Basic study on SH2 domain of Grb2 as a molecular probe for detection of RTK activation.
Basic study on SH2 domain of Grb2 as a molecular probe for detection of RTK activation.
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DOI：
10.3892/ijo_00000676
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发表时间：
2010-08
影响因子：
5.2
通讯作者：
Yuriko Saito;T. Furukawa;Y. Arano;Y. Fujibayashi;T. Saga
中科院分区：
文献类型：
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作者：
Yuriko Saito;T. Furukawa;Y. Arano;Y. Fujibayashi;T. Saga
表皮生长因子受体（EGFR）和其他受体酪氨酸激酶（RTKs）在多种癌症中过度表达和/或突变，其异常激活与癌变有关。我们探索了利用Grb2的SH2结构域生成RTK激活成像探针用于癌症表征的可能性。为了进行细胞渗透、分子分析和放射性标记，SH2结构域分别与TAT、flag和酪氨酸残基（称为TSF）融合。我们分析了细胞中TSF的特征，如细胞摄取、稳定性和定位。在摄取到表达egfr的细胞后，发现TSF与磷酸化的egfr结合，并在EGF的刺激下增加。在EGFR激活的细胞中，TSF与EGFR共定位，而在EGFR未激活的细胞中，TSF以点的形式定位在细胞质中。在EGFR激活和EGF刺激下，TSF的细胞滞留时间显著延长，在酪氨酸激酶抑制剂Tyrphostin AG1478处理下，TSF的细胞滞留时间缩短。综上所述，Grb2的SH2结构域有潜力作为探针的结合成分来检测活化的RTK，并评估激酶抑制剂对RTK活化的影响。
Epidermal growth factor receptor (EGFR) and other receptor tyrosine kinases (RTKs) are overexpressed and/or mutated in various cancers and their abnormal activation is implicated in carcinogenesis. We explored the possibility of generating an imaging probe for RTK activation using the SH2 domain of Grb2 for cancer characterization. For cell penetration, molecular analysis and radiolabeling, the SH2 domain was fused with TAT, flag and tyrosine residue, respectively (termed TSF). We analyzed TSF characteristics in cells such as cellular uptake, stability and localization. After uptake into EGFR-expressing cells, TSF was found to be binding to phosphorylated-EGFR, which increased by stimulation with EGF. TSF was co-localized with EGFR in EGFR-activated cells, while it was localized as dots in cytosol in EGFR-non-activated cells. Cellular retention time of TSF was significantly extended under EGFR activation with EGF stimuli and reduced under the treatment with a tyrosine kinase inhibitor, Tyrphostin AG1478. In conclusion, the SH2 domain of Grb2 has the potential to be used as a binding component of a probe to detect activated-RTK and to evaluate the effect of kinase inhibitors on RTK activation.