PHYSICOCHEMICAL CHARACTERIZATION OF THE CYTOPLASMIC DOMAIN OF THE EPIDERMAL GROWTH-FACTOR RECEPTOR AND EVIDENCE FOR CONFORMATIONAL-CHANGES ASSOCIATED WITH ITS ACTIVATION BY AMMONIUM-SULFATE

The physicochemical properties of the purified cytoplasmic domain of t he epidermal growth factor (EGF) receptor, its self-phosphorylation an d peptide phosphorylation activities, and its activation by ammonium s ulphate have been studied. Highly efficient purification procedures fo r the isolation of the recombinant cytoplasmic domain (Met(644)-Ala(11 86)) of the EGF receptor, expressed in the baculovirus/insect cell sys tem, are described. Physicochemical characterization of the protein in cluded investigation of its isoelectric and hydrodynamic properties, s tability, oligomeric status, and secondary structure using far-u.v. ci rcular dichroism. The recombinant protein was not recognized by antiph osphotyrosine antibodies, unless first self-phosphorylated in vitro. T ryptic phosphopeptide maps of self-phosphorylated recombinant cytoplas mic domain and the EGF-stimulated A431-membrane receptor were very sim ilar, suggesting that the recombinant had similar self-phosphorylation capacity and specificity. The preparations were characterized by high specific activity towards peptide tyrosine phosphorylation. Although the cytoplasmic domain was isolated as a homogeneously monomeric prote in, storage at 4 degrees C led to slow, spontaneous aggregation with r eduction in specific activity. Both high activity and monomeric state were maintained by storage below 0 degrees C. The dependence of the in itial rate of self-phosphorylation on protein concentration was consis tent with cross-phosphorylation but not with the known oligomerization -induced activation of holoreceptor. The peptide phosphorylation activ ity was stimulated by Mn2+, Mg2+ and (NH4)(2)SO4 at high concentration s. The substrate specificity of (NH4)(2)SO4 activation was studied usi ng synthetic peptides. Self-phosphorylation was inhibited by (NH4)(2)S O4 in the range 0-0.25 M but activated at 1.0-1.5 M, possibly as a res ult of ionic and hydrophobic protein interactions respectively. Phosph opeptide maps of cytoplasmic domain phosphorylated in the presence of high (NH4)(2)SO4 showed that the protein was more extensively phosphor ylated than in the absence of salt, or than the native receptor. Far-u .v. circulardichroism spectra of the cytoplasmic domain changed dramat ically at 1 M (NH4)(2)SO4, raising the possibility that (NH4)(2)SO4 ac tivates the kinase catalytic domain by inducing conformational changes .