Gregoriou M, Jones P F, Timms J F, Yang J J, Radford S E, Rees A R
Department of Biochemistry, University of Oxford, UK.
Biochem J. 1995 Mar 15;306 ( Pt 3)(Pt 3):667-78. doi: 10.1042/bj3060667.
The physiochemical properties of the purified cytoplasmic domain of the epidermal growth factor (EGF) receptor, its self-phosphorylation and peptide phosphorylation activities, and its activation by ammonium sulphate have been studied. Highly efficient purification procedures for the isolation of the recombinant cytoplasmic domain (Met644-Ala1186) of the EGF receptor, expressed in the baculovirus/insect cell system, are described. Physicochemical characterization of the protein included investigation of its isoelectric and hydrodynamic properties, stability, oligomeric status, and secondary structure using far-u.v. circular dichroism. The recombinant protein was not recognized by anti-phosphotyrosine antibodies, unless first self-phosphorylated in vitro. Tryptic phosphopeptide maps of self-phosphorylated recombinant cytoplasmic domain and the EGF-stimulated A431-membrane receptor were very similar, 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 protein, storage at 4 degrees C led to slow, spontaneous aggregation with reduction in specific activity. Both high activity and monomeric state were maintained by storage below 0 degree C. The dependence of the initial rate of self-phosphorylation on protein concentration was consistent with cross-phosphorylation but not with the known oligomerization-induced activation of holoreceptor. The peptide phosphorylation activity was stimulated by Mn2+, Mg2+ and (NH4)2SO4 at high concentrations. The substrate specificity of (NH4)2SO4 activation was studied using synthetic peptides. Self-phosphorylation was inhibited by (NH4)2SO4 in the range 0-0.25 M but activated at 1.0-1.5 M, possibly as a result of ionic and hydrophobic protein interactions respectively. Phosphopeptide maps of cytoplasmic domain phosphorylated in the presence of high (NH4)2SO4 showed that the protein was more extensively phosphorylated than in the absence of salt, or than the native receptor. Far-u.v. circular-dichroism spectra of the cytoplasmic domain changed dramatically at 1 M (NH4)2SO4, raising the possibility that (NH4)2SO4 activates the kinase catalytic domain by inducing conformational changes.
对表皮生长因子（EGF）受体纯化的胞质结构域的理化性质、自身磷酸化和肽磷酸化活性以及硫酸铵对其的激活作用进行了研究。描述了在杆状病毒/昆虫细胞系统中表达的EGF受体重组胞质结构域（Met644 - Ala1186）的高效纯化程序。对该蛋白质的理化特性进行了表征，包括使用远紫外圆二色性研究其等电和流体动力学性质、稳定性、寡聚状态及二级结构。除非先在体外进行自身磷酸化，否则重组蛋白不能被抗磷酸酪氨酸抗体识别。自身磷酸化的重组胞质结构域和EGF刺激的A431膜受体的胰蛋白酶磷酸肽图谱非常相似，表明重组蛋白具有相似的自身磷酸化能力和特异性。这些制剂对肽酪氨酸磷酸化具有高比活性。尽管胞质结构域被分离为均匀的单体蛋白，但在4℃储存会导致缓慢的自发聚集，比活性降低。在0℃以下储存可维持高活性和单体状态。自身磷酸化的初始速率对蛋白质浓度的依赖性与交叉磷酸化一致，但与已知的全受体寡聚化诱导激活不一致。高浓度的Mn2 +、Mg2 +和（NH4）2SO4刺激肽磷酸化活性。使用合成肽研究了（NH4）2SO4激活的底物特异性。在0 - 0.25 M范围内，（NH4）2SO4抑制自身磷酸化，但在1.0 - 1.5 M时激活，这可能分别是离子和疏水蛋白相互作用的结果。在高浓度（NH4）2SO4存在下磷酸化的胞质结构域的磷酸肽图谱表明，该蛋白比无盐时或天然受体时磷酸化程度更高。胞质结构域的远紫外圆二色性光谱在1 M（NH4）2SO4时发生显著变化，增加了（NH4）2SO4通过诱导构象变化激活激酶催化结构域的可能性。
