Helin K, Velu T, Martin P, Vass W C, Allevato G, Lowy D R, Beguinot L
Microbiology Institute, University of Copenhagen, Denmark.
Oncogene. 1991 May;6(5):825-32.
The epidermal growth factor receptor (EGF-R) C-terminus contains three conserved tyrosines (Y-1068, Y-1148, Y-1173) which are phosphorylated upon EGF activation. To clarify the functional role of these tyrosines, each has been mutated to phenylalanine and studied as single, double and triple mutants in the full length receptor. EGF-dependent transforming ability of the single point mutants is similar to that of the wild type, while that of double mutants is decreased and an even lower activity is present in the triple mutant. In each bioassay, including EGF-dependent focal transformation, growth in agar and growth in low serum, mutant receptors display a similar hierarchy of activity. The lower activity is intrinsic in the mutants since they are expressed at similar level as the wild type and bind EGF with similar affinity. Deletion mutants lacking the last 19 or 63 amino acids (Velu et al., 1989a) show a similar decline in biological activity when compared to the corresponding point mutants, although the reduction is more pronounced than with the point mutants. Deletion of the last 123 aa, which removes all three tyrosines (Dc123), results in a receptor that is almost inactive biologically. The EGF-R kinase activity is affected by tyrosine substitution since in vitro phosphorylation of exogenous substrates is reduced in the double and triple mutants. Autophosphorylation, in vivo and in vitro, is also reduced, but not totally abolished in the triple point mutant and Dc123 indicating the existence of other autophosphorylation sites. A new site of autophosphorylation is found in the Dc123 mutant. We conclude, therefore, that the tyrosines at the extreme C-terminus positively regulate the biological and transforming activity of the EGF-R, probably via autophosphorylation.
表皮生长因子受体（EGF-R）的C末端包含三个保守的酪氨酸（Y-1068、Y-1148、Y-1173），它们在EGF激活后会被磷酸化。为了阐明这些酪氨酸的功能作用，每个酪氨酸都被突变为苯丙氨酸，并在全长受体中作为单突变体、双突变体和三突变体进行研究。单点突变体的EGF依赖性转化能力与野生型相似，而双突变体的转化能力降低，三突变体的活性更低。在包括EGF依赖性灶性转化、琼脂中生长和低血清中生长的每种生物测定中，突变受体都表现出相似的活性等级。较低的活性是突变体固有的，因为它们的表达水平与野生型相似，并且与EGF的结合亲和力也相似。与相应的点突变体相比，缺失最后19或63个氨基酸的缺失突变体（Velu等人，1989a）的生物活性也有类似的下降，尽管这种下降比点突变体更明显。缺失最后123个氨基酸（Dc123），从而去除了所有三个酪氨酸，导致一种在生物学上几乎无活性的受体。EGF-R激酶活性受到酪氨酸替代的影响，因为在双突变体和三突变体中外源底物的体外磷酸化减少。体内和体外的自磷酸化也减少，但在三突变体和Dc123中并未完全消除，这表明存在其他自磷酸化位点。在Dc123突变体中发现了一个新的自磷酸化位点。因此，我们得出结论，极端C末端的酪氨酸可能通过自磷酸化正向调节EGF-R的生物学和转化活性。
