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Global phosphorylation analysis of beta-arrestin-mediated signaling downstream of a seven transmembrane receptor (7TMR).

Publication ,  Journal Article
Xiao, K; Sun, J; Kim, J; Rajagopal, S; Zhai, B; Villén, J; Haas, W; Kovacs, JJ; Shukla, AK; Hara, MR; Hernandez, M; Lachmann, A; Zhao, S ...
Published in: Proc Natl Acad Sci U S A
August 24, 2010

beta-Arrestin-mediated signaling downstream of seven transmembrane receptors (7TMRs) is a relatively new paradigm for signaling by these receptors. We examined changes in protein phosphorylation occurring when HEK293 cells expressing the angiotensin II type 1A receptor (AT1aR) were stimulated with the beta-arrestin-biased ligand Sar(1), Ile(4), Ile(8)-angiotensin (SII), a ligand previously found to signal through beta-arrestin-dependent, G protein-independent mechanisms. Using a phospho-antibody array containing 46 antibodies against signaling molecules, we found that phosphorylation of 35 proteins increased upon SII stimulation. These SII-mediated phosphorylation events were abrogated after depletion of beta-arrestin 2 through siRNA-mediated knockdown. We also performed an MS-based quantitative phosphoproteome analysis after SII stimulation using a strategy of stable isotope labeling of amino acids in cell culture (SILAC). We identified 1,555 phosphoproteins (4,552 unique phosphopeptides), of which 171 proteins (222 phosphopeptides) showed increased phosphorylation, and 53 (66 phosphopeptides) showed decreased phosphorylation upon SII stimulation of the AT1aR. This study identified 38 protein kinases and three phosphatases whose phosphorylation status changed upon SII treatment. Using computational approaches, we performed system-based analyses examining the beta-arrestin-mediated phosphoproteome including construction of a kinase-substrate network for beta-arrestin-mediated AT1aR signaling. Our analysis demonstrates that beta-arrestin-dependent signaling processes are more diverse than previously appreciated. Notably, our analysis identifies an AT1aR-mediated cytoskeletal reorganization network whereby beta-arrestin regulates phosphorylation of several key proteins, including cofilin and slingshot. This study provides a system-based view of beta-arrestin-mediated phosphorylation events downstream of a 7TMR and opens avenues for research in a rapidly evolving area of 7TMR signaling.

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Published In

Proc Natl Acad Sci U S A

DOI

EISSN

1091-6490

Publication Date

August 24, 2010

Volume

107

Issue

34

Start / End Page

15299 / 15304

Location

United States

Related Subject Headings

  • beta-Arrestins
  • beta-Arrestin 2
  • Systems Biology
  • Signal Transduction
  • Receptors, G-Protein-Coupled
  • Receptor, Angiotensin, Type 1
  • RNA, Small Interfering
  • Proteome
  • Protein Kinases
  • Phosphorylation
 

Citation

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Xiao, K., Sun, J., Kim, J., Rajagopal, S., Zhai, B., Villén, J., … Lefkowitz, R. J. (2010). Global phosphorylation analysis of beta-arrestin-mediated signaling downstream of a seven transmembrane receptor (7TMR). Proc Natl Acad Sci U S A, 107(34), 15299–15304. https://doi.org/10.1073/pnas.1008461107
Xiao, Kunhong, Jinpeng Sun, Jihee Kim, Sudarshan Rajagopal, Bo Zhai, Judit Villén, Wilhelm Haas, et al. “Global phosphorylation analysis of beta-arrestin-mediated signaling downstream of a seven transmembrane receptor (7TMR).Proc Natl Acad Sci U S A 107, no. 34 (August 24, 2010): 15299–304. https://doi.org/10.1073/pnas.1008461107.
Xiao K, Sun J, Kim J, Rajagopal S, Zhai B, Villén J, et al. Global phosphorylation analysis of beta-arrestin-mediated signaling downstream of a seven transmembrane receptor (7TMR). Proc Natl Acad Sci U S A. 2010 Aug 24;107(34):15299–304.
Xiao, Kunhong, et al. “Global phosphorylation analysis of beta-arrestin-mediated signaling downstream of a seven transmembrane receptor (7TMR).Proc Natl Acad Sci U S A, vol. 107, no. 34, Aug. 2010, pp. 15299–304. Pubmed, doi:10.1073/pnas.1008461107.
Xiao K, Sun J, Kim J, Rajagopal S, Zhai B, Villén J, Haas W, Kovacs JJ, Shukla AK, Hara MR, Hernandez M, Lachmann A, Zhao S, Lin Y, Cheng Y, Mizuno K, Ma’ayan A, Gygi SP, Lefkowitz RJ. Global phosphorylation analysis of beta-arrestin-mediated signaling downstream of a seven transmembrane receptor (7TMR). Proc Natl Acad Sci U S A. 2010 Aug 24;107(34):15299–15304.
Journal cover image

Published In

Proc Natl Acad Sci U S A

DOI

EISSN

1091-6490

Publication Date

August 24, 2010

Volume

107

Issue

34

Start / End Page

15299 / 15304

Location

United States

Related Subject Headings

  • beta-Arrestins
  • beta-Arrestin 2
  • Systems Biology
  • Signal Transduction
  • Receptors, G-Protein-Coupled
  • Receptor, Angiotensin, Type 1
  • RNA, Small Interfering
  • Proteome
  • Protein Kinases
  • Phosphorylation