Publication Date:
2014-07-22
Description:
G-protein-coupled receptors (GPCRs) are critically regulated by beta-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 beta2 adrenergic receptor (beta2AR)-G-protein complex, has provided novel insights into the structural basis of receptor activation. However, complementary information has been lacking on the recruitment of beta-arrestins to activated GPCRs, primarily owing to challenges in obtaining stable receptor-beta-arrestin complexes for structural studies. Here we devised a strategy for forming and purifying a functional human beta2AR-beta-arrestin-1 complex that allowed us to visualize its architecture by single-particle negative-stain electron microscopy and to characterize the interactions between beta2AR and beta-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 beta-arrestin 1 to the beta2AR, 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 beta-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 beta-arrestin 1 when coupled to the beta2AR. A molecular model of the beta2AR-beta-arrestin signalling complex was made by docking activated beta-arrestin 1 and beta2AR 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.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4134437/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉 〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4134437/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉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 Jr -- Kobilka, Brian K -- Skiniotis, Georgios -- Lefkowitz, Robert J -- DK090165/DK/NIDDK NIH HHS/ -- GM072688/GM/NIGMS NIH HHS/ -- GM087519/GM/NIGMS NIH HHS/ -- GM60635/GM/NIGMS NIH HHS/ -- HL075443/HL/NHLBI NIH HHS/ -- HL16037/HL/NHLBI NIH HHS/ -- HL70631/HL/NHLBI NIH HHS/ -- MOP-93725/Canadian Institutes of Health Research/Canada -- NS028471/NS/NINDS NIH HHS/ -- R01 DK090165/DK/NIDDK NIH HHS/ -- R01 GM060635/GM/NIGMS NIH HHS/ -- R01 GM072688/GM/NIGMS NIH HHS/ -- R01 HL016037/HL/NHLBI NIH HHS/ -- R01 HL070631/HL/NHLBI NIH HHS/ -- R01 NS028471/NS/NINDS NIH HHS/ -- UL1 TR000430/TR/NCATS NIH HHS/ -- Howard Hughes Medical Institute/ -- England -- Nature. 2014 Aug 14;512(7513):218-22. doi: 10.1038/nature13430. Epub 2014 Jun 22.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉1] Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710, USA [2] Department of Biological Sciences and Bioengineering, Indian Institute of Technology, Kanpur 208016, India. [3]. ; 1] Life Sciences Institute and Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA [2]. ; 1] Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710, USA [2]. ; Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710, USA. ; Department of Chemistry, University of California at San Diego, La Jolla, California 92093, USA. ; Life Sciences Institute and Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA. ; School of Pharmaceutical &Life Sciences, Changzhou University, Changzhou, Jiangsu 213164, China. ; Terrence Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario M5S 3E1, Canada. ; Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA. ; Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois 60637, USA. ; Department of Biochemistry and Molecular Biology, The University of Texas Medical School at Houston, Houston, Texas 77054, USA. ; 1] Department of Chemistry, University of California at San Diego, La Jolla, California 92093, USA [2]. ; Department of Molecular and Cellular Physiology, Stanford University School of Medicine, 279 Campus Drive, Stanford, California 94305, USA. ; 1] Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710, USA [2] Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA [3] Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina 27710, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/25043026" target="_blank"〉PubMed〈/a〉
Keywords:
Animals
;
Arrestins/*chemistry/*metabolism
;
GTP-Binding Proteins/chemistry/metabolism
;
*Models, Molecular
;
Protein Structure, Quaternary
;
Receptors, Adrenergic, beta-2/chemistry/metabolism
;
Receptors, G-Protein-Coupled/*chemistry/*metabolism
;
Sf9 Cells
Print ISSN:
0028-0836
Electronic ISSN:
1476-4687
Topics:
Biology
,
Chemistry and Pharmacology
,
Medicine
,
Natural Sciences in General
,
Physics
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