Effect of phosphorylation on EGFR dimer stability probed by single-molecule dynamics and FRET/FLIM.
Effect of phosphorylation on EGFR dimer stability probed by single-molecule dynamics and FRET/FLIM.
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DOI：
10.1016/j.bpj.2015.01.005
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发表时间：
2015-03-10
影响因子：
3.4
通讯作者：
Ng, Tony
中科院分区：
文献类型：
--
作者：
Coban, Oana;Zanetti-Dominguez, Laura C.;Matthews, Daniel R.;Rolfe, Daniel J.;Weitsman, Gregory;Barber, Paul R.;Barbeau, Jody;Devauges, Viviane;Kampmeier, Florian;Winn, Martyn;Vojnovic, Borivoj;Parker, Peter J.;Lidke, Keith A.;Lidke, Diane S.;Ameer-Beg, Simon M.;Martin-Fernandez, Marisa L.;Ng, Tony
表皮生长因子受体（EGFR）信号的失调与多种人类癌症的发展有关。egf诱导的受体二聚化和随后的反式自磷酸化是信号转导中最早的事件之一。EGF的结合被认为可以诱导构象变化，从而间接地展开二聚化所需的外域环。它也可能在细胞质域引起重要的变构变化。尽管对EGFR的生理激活有广泛的了解，但靶向治疗对受体构象的影响尚不清楚，受体功能的这一特殊方面可能受到药物治疗的影响，这可能部分解释了癌症患者的异质临床反应。在这里，我们使用Förster共振能量转移/荧光寿命成像显微镜（FRET/FLIM）结合双色单分子跟踪来研究atp竞争小分子酪氨酸激酶抑制剂（TKIs）和基于磷酸酶的EGFR磷酸化操作对活细胞的影响。二聚体的接通时间分布拟合为单指数，提取二聚体的接通率（koff）。我们的数据表明，用吉非替尼（活性构象粘合剂）预处理可以稳定EGFR配体结合的同型二聚体。egfr特异性DEP-1磷酸酶的过表达也被发现对同型二聚体具有稳定作用。当使用抗egfr抗体（425 Snap单链可变片段）时，可以检测到二聚体的koff没有显着差异，该抗体允许配体结合受体的二聚化，但不能磷酸化。这些结果表明，细胞外结构域的构象和受体的磷酸化状态都参与调节二聚体的稳定性。这两个EGFR亚群（相互作用与自由）的相对分数是通过对集合FRET/FLIM图像的分数强度分析获得的。我们的联合成像方法显示，在吉非替尼预处理或DEP-1磷酸酶过表达后，分数和亲和力（单分子水平的构象替代品）都增加了。利用干扰EGFR在细胞内二聚能力的EGFR突变（I706Q, V948R），我们发现适度的药物诱导的EGFR同型二聚体的分数/稳定性的增加可能对肿瘤细胞的增殖潜力有显著的生物学影响。
Deregulation of epidermal growth factor receptor (EGFR) signaling has been correlated with the development of a variety of human carcinomas. EGF-induced receptor dimerization and consequent trans- auto-phosphorylation are among the earliest events in signal transduction. Binding of EGF is thought to induce a conformational change that consequently unfolds an ectodomain loop required for dimerization indirectly. It may also induce important allosteric changes in the cytoplasmic domain. Despite extensive knowledge on the physiological activation of EGFR, the effect of targeted therapies on receptor conformation is not known and this particular aspect of receptor function, which can potentially be influenced by drug treatment, may in part explain the heterogeneous clinical response among cancer patients. Here, we used Förster resonance energy transfer/fluorescence lifetime imaging microscopy (FRET/FLIM) combined with two-color single-molecule tracking to study the effect of ATP-competitive small molecule tyrosine kinase inhibitors (TKIs) and phosphatase-based manipulation of EGFR phosphorylation on live cells. The distribution of dimer on-times was fitted to a monoexponential to extract dimer off-rates (koff). Our data show that pretreatment with gefitinib (active conformation binder) stabilizes the EGFR ligand-bound homodimer. Overexpression of EGFR-specific DEP-1 phosphatase was also found to have a stabilizing effect on the homodimer. No significant difference in the koff of the dimer could be detected when an anti-EGFR antibody (425 Snap single-chain variable fragment) that allows for dimerization of ligand-bound receptors, but not phosphorylation, was used. These results suggest that both the conformation of the extracellular domain and phosphorylation status of the receptor are involved in modulating the stability of the dimer. The relative fractions of these two EGFR subpopulations (interacting versus free) were obtained by a fractional-intensity analysis of ensemble FRET/FLIM images. Our combined imaging approach showed that both the fraction and affinity (surrogate of conformation at a single-molecule level) increased after gefitinib pretreatment or DEP-1 phosphatase overexpression. Using an EGFR mutation (I706Q, V948R) that perturbs the ability of EGFR to dimerize intracellularly, we showed that a modest drug-induced increase in the fraction/stability of the EGFR homodimer may have a significant biological impact on the tumor cell’s proliferation potential.
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DOI：
10.1083/jcb.109.5.2495
发表时间：
1989-11
期刊：
The Journal of cell biology
影响因子：
--
作者：
Defize LH;Boonstra J;Meisenhelder J;Kruijer W;Tertoolen LG;Tilly BC;Hunter T;van Bergen en Henegouwen PM;Moolenaar WH;de Laat SW
通讯作者：
de Laat SW
影响因子：
8
作者：
Hartman, Z.;Zhao, H.;Agazie, Y. M.
通讯作者：
Agazie, Y. M.
缺乏血型A抗原显示的A431细胞变体增加了高亲和力表皮生长因子受体数，蛋白质酪氨酸激酶活性和受体更新。
影响因子：
7.8
作者：
Defize, L H;Arndt-Jovin, D J;Jovin, T M;Boonstra, J;Meisenhelder, J;Hunter, T;de Hey, H T;de Laat, S W
通讯作者：
de Laat, S W
影响因子：
11.5
作者：
Hickinson, D. Mark;Klinowska, Teresa;Ogilvie, Donald
通讯作者：
Ogilvie, Donald
影响因子：
16
作者：
Garrett, TPJ;McKern, NM;Ward, CW
通讯作者：
Ward, CW