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

Abstract

The type 1 angiotensin II (AngII) receptor (AT 1 R) 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 AT 1 R was able to transactivate HER2, but not HER3. Mechanistically, AT 1 R-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 AT 1 R mutants and in CRISPR cell lines lacking G q/11. EGFR transactivation was also apparently independent of βarrestins. Finally, we used additional BRET-based assays and confocal microscopy to provide evidence that both AngII-and EGF-stimulation promoted AT 1 R-EGFR heteromerization. In summary, we report an alternative approach to monitoring AT 1 R-EGFR transactivation in live cells, which provides a more direct and proximal view of this process, including the potential for complexes between the AT 1 R and EGFR.

Investigation of Receptor Heteromers Using NanoBRET Ligand Binding

“…In order to address this issue, we have developed the Receptor-Heteromer Investigation Technology (Receptor-HIT) [2,3], which has been used to investigate several G protein-coupled receptor (GPCR) heteromers [2,[4][5][6][7][8][9][10][11]. In addition, the Receptor-HIT assay has been used to characterize receptor tyrosine kinase (RTK) heteromers [12], as well as heteromers composed of both GPCRs and RTKs [13][14][15] or a GPCR and the Receptor for Advanced Glycation Endproducts [16].…”

“…2021, 22, x 2 of 18 receptor (GPCR) heteromers [2,[4][5][6][7][8][9][10][11]. In addition, the Receptor-HIT assay has been used to characterize receptor tyrosine kinase (RTK) heteromers [12], as well as heteromers composed of both GPCRs and RTKs [13][14][15] or a GPCR and the Receptor for Advanced Glycation End-products [16]. The Receptor-HIT assay enables rapid identification, screening and profiling of receptor heteromers through ligand-dependent modulation of interactions between the receptors and specific biomolecules.…”

Profiling novel pharmacology of receptor complexes using Receptor-HIT

“…Receptor-HIT has also been used to investigate heteromers composed of GPCRs and other receptors, including RTKs such as epidermal growth factor receptor (EGFR) [ 36 , 37 ], vascular endothelial growth factor receptor 2 (VEGFR2) [ 38 ], and the insulin receptor [ 39 ], as well as the receptor for advanced glycation end-products (RAGE) [ 40 ].…”

“…A good example is the transactivation of the EGFR by the AT 1 receptor, which mediates cell growth and survival, and has been implicated in numerous cardiovascular pathologies [ 42 ]. Using the Receptor-HIT assay in various immortalised and primary cells, we demonstrated AngII-induced recruitment of Grb2/Venus proximal to EGFR/Rluc8 and showed that the transactivation was independent of G q/11 and β-arrestin, and only partially dependent on EGFR tyrosine kinase activity [ 36 ]. We then followed up this study by investigating the involvement of a protein called TRIO in AT 1 -mediated EGFR transactivation, as it had previously been identified as an intermediate specific to AngII/AT 1 -mediated EGFR transactivation rather than EGF-mediated EGFR transactivation [ 43 ].…”

Roles of renin-angiotensin system in the regulation of prostate cancer bone metastasis: a critical review

“…Moreover, it also mediates the generation of reactive oxygen species (ROS) [13] and the modulation of various ion channels [14][15][16]. The mechanisms underlying the AT1R-triggered regulation of tyrosine kinaserelated pathways are thought to initiate from (1) the de novo synthesis of cytokine and growth factor ligands, and/or (2) the binding of the AT1R with the upstream activators and scaffolds [17], (3) and/or the transactivation of growth factor receptors such as the epidermal growth factor receptor (EGFR) [18]. EGFR transactivation involves the capacity of GPCRs to activate metalloproteinases (MMPs) that cleave cell surface precursors of the EGF ligands, yielding the shed mature ligand that subsequently binds and activates the EGFR to pioneer various cellular signals [19], which are crucial for Ang II/AT1R-mediated hypertrophy and/ or proliferation of cardiac, vascular and renal cells.…”