Receptor Biology Group, The School of Biomedical Sciences, Faculty of Medicine, The University of Queensland, St Lucia 4072, Queensland, Australia.
Molecular Endocrinology and Pharmacology, Harry Perkins Institute of Medical Research, QEII Medical Centre, Nedlands and Centre for Medical Research, The University of Western Australia, Crawley, Western Australia 6009, Australia.
Biochem Pharmacol. 2018 Dec;158:232-242. doi: 10.1016/j.bcp.2018.10.017. Epub 2018 Oct 19.
The type 1 angiotensin II (AngII) receptor (ATR) transactivates the epidermal growth factor receptor (EGFR), which leads to pathological remodeling of heart, blood vessels and kidney. End-point assays are used as surrogates of EGFR activation, however these downstream readouts are not applicable to live cells, in real-time. Herein, we report the use of a bioluminescence resonance energy transfer (BRET)-based assay to assess recruitment of the EGFR adaptor protein, growth factor receptor-bound protein 2 (Grb2), to the EGFR. In a variety of cell lines, both epidermal growth factor (EGF) and AngII stimulated Grb2 recruitment to EGFR. The BRET assay was used to screen a panel of 9 G protein-coupled receptors (GPCRs) and further developed for other EGFR family members (HER2 and HER3); the ATR was able to transactivate HER2, but not HER3. Mechanistically, ATR-mediated ERK1/2 activation was dependent on G and EGFR tyrosine kinase activity, whereas the recruitment of Grb2 to the EGFR was independent of G and only partially dependent on EGFR tyrosine kinase activity. This G independence of EGFR transactivation was confirmed using ATR mutants and in CRISPR cell lines lacking G. EGFR transactivation was also apparently independent of β-arrestins. Finally, we used additional BRET-based assays and confocal microscopy to provide evidence that both AngII- and EGF-stimulation promoted ATR-EGFR heteromerization. In summary, we report an alternative approach to monitoring ATR-EGFR transactivation in live cells, which provides a more direct and proximal view of this process, including the potential for complexes between the ATR and EGFR.
1 型血管紧张素 II（AngII）受体（ATR）可激活表皮生长因子受体（EGFR），导致心脏、血管和肾脏的病理性重塑。终点测定法被用作 EGFR 激活的替代物，然而这些下游的检测方法不适用于实时的活细胞。在此，我们报告了使用生物发光共振能量转移（BRET）测定法来评估 EGFR 衔接蛋白生长因子受体结合蛋白 2（Grb2）向 EGFR 的募集。在各种细胞系中，表皮生长因子（EGF）和 AngII 均能刺激 Grb2 向 EGFR 的募集。该 BRET 测定法用于筛选一组 9 个 G 蛋白偶联受体（GPCR），并进一步开发用于其他 EGFR 家族成员（HER2 和 HER3）；ATR 能够激活 HER2，但不能激活 HER3。从机制上讲，ATR 介导的 ERK1/2 激活依赖于 G 和 EGFR 酪氨酸激酶活性，而 Grb2 向 EGFR 的募集则不依赖于 G，仅部分依赖于 EGFR 酪氨酸激酶活性。这一发现通过 ATR 突变体和缺乏 G 的 CRISPR 细胞系得到了证实。EGFR 的这种 G 独立性激活也明显独立于β-arrestin。最后，我们使用了其他 BRET 测定法和共聚焦显微镜，提供了证据表明 AngII 和 EGF 刺激均可促进 ATR-EGFR 异源二聚体的形成。总之，我们报告了一种在活细胞中监测 ATR-EGFR 转激活的替代方法，该方法提供了对该过程的更直接和更接近的观察，包括 ATR 和 EGFR 之间可能形成复合物的情况。
