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: London Nature Publishing Group UK 14.08.2014; Nature Publishing Group
• Subjects: 631/535/1258; 631/92/612/194; Animals; Arrestins - chemistry; Arrestins - metabolism; beta-Arrestin 1; beta-Arrestins; Chromatography; Crosslinked polymers; Crystals; Deuterium; Efficiency; Electron microscopy; G proteins; GTP-Binding Proteins - chemistry; GTP-Binding Proteins - metabolism; Humanities and Social Sciences; letter; Ligands; Mass spectrometry; Models, Molecular; multidisciplinary; Protein Structure, Quaternary; Proteins; Receptors, Adrenergic, beta-2 - chemistry; Receptors, Adrenergic, beta-2 - metabolism; Receptors, G-Protein-Coupled - chemistry; Receptors, G-Protein-Coupled - metabolism; Science; Sf9 Cells; Signal transduction; Structure; United States
• ISSN: 0028-0836; 1476-4687
• DOI: 10.1038/nature13430

Single-particle electron microscopy and hydrogen–deuterium exchange mass spectrometry are used to characterize the structure and dynamics of a G-protein-coupled receptor–arrestin complex. An arrestin–GPCR complex structure Much has been learned about the structure of G-protein-coupled receptors (GCPRs) over the past seven years, but we still don't know what an activated GPCR looks like when it is bound to a β-arrestin. (Arrestins are cellular mediators with a broad range of functions, many of them involving GPCRs.) In this study the authors use single-particle electron microscopy and hydrogen–deuterium exchange mass spectrometry to characterize the structure and dynamics of a GPCR–arrestin complex. Their data support a 'biphasic' mechanism, in which the arrestin initially interacts with the phosphorylated carboxy terminus of the GPCR before re-arranging to more fully engage the membrane protein in a signalling-competent conformation. 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 1 , 2 , 3 , 4 , 5 . A recent surge of structural data on a number of GPCRs, including the β 2 adrenergic receptor (β 2 AR)–G-protein complex, has provided novel insights into the structural basis of receptor activation 6 , 7 , 8 , 9 , 10 , 11 . 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 β 2 AR–β-arrestin-1 complex that allowed us to visualize its architecture by single-particle negative-stain electron microscopy and to characterize the interactions between β 2 AR 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 β 2 AR, 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 β 2 AR. A molecular model of the β 2 AR–β-arrestin signalling complex was made by docking activated β-arrestin 1 and β 2 AR 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.