title: 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.
authors.affiliation: Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710, USA.
authors.forename: Arun K
authors.initials: AK
authors.lastname: Shukla
authors.affiliation: Life Sciences Institute and Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI 48109, USA.
authors.forename: Gerwin H
authors.initials: GH
authors.lastname: Westfield
authors.affiliation: Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710, USA.
authors.forename: Kunhong
authors.initials: K
authors.lastname: Xiao
authors.affiliation: Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710, USA.
authors.forename: Rosana I
authors.initials: RI
authors.lastname: Reis
authors.affiliation: Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710, USA.
authors.forename: Li-Yin
authors.initials: LY
authors.lastname: Huang
authors.affiliation: Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710, USA.
authors.forename: Prachi
authors.initials: P
authors.lastname: Tripathi-Shukla
authors.affiliation: Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710, USA.
authors.forename: Jiang
authors.initials: J
authors.lastname: Qian
authors.affiliation: Department of Chemistry, University of California at San Diego, La Jolla, CA 92093, USA.
authors.forename: Sheng
authors.initials: S
authors.lastname: Li
authors.affiliation: Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710, USA.
authors.forename: Adi
authors.initials: A
authors.lastname: Blanc
authors.affiliation: Life Sciences Institute and Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI 48109, USA.
authors.forename: Austin N
authors.initials: AN
authors.lastname: Oleskie
authors.affiliation: Life Sciences Institute and Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI 48109, USA.
authors.forename: Anne M
authors.initials: AM
authors.lastname: Dosey
authors.affiliation: Life Sciences Institute and Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI 48109, USA.
authors.forename: Min
authors.initials: M
authors.lastname: Su
authors.affiliation: School of Pharmaceutical & Life Sciences, Changzhou University, Changzhou, Jiangsu 213164, China.
authors.forename: Cui-Rong
authors.initials: CR
authors.lastname: Liang
authors.affiliation: School of Pharmaceutical & Life Sciences, Changzhou University, Changzhou, Jiangsu 213164, China.
authors.forename: Ling-Ling
authors.initials: LL
authors.lastname: Gu
authors.affiliation: School of Pharmaceutical & Life Sciences, Changzhou University, Changzhou, Jiangsu 213164, China.
authors.forename: Jin-Ming
authors.initials: JM
authors.lastname: Shan
authors.affiliation: School of Pharmaceutical & Life Sciences, Changzhou University, Changzhou, Jiangsu 213164, China.
authors.forename: Xin
authors.initials: X
authors.lastname: Chen
authors.affiliation: Terrence Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, ON, Canada.
authors.forename: Rachel
authors.initials: R
authors.lastname: Hanna
authors.affiliation: Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
authors.forename: Minjung
authors.initials: M
authors.lastname: Choi
authors.affiliation: Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710, USA.
authors.forename: Xiao Jie
authors.initials: XJ
authors.lastname: Yao
authors.affiliation: Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710, USA.
authors.forename: Bjoern U
authors.initials: BU
authors.lastname: Klink
authors.affiliation: Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710, USA.
authors.forename: Alem W
authors.initials: AW
authors.lastname: Kahsai
authors.affiliation: Terrence Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, ON, Canada.
authors.forename: Sachdev S
authors.initials: SS
authors.lastname: Sidhu
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois 60637, USA.
authors.forename: Shohei
authors.initials: S
authors.lastname: Koide
authors.affiliation: Department of Biochemistry and Molecular Biology, The University of Texas Medical School at Houston, Houston, TX 77054, USA.
authors.forename: Pawel A
authors.initials: PA
authors.lastname: Penczek
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois 60637, USA.
authors.forename: Anthony A
authors.initials: AA
authors.lastname: Kossiakoff
authors.affiliation: Department of Chemistry, University of California at San Diego, La Jolla, CA 92093, USA.
authors.forename: Virgil L
authors.initials: VL
authors.lastname: Woods
authors.affiliation: Department of Molecular and Cellular Physiology, Stanford University School of Medicine, 279 Campus Drive, Stanford, California 94305, USA.
authors.forename: Brian K
authors.initials: BK
authors.lastname: Kobilka
authors.affiliation: Life Sciences Institute and Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI 48109, USA.
authors.forename: Georgios
authors.initials: G
authors.lastname: Skiniotis
authors.affiliation: Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710, USA.|Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.|Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina 27710, USA.
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Lefkowitz
mesh_terms: D000818:Animals; D019390:Arrestins / Q000737:chemistry* / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D020836:Protein Structure, Quaternary; D018343:Receptors, Adrenergic, beta-2 / Q000737:chemistry / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry* / Q000378:metabolism*; D061987:Sf9 Cells; D000071559:beta-Arrestin 1; D000071557:beta-Arrestins
chemical_list: C000605253:ARRB1 protein, human; D019390:Arrestins; D018343:Receptors, Adrenergic, beta-2; D043562:Receptors, G-Protein-Coupled; D000071559:beta-Arrestin 1; D000071557:beta-Arrestins; D019204:GTP-Binding Proteins