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

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Date
2014-08-14
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, Virgil L
Kobilka, Brian K
Skiniotis, Georgios
Lefkowitz, Robert J
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Abstract
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.
Type
Journal article
Subject
Animals
Arrestins
GTP-Binding Proteins
Models, Molecular
Protein Structure, Quaternary
Receptors, Adrenergic, beta-2
Receptors, G-Protein-Coupled
Sf9 Cells
beta-Arrestin 1
beta-Arrestins
Permalink
https://hdl.handle.net/10161/13107
Published Version (Please cite this version)
10.1038/nature13430
Publication Info
Shukla, Arun K; Westfield, Gerwin H; Xiao, Kunhong; Reis, Rosana I; Huang, Li-Yin; Tripathi-Shukla, Prachi; ... Lefkowitz, Robert J (2014). Visualization of arrestin recruitment by a G-protein-coupled receptor. Nature, 512(7513). pp. 218-222. 10.1038/nature13430. Retrieved from https://hdl.handle.net/10161/13107.
This is constructed from limited available data and may be imprecise. To cite this article, please review & use the official citation provided by the journal.
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Scholars@Duke

Kahsai

Alem W Kahsai

Assistant Professor in Medicine
Lefkowitz

Robert J. Lefkowitz

James B. Duke Distinguished Professor of Medicine
Dr. Lefkowitz’s memoir, A Funny Thing Happened on the Way to Stockholm, recounts his early career as a cardiologist and his transition to biochemistry, which led to his Nobel Prize win. Robert J. Lefkowitz, M.D. is James B. Duke Professor of Medicine and Professor of Biochemistry and Chemistry at the Duke University Medical Center. He has been an Investigator of the

Kunhong Xiao

Adjunct Assistant Professor in the Department of Medicine
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