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

机译：通过G蛋白偶联受体观察抑制素募集

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摘要

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 β_2 adrenergic receptor (β_2AR)-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 β_2AR-β-arrestin-1 complex that allowed us to visualize its architecture by single-particle negative-stain electron microscopy and to characterize the interactions between β_2AR 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 β_2AR, involving two separate sets of interactions, one with the phosphoryiated 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 β_2AR. A molecular model of the β_2AR-β-arrestin signalling complex was made by docking activated β-arrestin 1 and β_2AR 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.
机译：G蛋白偶联受体（GPCR）受到β-arrestin的严格调节，不仅使G蛋白信号脱敏，而且引发了G蛋白独立的信号波。最近在许多GPCR上的结构数据激增，包括β_2肾上腺素能受体（β_2AR）-G蛋白复合物，为受体激活的结构基础提供了新见解。但是，关于将β-arrestin募集至活化GPCR的补充信息一直缺乏，这主要是由于在获得用于结构研究的稳定受体-β-arrestin复合物方面的挑战。在这里，我们设计了一种形成和纯化功能性人β_2AR-β-arrestin-1复合物的策略，该复合物使我们能够通过单颗粒负染色电子显微镜观察其结构，并利用氢表征β_2AR和β-arrestin-1之间的相互作用。 -氘交换质谱法（HDX-MS）和化学交联。电子显微镜的二维平均和三维重建显示了β-arrestin1与β_2AR的双峰结合，涉及两组独立的相互作用，一组与受体的磷酸化羧基末端相互作用，另一组与其7个跨膜核心相互作用。 HDX减少的区域以及交联残基的鉴定表明β-arrestin1的指环与受体的七跨膜核心结合。相反，HDX水平升高的焦点区域表明，当与β_2AR偶联时，β-arrestin1的N和C结构域的动力学增强区域。 β_2AR-β-arrestin信号复合物的分子模型是通过将活化的β-arrestin1和β_2AR的晶体结构对接到电子显微镜图密度中并由HDX-MS和交联作用制得的，从而使我们可以获得对整体的有价值的见解。 -抑制蛋白复合物的结构。本文提供的动态和结构信息提供了一个框架，可让您更好地了解抑制蛋白对GPCR调控的基础。

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  • 来源
    《Nature》 |2014年第7513期|218-222|共5页
  • 作者

    Arun K. Shukla; Gerwin H. Westfield; Kunhong Xiao; Rosana I. Reis; Li-Yin Huang; Prachi Tripathi-Shukla; Jiang Qian; Sheng Li; Adi Blanc; Austin N. Oleskie; Anne M. Dosey; Min Su; Cui-Rong Liang; Ling-Ling Gu; Jin-Ming Shan; Xin Chen; Rachel Hanna; Minjung Choi; Xiao Jie Yao; Bjoern U. Klink; Alem W. Kahsai; Sachdev S. Sidhu; Shohei Koide; Pawel A. Penczek; Anthony A. Kossiakoff; Virgil L. Woods Jr; Brian K. Kobilka; Georgios Skiniotis; Robert J. Lefkowitz;

  • 作者单位

    Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710, USA,Department of Biological Sciencesand Bioengineering, Indian Institute of Technology, Kanpur 208016, India;

    Life Sciences Institute and Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA;

    Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710, USA;

    Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710, USA;

    Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710, USA;

    Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710, USA;

    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;

    Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710, USA;

    Life Sciences Institute and Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA;

    Life Sciences Institute and Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109, 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;

    School of Pharmaceutical & Life Sciences, Changzhou University, Changzhou, Jiangsu 213164, China;

    School of Pharmaceutical & Life Sciences, Changzhou University, Changzhou, Jiangsu 213164, China;

    School of Pharmaceutical & Life Sciences, Changzhou University, Changzhou, Jiangsu 213164, China;

    Terrence Donnelly Centre for Cellularand Biomolecular Research, University of Toronto, Toronto, Ontario M5S 3E1, Canada;

    Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA;

    Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710, USA;

    Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710, USA;

    Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710, USA;

    Terrence Donnelly Centre for Cellularand Biomolecular Research, University of Toronto, Toronto, Ontario M5S 3E1, Canada;

    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;

    Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois 60637, USA;

    Department of Chemistry, University of California at San Diego, La Jolla, California 92093, USA;

    Department of Molecular and Cellular Physiology, Stanford University School of Medicine, 279 Campus Drive, Stanford, California 94305, USA;

    Life Sciences Institute and Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA;

    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;

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