TY - JOUR
T1 - GPCR-mediated β-arrestin activation deconvoluted with single-molecule precision
AU - Asher, Wesley B.
AU - Terry, Daniel S.
AU - Gregorio, G. Glenn A.
AU - Kahsai, Alem W.
AU - Borgia, Alessandro
AU - Xie, Bing
AU - Modak, Arnab
AU - Zhu, Ying
AU - Jang, Wonjo
AU - Govindaraju, Alekhya
AU - Huang, Li Yin
AU - Inoue, Asuka
AU - Lambert, Nevin A.
AU - Gurevich, Vsevolod V.
AU - Shi, Lei
AU - Lefkowitz, Robert J.
AU - Blanchard, Scott C.
AU - Javitch, Jonathan A.
N1 - Funding Information:
This work was supported by NIH grants R21NS102694 (S.C.B.), R01MH054137 (J.A.J.), R01HL016037 (R.J.L.), R35 GM122491 (V.V.G.), and R01 GM130142 (N.A.L.); the Hope for Depression Research Foundation (J.A.J.); the Brain and Behavior Research Foundation NARSAD Young Investigator Award (W.B.A.); the National Institute on Drug Abuse – Intramural Research Program (Z1A DA000606 , L.S.); the PhRMA Foundation (W.J.); and the Single-Molecule Imaging Center at St. Jude Children’s Research Hospital . R.J.L. is an HHMI Investigator. This work utilized the computational resources of the NIH HPC Biowulf cluster ( http://hpc.nih.gov ). We thank Michael Berne ( Tufts University Core Facility ) for preparing the synthetic peptides and related discussions. We thank Harel Weinstein and Giulia Morra for helpful discussion about βarr dynamics; Xuanzhi Zhan and Sergey Vishnivetskiy for helpful discussions concerning βarr expression; and Marina Dawoud, Lisa Lin, and Mel Nelson for technical assistance. We thank Kuo-Hao Lee for help with Rosetta modeling. We thank Ines Chen for editing the manuscript and Zhaowen Luo for assistance rendering protein structures and schematics. This work is dedicated to the life and memory of Miriam Javitch, who loved hearing the story and looking at the figures as they emerged.
Funding Information:
This work was supported by NIH grants R21NS102694 (S.C.B.), R01MH054137 (J.A.J.), R01HL016037 (R.J.L.), R35 GM122491 (V.V.G.), and R01 GM130142 (N.A.L.); the Hope for Depression Research Foundation (J.A.J.); the Brain and Behavior Research Foundation NARSAD Young Investigator Award (W.B.A.); the National Institute on Drug Abuse – Intramural Research Program (Z1A DA000606, L.S.); the PhRMA Foundation (W.J.); and the Single-Molecule Imaging Center at St. Jude Children's Research Hospital. R.J.L. is an HHMI Investigator. This work utilized the computational resources of the NIH HPC Biowulf cluster (http://hpc.nih.gov). We thank Michael Berne (Tufts University Core Facility) for preparing the synthetic peptides and related discussions. We thank Harel Weinstein and Giulia Morra for helpful discussion about βarr dynamics; Xuanzhi Zhan and Sergey Vishnivetskiy for helpful discussions concerning βarr expression; and Marina Dawoud, Lisa Lin, and Mel Nelson for technical assistance. We thank Kuo-Hao Lee for help with Rosetta modeling. We thank Ines Chen for editing the manuscript and Zhaowen Luo for assistance rendering protein structures and schematics. This work is dedicated to the life and memory of Miriam Javitch, who loved hearing the story and looking at the figures as they emerged. W.B.A. D.S.T. S.C.B. and J.A.J. wrote the manuscript, with contributions from all the authors. W.B.A. designed and optimized the βarr samples for smFRET studies. W.B.A. and A.G. prepared and labeled the βarr samples. W.B.A. G.G.A.G. D.S.T. A.B. S.C.B. and J.A.J. designed the single-molecule experiments and interpreted the results with critical input from A.W.K. and R.J.L. W.B.A. and G.G.A.G. performed groundwork TIRF-based smFRET experiments and analysis, and D.S.T. performed the TIRF-based smFRET experiments and analyzed the data shown in the manuscript. A.M. performed the smFRET experiments with IP6 and PIP2. A.B. designed and performed the confocal single-molecule FRET and FCS experiments and the corresponding data analysis. W.B.A. and Y.Z. performed the live-cell βarr recruitment assays. N.A.L. and W.J. designed and W.J. performed live-cell mini-Gs recruitment assays. L.S. and B.X. performed molecular dynamics simulations and the corresponding analysis. A.W.K. prepared β2V2R, synthesized Cmpd-6FA, and performed the ITC experiments. V.V.G. provided cysteine-less βarr1 constructs and helped guide sensor development. A.I. provided the HEK GRK KO cells. S.C.B. and J.A.J. supervised the project. S.C.B. has an equity interest in Lumidyne Technologies.
Publisher Copyright:
© 2022 The Authors
PY - 2022/5/12
Y1 - 2022/5/12
N2 - β-arrestins bind G protein-coupled receptors to terminate G protein signaling and to facilitate other downstream signaling pathways. Using single-molecule fluorescence resonance energy transfer imaging, we show that β-arrestin is strongly autoinhibited in its basal state. Its engagement with a phosphopeptide mimicking phosphorylated receptor tail efficiently releases the β-arrestin tail from its N domain to assume distinct conformations. Unexpectedly, we find that β-arrestin binding to phosphorylated receptor, with a phosphorylation barcode identical to the isolated phosphopeptide, is highly inefficient and that agonist-promoted receptor activation is required for β-arrestin activation, consistent with the release of a sequestered receptor C tail. These findings, together with focused cellular investigations, reveal that agonism and receptor C-tail release are specific determinants of the rate and efficiency of β-arrestin activation by phosphorylated receptor. We infer that receptor phosphorylation patterns, in combination with receptor agonism, synergistically establish the strength and specificity with which diverse, downstream β-arrestin-mediated events are directed.
AB - β-arrestins bind G protein-coupled receptors to terminate G protein signaling and to facilitate other downstream signaling pathways. Using single-molecule fluorescence resonance energy transfer imaging, we show that β-arrestin is strongly autoinhibited in its basal state. Its engagement with a phosphopeptide mimicking phosphorylated receptor tail efficiently releases the β-arrestin tail from its N domain to assume distinct conformations. Unexpectedly, we find that β-arrestin binding to phosphorylated receptor, with a phosphorylation barcode identical to the isolated phosphopeptide, is highly inefficient and that agonist-promoted receptor activation is required for β-arrestin activation, consistent with the release of a sequestered receptor C tail. These findings, together with focused cellular investigations, reveal that agonism and receptor C-tail release are specific determinants of the rate and efficiency of β-arrestin activation by phosphorylated receptor. We infer that receptor phosphorylation patterns, in combination with receptor agonism, synergistically establish the strength and specificity with which diverse, downstream β-arrestin-mediated events are directed.
KW - G protein-coupled receptor
KW - GPCR
KW - agonist
KW - arrestin
KW - conformational dynamics
KW - phosphorylation
KW - phosphorylation barcode
KW - receptor signaling
KW - single-molecule FRET
KW - β-arrestin
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U2 - 10.1016/j.cell.2022.03.042
DO - 10.1016/j.cell.2022.03.042
M3 - Article
C2 - 35483373
AN - SCOPUS:85129971652
SN - 0092-8674
VL - 185
SP - 1661-1675.e16
JO - Cell
JF - Cell
IS - 10
ER -