Klein O, Kegler-Ebo D, Su J, Smith S, DiMaio D
Department of Genetics, Yale University School of Medicine, New Haven, Connecticut 06510, USA.
J Virol. 1999 Apr;73(4):3264-72. doi: 10.1128/JVI.73.4.3264-3272.1999.
The bovine papillomavirus E5 gene encodes a 44-amino-acid, homodimeric transmembrane protein that is the smallest known transforming protein. The E5 protein transforms cultured fibroblasts by forming a stable complex with the endogenous platelet-derived growth factor (PDGF) beta receptor through transmembrane and juxtamembrane interactions, leading to sustained receptor activation. Aspartic acid 33 in the extracellular juxtamembrane region of the E5 protein is important for cell transformation and interaction with the PDGF beta receptor. A. N. Meyer et al. (Proc. Natl. Acad. Sci USA 91:4634-4638, 1994) speculated that this residue interacted with lysine 499 on the receptor. We constructed E5 mutants containing all possible substitutions at position 33, as well as several double mutants containing substitutions at aspartic acid 33 and at glutamic acid 36, and we examined the ability of these mutants to transform C127 mouse fibroblasts and to bind to and induce activation of the PDGF beta receptor. There was an excellent correlation between the transformation activities of the various mutants and their ability to bind to and activate the PDGF beta receptor. Analysis of the mutants demonstrated that a juxtamembrane negative charge on the E5 protein was required for cell transformation and for productive interaction with the PDGF beta receptor and indicated that aspartic acid 33 was more important for these activities than was glutamic acid 36. These results are consistent with the existence of an essential juxtamembrane salt bridge between lysine 499 on the PDGF beta receptor and an acidic residue in the C terminus of the E5 protein and lend support to our proposed model for the complex between the E5 dimer and the PDGF beta receptor.
牛乳头瘤病毒E5基因编码一种由44个氨基酸组成的同二聚体跨膜蛋白，它是已知最小的转化蛋白。E5蛋白通过跨膜和近膜相互作用与内源性血小板衍生生长因子（PDGF）β受体形成稳定复合物，从而转化培养的成纤维细胞，导致受体持续激活。E5蛋白细胞外近膜区域的天冬氨酸33对于细胞转化以及与PDGFβ受体的相互作用很重要。A. N. 迈耶等人（《美国国家科学院院刊》91:4634 - 4638, 1994）推测该残基与受体上的赖氨酸499相互作用。我们构建了在第33位含有所有可能替代的E5突变体，以及几个在天冬氨酸33和谷氨酸36处含有替代的双突变体，并检测了这些突变体转化C127小鼠成纤维细胞以及结合并诱导PDGFβ受体激活的能力。各种突变体的转化活性与其结合并激活PDGFβ受体的能力之间存在极好的相关性。对突变体的分析表明，E5蛋白近膜区域的负电荷对于细胞转化以及与PDGFβ受体的有效相互作用是必需的，并且表明天冬氨酸33对于这些活性比谷氨酸36更重要。这些结果与PDGFβ受体上的赖氨酸499与E5蛋白C末端的酸性残基之间存在必需的近膜盐桥相一致，并支持了我们提出的E5二聚体与PDGFβ受体之间复合物的模型。
