AbstractEpidermal growth factor receptors (EGF‐R) have been shown to be associated with the detergent‐insoluble cytoskeleton of A431 cells, where they retained both a functional ligand‐binding domain and tyrosine kinase activity. In the present study we have characterized the tyrosine kinase and ligand binding activities of this cytoskeletally associated EGF‐R. The tyrosine kinase activity of the cytoskeletally associated EGF‐R was stimulated by EGF treatment of intact cells as evidenced by increased autophosphorylation and phosphorylation of the exogenous substrate angiotensin II (AII). The kinetic behavior of the EGF‐R associated with cytoskeletons of EGF‐treated cells was similar to that of purified receptors. The stimulation of the receptor kinase activity required EGF treatment of intact cells prior to Triton extraction. If cytoskeletons were prepared from untreated cells and then incubated with EGF, there was no stimulation of the detergent‐insoluble receptor kinase activity, indicating that the immobilized receptor was unable to undergo EGF‐stimulated activation. Comparison of peptide maps from soluble and cytoskeletally associated EGF‐R revealed qualitatively similar patterns; however, they are distinguished by a prominent 46 kD band in digests of the cytoskeletal EGF‐R. Saturable binding of125I‐EGF to A431 cytoskeletons prepared from adherent and suspended cells demonstrated the presence of specific receptors on the cytoskeleton. High‐affinity EGF‐R were preferentially retained upon detergent extraction of adherent cells, whereas both low‐ and high‐affinity receptors were solubilized from the cytoskeletons of suspended cells. Suspension of cells resulted in the solubilization of an additional 15% of the EGF‐R to that solubilized in adherent cells, indicating that EGF‐R can reversibly associate with
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