AGGREGATION-INDUCED ACTIVATION OF THE EPIDERMAL GROWTH-FACTOR RECEPTOR PROTEIN-TYROSINE KINASE

Various agents are able to stimulate the EGF receptor protein tyrosine kinase in the absence of ligand binding. To characterize their mechan ism of action, we investigated their effects on the kinase activity of the intracellular domain of the EGF receptor (EGFR-IC). EGFR-IC (67 k Da) lacking the extracellular domain and transmembrane segment of the EGF receptor, but retaining kinase and autophosphorylation domains, wa s produced and purified as a soluble, cytoplasmic protein from Sf9 ins ect cells infected with a recombinant baculovirus. EGFR-IC was able to undergo autophosphorylation in a manner similar to full-length EGFR. Synthetic substrate peptides showed similar affinity to EGFR-IC as to the full-length receptor. The activity of the EGFR-IC was found to be dependent on divalent cations, Mn2+ being a more potent activator than Mg2+. Agents capable of aggregating the kinase by direct interaction (cross-linking antibodies, polycations) or through altering the surrou nding solvent structure and thereby decreasing protein solubility [amm onium sulfate, poly(ethylene glycol), 2-methyl-2,4-pentanediol] activa ted the kinase in a manner which correlated with their ability to prec ipitate the EGFR intracellular domain. The widely different chemical n ature of these agents suggests that they do not act by direct interact ion with specific allosteric regulatory sites, but rather by facilitat ing the interactions between kinase molecules. These results support t he hypothesis that full-length receptor aggregation itself, induced by ligand binding to the extracellular domain, results in intracellular domain interactions and the activation of kinase activity.