Structure-function studies of ligand-induced epidermal growth factor receptor dimerization
Structure-function studies of ligand-induced epidermal growth factor receptor dimerization
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DOI：
10.1021/bi972548x
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发表时间：
1998-04-07
期刊：
影响因子：
2.9
通讯作者：
Davies, DE
中科院分区：
文献类型：
--
作者：
Neelam, B;Richter, A;Davies, DE
我们提出了一种新的96孔分析，我们已应用于表皮生长因子受体二聚化的结构-功能研究。该测定的基础在于当EGFR在同源配体存在下固定时，EGFR被二价抗体捕获为二聚体的可能性增加。一旦固定，抗体就充当系链，将受体保持在其二聚体状态，从而使放射性标记的配体探针的结合增加5-7倍。当该测定应用于EGF配体家族的成员时，鼠EGF、转化生长因子α和肝素结合EGF样生长因子与人EGF（EC 50 = 2nM）相当;具有更广泛受体特异性的β细胞素的效果略差。相反，双调蛋白（AR（1-84））具有截短的C-尾并且缺乏保守的亮氨酸残基，除非以>1 μ M使用，否则是无效的。我们通过比较两种遗传修饰的EGF（嵌合体mEGF/TGF α（44-50）和EGF点突变体L47 A）和AR的C末端延伸形式（AR（1-90））与其他两种无关的EGF突变体（I23 T和L15 A）的活性，进一步探索了C-尾和保守的亮氨酸残基在受体二聚化中的参与。这些配体的效力的顺序为EGF > I23 T> mEGF/TGF α（44-50）> L47 A = L15 A，远大于AR（1-90）> AR（1-84）。虽然AR比从其亲和力预测的要差得多，但这种缺陷可以通过将固定化抗体与肝素共定位来部分纠正。因此，AR似乎不能使EGFR二聚化，除非存在其他辅助分子以稳定其与EGFR的功能性缔合。
We present a novel 96-well assay which we have applied to a structure-function study of epidermal growth factor receptor dimerization. The basis of the assay lies in the increased probability of EGFRs being captured as dimers by a bivalent antibody when they are immobilized in the presence of a cognate ligand. Once immobilized, the antibody acts as a tether, retaining the receptor in its dimeric state with a resultant 5-7-fold increase in binding of a radiolabeled ligand probe. When the assay was applied to members of the EGF ligand family, murine EGF, transforming growth factor alpha, and heparin-binding EGF-like growth factor were comparable with human EGF (EC50 = 2nM); betacellulin, which has a broader receptor specificity, was slightly less effective. In contrast, amphiregulin (AR(1-84)), which has a truncated C-tail and lacks a conserved leucine residue, was ineffective unless used at >1 mu M. We further probed the involvement of the C-tail and the conserved leucine residue in receptor dimerization by comparing the activities of two genetically modified EGFs (the chimera mEGF/TGF alpha(44-50) and the EGF point mutant L47A) and a C-terminally extended form of AR (AR(1-90)) with those of two other unrelated EGF mutants (I23T and L15A). The potency of these ligands was in the order EGF > I23T > mEGF/TGF alpha(44-50) > L47A = L15A much greater than AR(1-90) > AR(1-84). Although AR was much worse than predicted from its affinity, this defect could be partially rectified by co-localization of the immobilizing antibody with heparin. Thus, it seems likely that AR cannot dimerize the EGFR unless other accessory molecules are present to stabilize its functional association with the EGFR.