EGF signalling in epithelial carcinoma cells utilizes preformed receptor homoclusters, with larger heteroclusters post activation
EGF signalling in epithelial carcinoma cells utilizes preformed receptor homoclusters, with larger heteroclusters post activation
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DOI：
10.1101/305292
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发表时间：
2018-04
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通讯作者：
C. Fournier;A. Wollman;I. Llorente-Garcia;Oliver L Harriman;Djamila Ouarat;J. Wilding;W. Bodmer;M. Leake
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作者：
C. Fournier;A. Wollman;I. Llorente-Garcia;Oliver L Harriman;Djamila Ouarat;J. Wilding;W. Bodmer;M. Leake
表皮生长因子 (EGF) 信号传导调节上皮细胞的生长、分化和增殖，据报道，EGF 受体 (EGFR) 在多种癌症类型中过度表达。结构和生化证据表明 EGF 结合刺激 EGFR 单体-二聚体转变，激活下游信号传导。然而，配体与活细胞中功能受体结合的机制细节仍然存在争议。我们报告了用 GFP 标记的 EGFR 转染的人上皮癌细胞（天然 EGFR 表达可忽略不计）在用 TMR 标记的 EGF 配体激活受体之前和之后的实时单分子 TIRF。以 40 nm 精度同时追踪荧光标记的 EGFR 和 EGF，以探索 EGF 结合时的化学计量和时空动力学。使用直接阻断与 EGFR 结合或通过 HER2 间接结合的抑制剂，我们的结果表明，预激活的 EGFR 由预先形成的同源簇组成，而包括 HER2 在内的较大异源簇在激活后形成。结合后 EGFR 与 EGF 的相对化学计量在 2 处达到峰值，表明 EGFR 激活的负协同性。
Epidermal growth factor (EGF) signalling regulates cell growth, differentiation and proliferation in epithelium and EGF receptor (EGFR) overexpression has been reported in several carcinoma types. Structural and biochemical evidence suggests EGF binding stimulates EGFR monomer-dimer transitions, activating downstream signalling. However, mechanistic details of ligand binding to functional receptors in live cells remain contentious. We report real time single-molecule TIRF of human epithelial carcinoma cells with negligible native EGFR expression, transfected with GFP-tagged EGFR, before and after receptor activation with TMR-labelled EGF ligand. Fluorescently labelled EGFR and EGF are simultaneously tracked to 40nm precision to explore stoichiometry and spatiotemporal dynamics upon EGF binding. Using inhibitors that block binding to EGFR directly, or indirectly through HER2, our results indicate that pre-activated EGFR consists of preformed homoclusters, while larger heteroclusters including HER2 form upon activation. The relative stoichiometry of EGFR to EGF after binding peaks at 2, indicating negative cooperativity of EGFR activation.