Conformational Profiling of the AT1 Angiotensin II Receptor Reflects Biased Agonism, G Protein Coupling, and Cellular Context
| Autor: | Dominic Devost, Alice Zhang, Rakan Okde, Terence E. Hébert, Junken Aoki, Yuji Shinjo, Rory Sleno, Asuka Inoue, Darlaine Pétrin |
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| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Cell signaling Protein Conformation G protein Biology Ligands Biochemistry Receptor Angiotensin Type 1 Cell Line 03 medical and health sciences 0302 clinical medicine GTP-Binding Proteins Fluorescence Resonance Energy Transfer Methods Functional selectivity Humans Binding site Receptor Cell Engineering Molecular Biology beta-Arrestins Angiotensin II receptor type 1 Angiotensin II HEK 293 cells Cell Biology 030104 developmental biology Biophysics 030217 neurology & neurosurgery Signal Transduction |
| Zdroj: | Journal of Biological Chemistry. 292:5443-5456 |
| ISSN: | 0021-9258 |
| DOI: | 10.1074/jbc.m116.763854 |
| Popis: | Here, we report the design and use of G protein-coupled receptor-based biosensors to monitor ligand-mediated conformational changes in receptors in intact cells. These biosensors use bioluminescence resonance energy transfer with Renilla luciferase (RlucII) as an energy donor, placed at the distal end of the receptor C-tail, and the small fluorescent molecule FlAsH as an energy acceptor, its binding site inserted at different positions throughout the intracellular loops and C-terminal tail of the angiotensin II type I receptor. We verified that the modifications did not compromise receptor localization or function before proceeding further. Our biosensors were able to capture effects of both canonical and biased ligands, even to the extent of discriminating between different biased ligands. Using a combination of G protein inhibitors and HEK 293 cell lines that were CRISPR/Cas9-engineered to delete Gαq, Gα11, Gα12, and Gα13 or β-arrestins, we showed that Gαq and Gα11 are required for functional responses in conformational sensors in ICL3 but not ICL2. Loss of β-arrestin did not alter biased ligand effects on ICL2P2. We also demonstrate that such biosensors are portable between different cell types and yield context-dependent readouts of G protein-coupled receptor conformation. Our study provides mechanistic insights into signaling events that depend on either G proteins or β-arrestin. |
| Databáze: | OpenAIRE |
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