van de Poll M L, Lenferink A E, van Vugt M J, Jacobs J J, Janssen J W, Joldersma M, van Zoelen E J
Department of Cell Biology, University of Nijmegen, The Netherlands.
J Biol Chem. 1995 Sep 22;270(38):22337-43. doi: 10.1074/jbc.270.38.22337.
The finding that human epidermal growth factor (hEGF) and human transforming growth factor (hTGF) alpha bind with similar affinity to the human EGF receptor but differ in their affinity for the chicken EGF receptor was used as a model system to study ligand-receptor interaction of EGF receptor agonists. We previously constructed domain-exchange mutants of hEGF and hTGF alpha and found that the region COOH-terminal of the sixth cysteine residue in hTGF alpha is important for high affinity binding to the chicken EGF receptor (Kramer, R. H., Lenferink, A. E. G., Lammerts van Bueren-Koornneef, I., van der Meer, A., van de Poll, M. L. M., and van Zoelen, E. J. J. (1994) J. Biol. Chem. 269, 8708-8711). To analyze this domain in more detail, we now constructed four additional chimeras in which either the region between the sixth cysteine residue and the highly conserved Leu-47 was exchanged or the region COOH-terminal of Leu-47. A mutant in which the latter region in hEGF was replaced by hTGF alpha (designated E6ET) showed intermediate binding affinity, whereas replacement of the former region in hEGF by hTGF alpha was sufficient to generate a mutant (designated E6TE) with a similar high affinity for the chicken EGF receptor as wild type hTGF alpha. Furthermore, a deletion mutant of hEGF lacking three COOH-terminal amino acids, EGF50, showed intermediate binding affinity for the chicken EGF receptor similar to E6ET, but upon additional deletions (EGF49 and EGF48), this initial gain in affinity was lost. A systematic analysis of the region between the sixth cysteine residue and Leu-47 showed that the low affinity of hEGF for the chicken EGF receptor is mainly due to the presence of Arg-45. Replacement of the positively charged Arg-45 by Ala, the corresponding amino acid in hTGF alpha, was sufficient to generate a mutant growth factor with high affinity for the chicken EGF receptor. This indicates that in hEGF Arg-45 may play an important role in receptor binding. A model is proposed in which positively charged amino acids close to or within the receptor recognition site of hEGF prohibit high affinity binding to the chicken EGF receptor due to electrostatic repulsion of positively charged amino acids in the putative ligand binding domain of the chicken EGF receptor.
人表皮生长因子（hEGF）和人转化生长因子（hTGF）α与人类表皮生长因子受体（EGF受体）具有相似的结合亲和力，但它们对鸡EGF受体的亲和力不同，这一发现被用作研究EGF受体激动剂配体 - 受体相互作用的模型系统。我们之前构建了hEGF和hTGFα的结构域交换突变体，发现hTGFα中第六个半胱氨酸残基的羧基末端区域对于与鸡EGF受体的高亲和力结合很重要（Kramer, R. H., Lenferink, A. E. G., Lammerts van Bueren-Koornneef, I., van der Meer, A., van de Poll, M. L. M., and van Zoelen, E. J. J. (1994) J. Biol. Chem. 269, 8708 - 8711）。为了更详细地分析该结构域，我们现在构建了另外四个嵌合体，其中要么交换了第六个半胱氨酸残基和高度保守的Leu - 47之间的区域，要么交换了Leu - 47的羧基末端区域。hEGF中后一个区域被hTGFα取代的突变体（命名为E6ET）表现出中等结合亲和力，而hEGF中前一个区域被hTGFα取代足以产生一个对鸡EGF受体具有与野生型hTGFα相似高亲和力的突变体（命名为E6TE）。此外，缺失三个羧基末端氨基酸的hEGF缺失突变体EGF50对鸡EGF受体表现出与E6ET相似的中等结合亲和力，但在进一步缺失（EGF49和EGF48）后，这种最初的亲和力增加消失了。对第六个半胱氨酸残基和Leu - 47之间区域的系统分析表明，hEGF对鸡EGF受体的低亲和力主要是由于Arg - 45的存在。将带正电荷的Arg - 45替换为hTGFα中的相应氨基酸Ala，足以产生对鸡EGF受体具有高亲和力的突变生长因子。这表明在hEGF中Arg - 45可能在受体结合中起重要作用。提出了一个模型，其中hEGF受体识别位点附近或内部的带正电荷氨基酸由于与鸡EGF受体假定配体结合域中带正电荷氨基酸的静电排斥而阻止与鸡EGF受体的高亲和力结合。
