Design and evaluation of engineered protein biosensors for live-cell imaging of EGFR phosphorylation.
Design and evaluation of engineered protein biosensors for live-cell imaging of EGFR phosphorylation.
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用于 EGFR 磷酸化活细胞成像的工程蛋白生物传感器的设计和评估。
DOI：
10.1126/scisignal.aap7584
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发表时间：
2019
影响因子：
7.3
通讯作者：
Rao,BalajiM
中科院分区：
文献类型：
--
作者：
Tiruthani,Karthik;Mischler,Adam;Ahmed,Shoeb;Mahinthakumar,Jessica;Haugh,JasonM;Rao,BalajiM
Live-cell fluorescence microscopy is broadly applied to study the dynamics of receptor-mediated cell signaling, but the availability of intracellular biosensors is limited. A biosensor based on the tandem SH2 domains from phospholipase C–γ1 (PLCγ1), tSH2-WT, has been used to measure phosphorylation of the epidermal growth factor receptor (EGFR). Here, we found that tSH2-WT lacked specificity for phosphorylated EGFR, consistent with the known promiscuity of SH2 domains. Further, EGF-stimulated membrane recruitment of tSH2-WT differed qualitatively from the expected kinetics of EGFR phosphorylation. Analysis of a mathematical model suggested, and experiments confirmed, that the high avidity of tSH2-WT resulted in saturation of its target and interference with EGFR endocytosis. To overcome the apparent target specificity and saturation issues, we implemented two protein engineering strategies. In the first approach, we screened a combinatorial library generated by random mutagenesis of the C-terminal SH2 domain (cSH2) of PLCγ1 and isolated a mutant form (mSH2) with enhanced specificity for phosphorylated Tyr992(pTyr992) of EGFR. A biosensor based on mSH2 closely reported the kinetics of EGFR phosphorylation but retained cross-reactivity similar to tSH2-WT. In the second approach, we isolated a pTyr992-binding protein (SPY992) from a combinatorial library generated by mutagenesis of the Sso7d protein scaffold. Compared to tSH2-WT and mSH2, SPY992 exhibited superior performance as a specific, moderate-affinity biosensor. We extended this approach to isolate a biosensor for EGFR pTyr1148(SPY1148). This approach of integrating theoretical considerations with protein engineering strategies can be generalized to design and evaluate suitable biosensors for various phospho-specific targets.
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DOI：
10.1016/s0021-9258(18)32085-4
发表时间：
1983-07
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
K. Heidenreich;N. Zahniser;P. Berhanu;D. Brandenburg;J. Olefsky
通讯作者：
K. Heidenreich;N. Zahniser;P. Berhanu;D. Brandenburg;J. Olefsky
影响因子：
15.9
作者：
CARO, JF;ITTOOP, O;SINHA, MK
通讯作者：
SINHA, MK
大鼠肝脏和肌肉来源的胰岛素受体之间的结构差异。
DOI：
--
发表时间：
1986
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Burant,CF;Treutelaar,MK;Block,NE;Buse,MG
通讯作者：
Buse,MG
人单核细胞系 U-937 胰岛素受体表观分子量的变化
影响因子：
7.7
作者：
A. Mcelduff;G. Grunberger;P. Gorden
通讯作者：
P. Gorden
影响因子：
56.9
作者：
O. Rosen
通讯作者：
O. Rosen