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

Shannon L. O'Brien, Elizabeth K.M. Johnstone, Dominic Devost, Jacinta Conroy, Melissa E. Reichelt, Brooke W. Purdue, Mohammed A. Ayoub, Tatsuo Kawai, Asuka Inoue, Satoru Eguchi, Terence E. Hébert, Kevin D.G. Pfleger, Walter G. Thomas

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2 Citations (Scopus)

Abstract

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 Gq/11 and EGFR tyrosine kinase activity, whereas the recruitment of Grb2 to the EGFR was independent of Gq/11 and only partially dependent on EGFR tyrosine kinase activity. This Gq/11 independence of EGFR transactivation was confirmed using AT1R mutants and in CRISPR cell lines lacking Gq/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.

Original languageEnglish
Pages (from-to)232-242
Number of pages11
JournalBiochemical Pharmacology
Volume158
DOIs
Publication statusPublished - 2018 Dec

Keywords

  • Angiotensin II
  • Bioluminescence resonance energy transfer
  • EGFR transactivation
  • Epidermal growth factor receptor
  • G protein-coupled receptor
  • Receptor heteromerization

ASJC Scopus subject areas

  • Biochemistry
  • Pharmacology

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    O'Brien, S. L., Johnstone, E. K. M., Devost, D., Conroy, J., Reichelt, M. E., Purdue, B. W., Ayoub, M. A., Kawai, T., Inoue, A., Eguchi, S., Hébert, T. E., Pfleger, K. D. G., & Thomas, W. G. (2018). BRET-based assay to monitor EGFR transactivation by the AT1R reveals Gq/11 protein-independent activation and AT1R-EGFR complexes. Biochemical Pharmacology, 158, 232-242. https://doi.org/10.1016/j.bcp.2018.10.017