The epidermal growth factor receptor (EGFR) is a membrane protein that regulates cell proliferation, differentiation and survival, and is a drug target for cancer therapy. Ligand-induced activation of the EGFR kinase is generally regarded to require ligand-bound-dimers, while phosphorylation and down-stream signalling is modulated by oligomers. Recent work has unveiled changes in EGFR dynamics from ligand-induced dimerization in membranes extracted from cells, however, less is known about the changes in EGFR dynamics that accompany the ligand-induced oligomerization in a live cell environment. Here, we determine the dynamics of a c-terminal GFP tag attached to EGFR in the unliganded dimer and in the liganded oligomers. By means of the single-frequency polarized phasor ellipse approach we extracted two correlation times on the sub-nanosecond and super-nanosecond timescales, respectively. EGF binding to the EGFR-GFP dimer lengthened the sub-nanosecond correlation time (from 0.1 to 1.3 ns) and shortened the super-nanosecond correlation time (from 210 to 56 ns) of the c-terminal GFP probe. The sub-nanosecond depolarization processes were assigned to electronic energy migration between proximal GFPs in the EGFR dimer or oligomer, while the super-nanosecond correlation times were assigned to nanosecond fluctuations of the GFP probe in the EGFR complex. Accordingly, these results show that ligand binding increased the average separation between the c-terminal tags and increased their rotational mobility. We propose that the dynamics are linked to an inhibitory function of the c-terminal tail in the un-liganded dimer and to the requirement of facile stochastic switching between kinase activation and cytoplasmic adaptor/effector binding in the active oligomers.

中文翻译：

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配体诱导的二聚体-寡聚体转变过程中 EGF 受体的细胞内结构动力学。

表皮生长因子受体（EGFR）是一种调节细胞增殖、分化和存活的膜蛋白，是癌症治疗的药物靶点。配体诱导的 EGFR 激酶激活通常被认为需要配体结合的二聚体，而磷酸化和下游信号传导由寡聚体调节。最近的工作揭示了从细胞中提取的膜中配体诱导二聚化导致 EGFR 动力学的变化，但是，关于在活细胞环境中伴随配体诱导寡聚化的 EGFR 动力学变化知之甚少。在这里，我们确定了在非配体二聚体和配体寡聚体中连接到 EGFR 的 c 末端 GFP 标签的动力学。通过单频极化相量椭圆方法，我们分别在亚纳秒和超纳秒时间尺度上提取了两个相关时间。EGF 与 EGFR-GFP 二聚体的结合延长了亚纳秒相关时间（从 0.1 到 1.3 ns）并缩短了 c 端 GFP 探针的超纳秒相关时间（从 210 到 56 ns）。亚纳秒去极化过程被指定为 EGFR 二聚体或寡聚体中近端 GFP 之间的电子能量迁移，而超纳秒相关时间被指定为 EGFR 复合物中 GFP 探针的纳秒波动。因此，这些结果表明配体结合增加了 c 端标签之间的平均间隔并增加了它们的旋转迁移率。