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.

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The angiotensin II (AngII) type 1 receptor (AT1R), a member of the G protein-coupled receptor (GPCR) family, signals through G proteins and β-arrestins, which act as adaptors to regulate AT1R internalization and mitogen-activated protein kinase (MAPK) ERK1/2 activation. β-arrestin-dependent ERK1/2 regulation is the subject of important studies because its spatiotemporal control remains poorly understood for many GPCRs, including AT1R. To study the link between β-arrestin-dependent trafficking and ERK1/2 signaling, we investigated three naturally occurring AT1R variants that show distinct receptor–β-arrestin interactions: A163T, T282M and C289W. Using bioluminescence resonance energy transfer (BRET)-based and conformational fluorescein arsenical hairpin–BRET sensors coupled with high-resolution fluorescence microscopy, we show that all AT1R variants form complexes with β-arrestin2 at the plasma membrane and efficiently internalize into endosomes upon AngII stimulation. However, mutant receptors imposed distinct conformations in β-arrestin2 and differentially impacted endosomal trafficking and MAPK signaling. Notably, T282M accumulated in endosomes, but its ability to form stable complexes following internalization was reduced, markedly impairing its ability to co-traffic with β-arrestin2. We also found that β-arrestin2 overexpression greatly reduced both T282M and C289W’s residency with β-arrestin2 in endosomes, leading to decreased β-arrestin-dependent ERK1/2 activation, faster recycling of receptors to the plasma membrane and impaired AngII-mediated proliferation. Our findings reveal that naturally occurring AT1R variants alter the patterns of receptor/β-arrestin2 trafficking and suggest conformationally-dependent β-arrestin-mediated MAPK activation as well as endosomal receptor–β-arrestin complex stabilization in the mitogenic response of AT1R.

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