Centre for Micro-Photonics, Faculty of Engineering and Industrial Sciences, Swinburne University of Technology , Hawthorn, Victoria 3122, Australia.
Biochemistry. 2014 Apr 29;53(16):2594-604. doi: 10.1021/bi500182x. Epub 2014 Apr 21.
Adaptor protein Grb2 binds phosphotyrosines in the epidermal growth factor (EGF) receptor (EGFR) and thereby links receptor activation to intracellular signaling cascades. Here, we investigated how recruitment of Grb2 to EGFR is affected by the spatial organization and quaternary state of activated EGFR. We used the techniques of image correlation spectroscopy (ICS) and lifetime-detected Förster resonance energy transfer (also known as FLIM-based FRET or FLIM-FRET) to measure ligand-induced receptor clustering and Grb2 binding to activated EGFR in BaF/3 cells. BaF/3 cells were stably transfected with fluorescently labeled forms of Grb2 (Grb2-mRFP) and EGFR (EGFR-eGFP). Following stimulation of the cells with EGF, we detected nanometer-scale association of Grb2-mRFP with EGFR-eGFP clusters, which contained, on average, 4 ± 1 copies of EGFR-eGFP per cluster. In contrast, the pool of EGFR-eGFP without Grb2-mRFP had an average cluster size of 1 ± 0.3 EGFR molecules per punctum. In the absence of EGF, there was no association between EGFR-eGFP and Grb2-mRFP. To interpret these data, we extended our recently developed model for EGFR activation, which considers EGFR oligomerization up to tetramers, to include recruitment of Grb2 to phosphorylated EGFR. The extended model, with adjustment of one new parameter (the ratio of the Grb2 and EGFR copy numbers), is consistent with a cluster size distribution where 2% of EGFR monomers, 5% of EGFR dimers, <1% of EGFR trimers, and 94% of EGFR tetramers are associated with Grb2. Together, our experimental and modeling results further implicate tetrameric EGFR as the key signaling unit and call into question the widely held view that dimeric EGFR is the predominant signaling unit.
衔接蛋白 Grb2 与表皮生长因子（EGF）受体（EGFR）的磷酸酪氨酸结合，从而将受体激活与细胞内信号级联联系起来。在这里，我们研究了 Grb2 与 EGFR 的募集如何受到激活的 EGFR 的空间组织和四级状态的影响。我们使用图像相关光谱（ICS）和寿命检测的Förster 共振能量转移（也称为基于 FLIM 的 FRET 或 FLIM-FRET）技术来测量配体诱导的受体聚类和 Grb2 与 BaF/3 细胞中激活的 EGFR 的结合。BaF/3 细胞稳定转染了荧光标记形式的 Grb2（Grb2-mRFP）和 EGFR（EGFR-eGFP）。在用 EGF 刺激细胞后，我们检测到 Grb2-mRFP 与 EGFR-eGFP 簇的纳米级关联，每个簇平均包含 4±1 个 EGFR-eGFP。相比之下，没有 Grb2-mRFP 的 EGFR-eGFP 池的平均簇大小为每个斑点 1±0.3 个 EGFR 分子。在没有 EGF 的情况下，EGFR-eGFP 与 Grb2-mRFP 之间没有关联。为了解释这些数据，我们扩展了我们最近开发的 EGFR 激活模型，该模型考虑了 EGFR 寡聚化至四聚体，以包括 Grb2 对磷酸化 EGFR 的募集。扩展模型，通过调整一个新参数（Grb2 和 EGFR 拷贝数的比值），与簇大小分布一致，其中 2%的 EGFR 单体、5%的 EGFR 二聚体、<1%的 EGFR 三聚体和 94%的 EGFR 四聚体与 Grb2 相关。总之，我们的实验和建模结果进一步表明四聚体 EGFR 是关键的信号单元，并对广泛持有的观点提出质疑，即二聚体 EGFR 是主要的信号单元。
