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Manifold roles of β-arrestins in GPCR signaling elucidated with siRNA and CRISPR/Cas9.

Publication ,  Journal Article
Luttrell, LM; Wang, J; Plouffe, B; Smith, JS; Yamani, L; Kaur, S; Jean-Charles, P-Y; Gauthier, C; Lee, M-H; Pani, B; Kim, J; Ahn, S ...
Published in: Sci Signal
September 25, 2018

G protein-coupled receptors (GPCRs) use diverse mechanisms to regulate the mitogen-activated protein kinases ERK1/2. β-Arrestins (βArr1/2) are ubiquitous inhibitors of G protein signaling, promoting GPCR desensitization and internalization and serving as scaffolds for ERK1/2 activation. Studies using CRISPR/Cas9 to delete βArr1/2 and G proteins have cast doubt on the role of β-arrestins in activating specific pools of ERK1/2. We compared the effects of siRNA-mediated knockdown of βArr1/2 and reconstitution with βArr1/2 in three different parental and CRISPR-derived βArr1/2 knockout HEK293 cell pairs to assess the effect of βArr1/2 deletion on ERK1/2 activation by four Gs-coupled GPCRs. In all parental lines with all receptors, ERK1/2 stimulation was reduced by siRNAs specific for βArr2 or βArr1/2. In contrast, variable effects were observed with CRISPR-derived cell lines both between different lines and with activation of different receptors. For β2 adrenergic receptors (β2ARs) and β1ARs, βArr1/2 deletion increased, decreased, or had no effect on isoproterenol-stimulated ERK1/2 activation in different CRISPR clones. ERK1/2 activation by the vasopressin V2 and follicle-stimulating hormone receptors was reduced in these cells but was enhanced by reconstitution with βArr1/2. Loss of desensitization and receptor internalization in CRISPR βArr1/2 knockout cells caused β2AR-mediated stimulation of ERK1/2 to become more dependent on G proteins, which was reversed by reintroducing βArr1/2. These data suggest that βArr1/2 function as a regulatory hub, determining the balance between mechanistically different pathways that result in activation of ERK1/2, and caution against extrapolating results obtained from βArr1/2- or G protein-deleted cells to GPCR behavior in native systems.

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Published In

Sci Signal

DOI

EISSN

1937-9145

Publication Date

September 25, 2018

Volume

11

Issue

549

Location

United States

Related Subject Headings

  • beta-Arrestins
  • Signal Transduction
  • Receptors, G-Protein-Coupled
  • Receptors, Adrenergic, beta-2
  • RNA, Small Interfering
  • Phosphorylation
  • MAP Kinase Signaling System
  • Humans
  • HEK293 Cells
  • Gene Knockdown Techniques
 

Citation

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Luttrell, L. M., Wang, J., Plouffe, B., Smith, J. S., Yamani, L., Kaur, S., … Lefkowitz, R. J. (2018). Manifold roles of β-arrestins in GPCR signaling elucidated with siRNA and CRISPR/Cas9. Sci Signal, 11(549). https://doi.org/10.1126/scisignal.aat7650
Luttrell, Louis M., Jialu Wang, Bianca Plouffe, Jeffrey S. Smith, Lama Yamani, Suneet Kaur, Pierre-Yves Jean-Charles, et al. “Manifold roles of β-arrestins in GPCR signaling elucidated with siRNA and CRISPR/Cas9.Sci Signal 11, no. 549 (September 25, 2018). https://doi.org/10.1126/scisignal.aat7650.
Luttrell LM, Wang J, Plouffe B, Smith JS, Yamani L, Kaur S, et al. Manifold roles of β-arrestins in GPCR signaling elucidated with siRNA and CRISPR/Cas9. Sci Signal. 2018 Sep 25;11(549).
Luttrell, Louis M., et al. “Manifold roles of β-arrestins in GPCR signaling elucidated with siRNA and CRISPR/Cas9.Sci Signal, vol. 11, no. 549, Sept. 2018. Pubmed, doi:10.1126/scisignal.aat7650.
Luttrell LM, Wang J, Plouffe B, Smith JS, Yamani L, Kaur S, Jean-Charles P-Y, Gauthier C, Lee M-H, Pani B, Kim J, Ahn S, Rajagopal S, Reiter E, Bouvier M, Shenoy SK, Laporte SA, Rockman HA, Lefkowitz RJ. Manifold roles of β-arrestins in GPCR signaling elucidated with siRNA and CRISPR/Cas9. Sci Signal. 2018 Sep 25;11(549).

Published In

Sci Signal

DOI

EISSN

1937-9145

Publication Date

September 25, 2018

Volume

11

Issue

549

Location

United States

Related Subject Headings

  • beta-Arrestins
  • Signal Transduction
  • Receptors, G-Protein-Coupled
  • Receptors, Adrenergic, beta-2
  • RNA, Small Interfering
  • Phosphorylation
  • MAP Kinase Signaling System
  • Humans
  • HEK293 Cells
  • Gene Knockdown Techniques