Genetic code expansion and photocross-linking identify different β-arrestin binding modes to the angiotensin II type 1 receptor

: The angiotensin II (AngII) type 1 receptor (AT1R) is a member of the G protein-coupled receptor (GPCR) family and binds β-arrestins (β-arrs), which regulate AT1R signaling and trafficking. These processes can be biased by different ligands or mutations in the AGTR1 gene. As for many GPCRs, the exact details for AT1R-β-arr interactions driven by AngII or β-arr-biased ligands remain largely unknown. Here, we used the amber-suppression technology to site-specifically introduce the unnatural amino acid (UAA) p-azido-l-phenylalanine (azF) into the intracellular loops (ICLs) and the C-tail of AT1R. Our goal was to generate competent photoreactive receptors that can be cross-linked to β-arrs in cells. We performed UV-mediated photolysis of 25 different azF-labeled AT1Rs to cross-link β-arr1 to AngII-bound receptors, enabling us to map important contact sites in the C-tail and in the ICL2 and ICL3 of the receptor. The extent of AT1R-β-arr1 cross-linking among azF-labeled receptors differed, revealing variability in β-arr's contact mode with the different AT1R domains. Moreover, the signature of ligated AT1R-β-arr complexes from a subset of azF-labeled receptors also differed between AngII and β-arr-biased ligand stimulation of receptors and between azF-labeled AT1R bearing and that lacking a bias signaling mutation. These observations further implied distinct interaction modalities of the AT1R-β-arr1 complex in biased signaling conditions. Our findings demonstrate that this photocross-linking approach is useful for understanding GPCR-β-arr complexes in different activation states and could be extended to study other protein-protein interactions in cells.