Intrapeptide autophosphorylation of the epidermal growth factor receptor: regulation of kinase catalytic function by receptor dimerization

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Abstract
The [mouse] epidermal growth factor (EGF) receptor is a transmembrane polypeptide of 170,000 daltons (Da) with a cytoplasmically facing protein kinase domain. The regulation of the tyrosine kinase activity of the EGF receptor by added EGF and by receptor association state was studied in an in vitro system. The rate of autophosphorylation of the solubilized and purified EGF receptor was found to be independent of receptor concentration. To determine whether the 0-order kinetics observed point to intrapeptide phosphorylation, the sedimentation characteristics of the undenatured solubilized receptor were measured. The receptor exists in 2 association-dissociation states-a monomeric 7.7S form and a dimeric 12S form. The 7.7S form is an active tyrosine kinase; it has high basal activity, and the activity is not further stimulated by EGF; it appears to be an EGF-independent form of the receptor kinase. The 12S form is devoid of catalytic activity, but in the presence of EGF it dissociates into the active monomeric form. Freshly purified receptor preparations contain mainly the monomeric receptor, have high basal kinase activity and show low EGF stimulatability (< 1.3-fold). Aging of the receptor results in progressive dimerization and decay of EGF-independent kinase activity (and increase in EGF stimulatability). All of these processes are reversed in the presence of EGF or dithiothreitol. The solubilized EGF receptor may be catalytically active only in its monomeric 170,000-Da form. Apparently autophosphorylation of the solubilized receptor is an intrapeptide function of the monomeric form. Bimolecular receptor-receptor interaction may lead to reversible inactivation of the protein kinase site and EGF appears to stimulate the kinase activity of the receptor by shifting the association equilibrium toward monomer formation. The pertinence of these results, obtained with solubilized and purified receptor, to the in vivo situation remains to be established.

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