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

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Abstract

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 (KD) of 0.7 nM (7.5 × 104 sites per cell) and a low-affinity site with a KD 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 (KD = 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 (KD = 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.

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