Walker F, Hibbs M L, Zhang H H, Gonez L J, Burgess A W
Ludwig Institute for Cancer Research, Melbourne Tumor Biology Branch, Victoria, Australia.
Growth Factors. 1998;16(1):53-67. doi: 10.3109/08977199809017491.
The Epidermal Growth Factor (EGF) receptor appears to require a fully active tyrosine kinase domain to transmit mitogenic signals. However, waved-2 mice carrying a mutation in the alpha-helix C of their EGF-R, which abolishes tyrosine kinase activity, only display a mild phenotype and are fully viable. This suggests that the mutant EGF-R signals through heterodimerization with endogenous, kinase active members of the EGF-R family such as ErbB-2 or ErbB-4. We have examined the biochemistry of EGF-Rs carrying mutations in the alpha-helix C of the human EGF-R (V741G and Y740F), in the ATP binding site (K721R) and at the C-terminus (CT957), by expression in BaF/3 cells which are devoid of EGF-R family members. The in vitro kinase activity of the alpha-helix C EGF-R mutants was severely impaired as a result of reduced phosphotransfer activity without appreciable changes in the affinity for either ATP or peptide substrate. Surprisingly, EGF stimulation of cells carrying the different mutant or wild type EGF-Rs resulted in tyrosine phosphorylation of EGF-R proteins; this phosphorylation was abolished in crude plasma membrane preparations, and appears to be due to activation of a membrane-associated or a cytosolic kinase. Receptor-mediated internalization of EGF was profoundly suppressed in the V741G, K721R and CT957 receptor mutant, and high affinity EGF binding was undetectable in the V741G and K721R receptors. We conclude that specific residues in the C-helix of the EGF-R kinase are essential for full kinase activity; mutations in this region do not affect ATP binding, but impair the receptors' phosphotransfer ability. High affinity binding of EGF is not dependent on tyrosine kinase activity or sequences in the C-terminus.
表皮生长因子（EGF）受体似乎需要一个完全活跃的酪氨酸激酶结构域来传递促有丝分裂信号。然而，携带EGF-R的α螺旋C突变（该突变消除了酪氨酸激酶活性）的waved-2小鼠仅表现出轻微的表型，并且完全可存活。这表明突变的EGF-R通过与EGF-R家族的内源性激酶活性成员（如ErbB-2或ErbB-4）异源二聚化来发出信号。我们通过在缺乏EGF-R家族成员的BaF/3细胞中表达，研究了携带人EGF-R的α螺旋C突变（V741G和Y740F）、ATP结合位点突变（K721R）以及C末端突变（CT957）的EGF-R的生物化学性质。由于磷酸转移活性降低，α螺旋C EGF-R突变体的体外激酶活性严重受损，而对ATP或肽底物的亲和力没有明显变化。令人惊讶的是，用EGF刺激携带不同突变型或野生型EGF-R的细胞会导致EGF-R蛋白的酪氨酸磷酸化；这种磷酸化在粗制质膜制剂中被消除，并且似乎是由于膜相关或胞质激酶的激活。在V741G、K721R和CT957受体突变体中，EGF的受体介导内化被显著抑制，并且在V741G和K721R受体中未检测到高亲和力EGF结合。我们得出结论，EGF-R激酶C螺旋中的特定残基对于完全激酶活性至关重要；该区域的突变不影响ATP结合，但损害受体的磷酸转移能力。EGF的高亲和力结合不依赖于酪氨酸激酶活性或C末端的序列。
