TY - JOUR
T1 - BRET-based assay to monitor EGFR transactivation by the AT1R reveals Gq/11 protein-independent activation and AT1R-EGFR complexes
AU - O'Brien, Shannon L.
AU - Johnstone, Elizabeth K.M.
AU - Devost, Dominic
AU - Conroy, Jacinta
AU - Reichelt, Melissa E.
AU - Purdue, Brooke W.
AU - Ayoub, Mohammed A.
AU - Kawai, Tatsuo
AU - Inoue, Asuka
AU - Eguchi, Satoru
AU - Hébert, Terence E.
AU - Pfleger, Kevin D.G.
AU - Thomas, Walter G.
N1 - Funding Information:
K.D.G.P. receives funding from Promega, B.M.G. Labtech and Dimerix as participating organizations of Australian Research Council Linkage Grant LP160100857. These organizations played no role in any aspect of the conception or design of the research, collection, analysis and interpretation of results, or writing and editing of the manuscript. K.D.G.P. is Chief Scientific Advisor of Dimerix, of which he maintains a shareholding. Dimerix has proprietary rights to the Receptor-HIT assay.
Funding Information:
We thank Ichiro Maruyama (Okinawa Institute of Science & Technology, Okinawa, Japan) for provision of the EGFR-Vc BiFC plasmid. Funding was provided by NHMRC Project Grant 1085996 awarded to W.G.T. and K.D.G.P. K.D.G.P. is an NHMRC RD Wright Fellow (1085842). This work was also funded by Japan Science and Technology Agency (JST; Grant Number JPMJPR1331 ), Japan Agency for Medical Research and Development (AMED; JP17gm5910013 ) and Japan Society for the Promotion of Science ( JSPS ) KAKENHI ( 17K08264 ) (to A.I.).
Publisher Copyright:
© 2018
PY - 2018/12
Y1 - 2018/12
N2 - 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.
AB - 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.
KW - Angiotensin II
KW - Bioluminescence resonance energy transfer
KW - EGFR transactivation
KW - Epidermal growth factor receptor
KW - G protein-coupled receptor
KW - Receptor heteromerization
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U2 - 10.1016/j.bcp.2018.10.017
DO - 10.1016/j.bcp.2018.10.017
M3 - Article
C2 - 30347205
AN - SCOPUS:85055740299
SN - 0006-2952
VL - 158
SP - 232
EP - 242
JO - Biochemical Pharmacology
JF - Biochemical Pharmacology
ER -