Abstract

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.

Highlights

  • The angiotensin II (AngII) type 1 receptor (AT1R) is a member of the G protein– coupled receptor (GPCR) family and binds ␤-arrestins (␤-arrs), which regulate AT1R signaling and trafficking

  • Other approaches, such as negative-stain EM combined with the use of a chimeric GPCR, have unnatural amino acid; azF, p-azido-L-phenylalanine; intracellular loops (ICLs), intracellular loop; GPCR kinases (GRKs), GPCR kinase; TM, transmembrane; aaRS, aminoacyl-tRNA synthetase; extracellular loops (ECLs), extracellular loop; BRET, bioluminescence resonance energy transfer; DMEM, Dulbecco’s modified Eagle’s medium; PEI, polyethylenimine; EDT, 1,2-ethanedithiol; ANOVA, analysis of variance; YFP, yellow fluorescent protein; Rluc, Renilla luciferase; DVG, [Asp1, Val5, Gly8]-AngII

  • Two residues in the extracellular loops (ECLs) of AT1R were selected as negative controls because these are not predicted to interact with ␤-arr

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Summary

Introduction

The angiotensin II (AngII) type 1 receptor (AT1R) is a member of the G protein– coupled receptor (GPCR) family and binds ␤-arrestins (␤-arrs), which regulate AT1R signaling and trafficking. The signature of ligated AT1R–␤-arr complexes from a subset of azFlabeled receptors differed between AngII and ␤-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–␤-arr complex in biased signaling conditions. Only a complex of rhodopsin bound to visual arrestin has been crystalized with high resolution [17] Other approaches, such as negative-stain EM combined with the use of a chimeric GPCR, have unnatural amino acid; azF, p-azido-L-phenylalanine; ICL, intracellular loop; GRK, GPCR kinase; TM, transmembrane; aaRS, aminoacyl-tRNA synthetase; ECL, extracellular loop; BRET, bioluminescence resonance energy transfer; DMEM, Dulbecco’s modified Eagle’s medium; PEI, polyethylenimine; EDT, 1,2-ethanedithiol; ANOVA, analysis of variance; YFP, yellow fluorescent protein; Rluc, Renilla luciferase; DVG, [Asp, Val, Gly8]-AngII. Because biased ligands and mutations within the receptor affect GPCR–␤-arr complex conformations [21, 22], such as in the case of AT1R [12, 14,15,16], intermolecular interactions within the complex may vary in biased signaling conditions

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