Gadella T W, Jovin T M
Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry, Goettingen, Federal Republic of Germany.
J Cell Biol. 1995 Jun;129(6):1543-58. doi: 10.1083/jcb.129.6.1543.
The aggregation states of the epidermal growth factor receptor (EGFR) on single A431 human epidermoid carcinoma cells were assessed with two new techniques for determining fluorescence resonance energy transfer: donor photobleaching fluorescence resonance energy transfer (pbFRET) microscopy and fluorescence lifetime imaging microscopy (FLIM). Fluorescein-(donor) and rhodamine-(acceptor) labeled EGF were bound to the cells and the extent of oligomerization was monitored by the spatially resolved FRET efficiency as a function of the donor/acceptor ratio and treatment conditions. An average FRET efficiency of 5% was determined after a low temperature (4 degrees C) incubation with the fluorescent EGF analogs for 40 min. A subsequent elevation of the temperature for 5 min caused a substantial increase of the average FRET efficiency to 14% at 20 degrees C and 31% at 37 degrees C. In the context of a two-state (monomer/dimer) model for the EGFR, these FRET efficiencies were consistent with minimal average receptor dimerizations of 13, 36, and 69% at 4, 20, and 37 degrees C, respectively. A431 cells were pretreated with the monoclonal antibody mAb 2E9 that specifically blocks EGF binding to the predominant population of low affinity EGFR (15). The average FRET efficiency increased dramatically to 28% at 4 degrees C, indicative of a minimal receptor dimerization of 65% for the subpopulation of high affinity receptors. These results are in accordance with prior studies indicating that binding of EGF leads to a fast and temperature-dependent microclustering of EGFR, but suggest in addition that the high affinity functional subclass of receptors on quiescent A431 cells are present in a predimerized or oligomerized state. We propose that the transmission of the external ligand-binding signal to the cytoplasmic domain is effected by a concerted relative rotational rearrangement of the monomeric units comprising the dimeric receptor, thereby potentiating a mutual activation of the tyrosine kinase domains.
采用两种测定荧光共振能量转移的新技术，即供体光漂白荧光共振能量转移（pbFRET）显微镜技术和荧光寿命成像显微镜技术（FLIM），评估了单个A431人表皮样癌细胞上表皮生长因子受体（EGFR）的聚集状态。将荧光素（供体）和罗丹明（受体）标记的表皮生长因子（EGF）与细胞结合，并根据供体/受体比例和处理条件，通过空间分辨的FRET效率监测寡聚化程度。在低温（4℃）下与荧光EGF类似物孵育40分钟后，测定平均FRET效率为5%。随后将温度升高5分钟，导致平均FRET效率在20℃时大幅增加至14%，在37℃时增加至31%。在EGFR的双态（单体/二聚体）模型背景下，这些FRET效率分别与4℃、20℃和37℃时最小平均受体二聚化率13%、36%和69%一致。用特异性阻断EGF与主要低亲和力EGFR群体结合的单克隆抗体mAb 2E9预处理A431细胞。在4℃时，平均FRET效率急剧增加至28%，表明高亲和力受体亚群的最小受体二聚化率为65%。这些结果与先前的研究一致，即EGF的结合导致EGFR快速且温度依赖性的微簇形成，但此外还表明，静止A431细胞上受体的高亲和力功能亚类以预二聚化或寡聚化状态存在。我们提出，外部配体结合信号向细胞质结构域的传递是由构成二聚体受体的单体单元协同相对旋转重排实现的，从而增强酪氨酸激酶结构域的相互激活。
