BRET-based assay to monitor EGFR transactivation by the AT₁R reveals G_q/11 protein-independent activation and AT₁R-EGFR complexes

The type 1 angiotensin II (AngII) receptor (AT₁R) 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 AT₁R was able to transactivate HER2, but not HER3. Mechanistically, AT₁R-mediated ERK1/2 activation was dependent on G_q/11 and EGFR tyrosine kinase activity, whereas the recruitment of Grb2 to the EGFR was independent of G_q/11 and only partially dependent on EGFR tyrosine kinase activity. This G_q/11 independence of EGFR transactivation was confirmed using AT₁R mutants and in CRISPR cell lines lacking G_q/11. 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 AT₁R-EGFR heteromerization. In summary, we report an alternative approach to monitoring AT₁R-EGFR transactivation in live cells, which provides a more direct and proximal view of this process, including the potential for complexes between the AT₁R and EGFR.