Unligated epidermal growth factor receptor forms higher order oligomers within microclusters on A431 cells that are sensitive to tyrosine kinase inhibitor binding
Unligated epidermal growth factor receptor forms higher order oligomers within microclusters on A431 cells that are sensitive to tyrosine kinase inhibitor binding
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DOI：
10.1021/bi700002b
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发表时间：
2007-04-17
期刊：
影响因子：
2.9
通讯作者：
Johns, Terrance G.
中科院分区：
文献类型：
--
作者：
Clayton, Andrew H. A.;Tavarnesi, Maria L.;Johns, Terrance G.
未连接的表皮生长因子受体（EGFR）的结合状态的表征对于了解肿瘤细胞环境中EGFR酪氨酸激酶激活的机制非常重要。我们详细分析了完整表皮样癌A431细胞表面未连接的免疫标记EGFR的缔合状态，使用AlexaFluor 488和AlexaFluor 546抗EGFR抗体，mAb 528作为探针。图像相关显微镜显示，在亚微米级簇中存在未连接的EGFR，平均含有10-30个受体（平均簇密度= 32 +/-9簇/平方微米）。基于寿命的福斯特共振能量转移（FRET）技术作为受体：供体标记比率的函数，公开了未连接的EGFR在纳米（< 10 nm）尺度上平均含有四个受体的簇中的聚集。纳米级和亚微米级协会之间的关系，确定使用一种新的分析，结合纳米级的信息，从寿命检测FRET成像与亚微米级的图像相关显微镜获得的信息。该分析揭示了单体（或小寡聚体）和含有15-30个受体的较大簇的存在，所述受体在亚10 nm尺度上部分缔合。用酪氨酸激酶抑制剂AG 1478预处理细胞引起亚微米簇的部分分散（平均簇密度= 85 +/-15簇/平方微米;平均缔合度= 4-10个受体/簇）并将FRET水平降低至我们的检测极限。这些结果与部分由激酶结构域控制的未连接受体群体的高阶纳米级受体组织一致。的结果，EGFR在肿瘤细胞环境中的激活机制的分歧进行了讨论。
Characterization of the association states of the unligated epidermal growth factor receptor (EGFR) is important in understanding the mechanism of EGFR tyrosine kinase activation in a tumor cell environment. We analyzed, in detail, the association states of unligated, immunotagged EGFR on the surface of intact epidermoid carcinoma A431 cells, using AlexaFluor488 and AlexaFluor546 anti-EGFR antibody, mAb528, as probes. Image correlation microscopy revealed the presence of unligated EGFR in submicron scale clusters containing an average of 10-30 receptors (mean cluster density = 32 +/- 9 clusters per square micron). Lifetime-based Forster resonance energy transfer (FRET) techniques as a function of acceptor:donor labeling ratio disclosed a clustering of the unligated EGFR in clusters containing an average of four receptors on the nanometer (< 10 nm) scale. The relationship between the nanoscale and submicron scale associations was determined using a new analysis that combines nanoscale information from lifetime-detected FRET imaging with submicron scale information obtained with image correlation microscopy. This analysis revealed the presence of monomers (or small oligomers) and larger clusters containing 15-30 receptors that were partially associated on the sub-10 nm scale. Pretreatment of the cells with the tyrosine kinase inhibitor AG1478 caused a partial dispersal of the submicron clusters (mean cluster density = 85 +/- 15 clusters per square micron; mean degree of association = 4-10 receptors per cluster) and reduced the level of FRET down to our limit of detection. These results are consistent with a higher order nanoscale receptor organization of the unligated receptor population that is partially controlled by the kinase domains. The ramifications of the results to mechanisms of EGFR activation in a tumor cell environment are discussed.