Visualization of arrestin recruitment by a G-protein-coupled receptor

• Published in: Nature (London) Vol. 512; no. 7513; pp. 218 - 222
• Main Authors: Shukla, Arun K, Westfield, Gerwin H, Xiao, Kunhong, Reis, Rosana I, Huang, Li-Yin, Tripathi-Shukla, Prachi, Qian, Jiang, Li, Sheng, Blanc, Adi, Oleskie, Austin N, Dosey, Anne M, Su, Min, Liang, Cui-Rong, Gu, Ling-Ling, Shan, Jin-Ming, Chen, Xin, Hanna, Rachel, Choi, Minjung, Yao, Xiao Jie, Klink, Bjoern U, Kahsai, Alem W, Sidhu, Sachdev S, Koide, Shohei, Penczek, Pawel A, Kossiakoff, Anthony A, Woods, Jr, Virgil L, Kobilka, Brian K, Skiniotis, Georgios, Lefkowitz, Robert J
• Format: Journal Article
• Language: English
• Published: England Nature Publishing Group 14.08.2014
• Subjects: Animals; Arrestins - chemistry; Arrestins - metabolism; beta-Arrestin 1; beta-Arrestins; Chromatography; Crosslinked polymers; Crystals; Efficiency; G proteins; GTP-Binding Proteins - chemistry; GTP-Binding Proteins - metabolism; Ligands; Models, Molecular; Protein Structure, Quaternary; Proteins; Receptors, Adrenergic, beta-2 - chemistry; Receptors, Adrenergic, beta-2 - metabolism; Receptors, G-Protein-Coupled - chemistry; Receptors, G-Protein-Coupled - metabolism; Sf9 Cells; Signal transduction; Structure; United States
• ISSN: 0028-0836; 1476-4687
• DOI: 10.1038/nature13430

G-protein-coupled receptors (GPCRs) are critically regulated by β-arrestins, which not only desensitize G-protein signalling but also initiate a G-protein-independent wave of signalling. A recent surge of structural data on a number of GPCRs, including the β2 adrenergic receptor (β2AR)-G-protein complex, has provided novel insights into the structural basis of receptor activation. However, complementary information has been lacking on the recruitment of β-arrestins to activated GPCRs, primarily owing to challenges in obtaining stable receptor-β-arrestin complexes for structural studies. Here we devised a strategy for forming and purifying a functional human β2AR-β-arrestin-1 complex that allowed us to visualize its architecture by single-particle negative-stain electron microscopy and to characterize the interactions between β2AR and β-arrestin 1 using hydrogen-deuterium exchange mass spectrometry (HDX-MS) and chemical crosslinking. Electron microscopy two-dimensional averages and three-dimensional reconstructions reveal bimodal binding of β-arrestin 1 to the β2AR, involving two separate sets of interactions, one with the phosphorylated carboxy terminus of the receptor and the other with its seven-transmembrane core. Areas of reduced HDX together with identification of crosslinked residues suggest engagement of the finger loop of β-arrestin 1 with the seven-transmembrane core of the receptor. In contrast, focal areas of raised HDX levels indicate regions of increased dynamics in both the N and C domains of β-arrestin 1 when coupled to the β2AR. A molecular model of the β2AR-β-arrestin signalling complex was made by docking activated β-arrestin 1 and β2AR crystal structures into the electron microscopy map densities with constraints provided by HDX-MS and crosslinking, allowing us to obtain valuable insights into the overall architecture of a receptor-arrestin complex. The dynamic and structural information presented here provides a framework for better understanding the basis of GPCR regulation by arrestins.