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. Steadystate EGF binding studies, analyzed according to the Scatchard method, showed that A43 1 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 x lo4 sites per cell) and a low-affinity site with a K D of 8.5 nM (1.9 x lo6 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-,) of 1.1. x 10-3s-1 (I.Gl.3 x lo6 sites per cell) and a slow-dissociating site, with a k-, of 3.5 x IO-'s-' ( 0 . 6 4 . 7 x lo6 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 fastdissociating component (k-l = 2.0 x 10-3s-1) and a slow-dissociating component (k-I = 9.1 x IO-'s-'). 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 4ยฐC 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 4ยฐC EGF induces the binding of low-affinity, fast-dissociating sites to the cytoskeleton of A431 cells.