Fluorescently tagged nanobodies and NanoBRET to study ligand-binding and agonist-induced conformational changes of full-length EGFR expressed in living cells.
Fluorescently tagged nanobodies and NanoBRET to study ligand-binding and agonist-induced conformational changes of full-length EGFR expressed in living cells.
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DOI：
10.3389/fimmu.2022.1006718
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发表时间：
2022
影响因子：
7.3
通讯作者：
Kilpatrick, Laura E. E.
中科院分区：
文献类型：
--
作者：
Comez, Dehan;Glenn, Jacqueline;Anbuhl, Stephanie M. M.;Heukers, Raimond;Smit, Martine J. J.;Hill, Stephen J. J.;Kilpatrick, Laura E. E.
表皮生长因子受体是Erb受体酪氨酸激酶家族的成员。它结合几种配体，包括EGF，β细胞素（BTC）和TGF-α，控制细胞增殖和侵袭，并在各种癌症类型中过表达。纳米抗体（VHH）是仅重链骆驼抗体的抗原结合片段。在本文中，我们使用NanoBRET来比较荧光EGF或两种不同的荧光标记的EGFR定向纳米抗体（Q44 c和Q86 c）与全长EGFR的结合特性。用N末端NLuc标记的EGFR稳定转染活HEK 293 T细胞。然后使用荧光标记的EGF配体（EGF-AF 488或EGF-AF 647）或荧光标记的EGFR靶向纳米抗体（Q44 c-HL 488和Q86 c-HL 488）进行NanoBRET饱和、置换或动力学实验。这些数据显示，EGFR纳米抗体Q44 c能够抑制EGF与全长EGFR的结合，而Q86 c能够识别激动剂结合的EGFR并充当构象传感器。荧光Q44 c-HL 488和EGF-AF 488的特异性结合被一系列EGFR配体抑制（EGF> BTC>TGF-α）。EGFR靶向纳米抗体是研究EGFR在健康和疾病中的作用的有力工具，并且允许配体结合和不同配体诱导的构象变化的真实的时间定量。
The Epidermal Growth Factor Receptor is a member of the Erb receptor tyrosine kinase family. It binds several ligands including EGF, betacellulin (BTC) and TGF-α, controls cellular proliferation and invasion and is overexpressed in various cancer types. Nanobodies (VHHs) are the antigen binding fragments of heavy chain only camelid antibodies. In this paper we used NanoBRET to compare the binding characteristics of fluorescent EGF or two distinct fluorescently labelled EGFR directed nanobodies (Q44c and Q86c) to full length EGFR. Living HEK293T cells were stably transfected with N terminal NLuc tagged EGFR. NanoBRET saturation, displacement or kinetics experiments were then performed using fluorescently labelled EGF ligands (EGF-AF488 or EGF-AF647) or fluorescently labelled EGFR targeting nanobodies (Q44c-HL488 and Q86c-HL488). These data revealed that the EGFR nanobody Q44c was able to inhibit EGF binding to full length EGFR, while Q86c was able to recognise agonist bound EGFR and act as a conformational sensor. The specific binding of fluorescent Q44c-HL488 and EGF-AF488 was inhibited by a range of EGFR ligands (EGF> BTC>TGF-α). EGFR targeting nanobodies are powerful tools for studying the role of the EGFR in health and disease and allow real time quantification of ligand binding and distinct ligand induced conformational changes.
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DOI：
10.1083/jcb.109.5.2495
发表时间：
1989-11
期刊：
The Journal of cell biology
影响因子：
--
作者：
Defize LH;Boonstra J;Meisenhelder J;Kruijer W;Tertoolen LG;Tilly BC;Hunter T;van Bergen en Henegouwen PM;Moolenaar WH;de Laat SW
通讯作者：
de Laat SW
影响因子：
4.8
作者：
Macdonald-Obermann, Jennifer L.;Pike, Linda J.
通讯作者：
Pike, Linda J.
影响因子：
4.7
作者：
Heukers, Raimond;Mashayekhi, Vida;Oliveira, Sabrina
通讯作者：
Oliveira, Sabrina
影响因子：
5.9
作者：
Schihada, Hannes;Vandenabeele, Sylvie;Maiellaro, Isabella
通讯作者：
Maiellaro, Isabella
影响因子：
64.5
作者：
Freed DM;Bessman NJ;Kiyatkin A;Salazar-Cavazos E;Byrne PO;Moore JO;Valley CC;Ferguson KM;Leahy DJ;Lidke DS;Lemmon MA
通讯作者：
Lemmon MA