to that of intact cells (65% fast and 35% slow sites). Incubation of A431 cells for 2 h at 4°C in the presence of EGF resulted in a dramatic increase in the number of EGF receptors associated to the cytoskeleton. These newly cytoskeleton‐associated receptors appeared to represent low‐affinity binding sites (K_D = 7 nM). Dissociation kinetics also revealed an increase of fast‐dissociating sites. These results indicate that at 4°C EGF induces the binding of low‐affinity, fast‐dissociating sites to the cytoskeleton of A431 cells.