Visualization of arrestin recruitment by a G-protein-coupled receptor.
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 articleSubject
AnimalsArrestins
GTP-Binding Proteins
Models, Molecular
Protein Structure, Quaternary
Receptors, Adrenergic, beta-2
Receptors, G-Protein-Coupled
Sf9 Cells
beta-Arrestin 1
beta-Arrestins
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https://hdl.handle.net/10161/13107Published Version (Please cite this version)
10.1038/nature13430Publication 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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Show full item recordScholars@Duke
Alem W Kahsai
Assistant Professor in Medicine
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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