Format

Send to

Choose Destination
Nature. 2014 Aug 14;512(7513):218-222. doi: 10.1038/nature13430. Epub 2014 Jun 22.

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

Author information

1
Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710, USA.
2
Life Sciences Institute and Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI 48109, USA.
3
Department of Chemistry, University of California at San Diego, La Jolla, CA 92093, USA.
4
School of Pharmaceutical & Life Sciences, Changzhou University, Changzhou, Jiangsu 213164, China.
5
Terrence Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, ON, Canada.
6
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
7
Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois 60637, USA.
8
Department of Biochemistry and Molecular Biology, The University of Texas Medical School at Houston, Houston, TX 77054, USA.
9
Department of Molecular and Cellular Physiology, Stanford University School of Medicine, 279 Campus Drive, Stanford, California 94305, USA.
10
Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina 27710, USA.
#
Contributed equally

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.

PMID:
25043026
PMCID:
PMC4134437
DOI:
10.1038/nature13430
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for Nature Publishing Group Icon for PubMed Central
Loading ...
Support Center