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Molecular Pharmacology

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Research ArticleArticle

Influence of the Accessory Protein SET on M3 Muscarinic Receptor Phosphorylation and G Protein Coupling

Violaine Simon, Sukru S. Oner, Joelle Cohen-Tannoudji, Andrew B. Tobin and Stephen M. Lanier
Molecular Pharmacology July 2012, 82 (1) 17-26; DOI: https://doi.org/10.1124/mol.111.075523
Violaine Simon
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Sukru S. Oner
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Joelle Cohen-Tannoudji
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Andrew B. Tobin
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Stephen M. Lanier
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Abstract

The proto-oncogene and inhibitor of protein phosphatase 2A (PP2A), SET, interacts with the third intracellular loop of the M3 muscarinic receptor (M3-MR), and SET knockdown with small interfering RNA (siRNA) in Chinese hamster ovary (CHO) cells augments M3-MR signaling. However, the mechanism of this action of SET on receptor signaling has not been defined, and we initiated studies to address this question. Knockdown of SET by siRNA in CHO cells stably expressing the M3-MR did not alter agonist-induced receptor phosphorylation or receptor internalization. Instead, it increased the extent of receptor dephosphorylation after agonist removal by ∼60%. In competition binding assays, SET knockdown increased high-affinity binding of agonist in intact cells and membrane preparations. Glutathione transferase pull-down assays and site-directed mutagenesis revealed a SET binding site adjacent to and perhaps overlapping the G protein-binding site within the third intracellular loop of the receptor. Mutation of this region in the M3-MR altered receptor coupling to G protein. These data indicate that SET decreases M3-MR dephosphorylation and regulates receptor engagement with G protein, both of which may contribute to the inhibitory action of SET on M3-MR signaling.

Footnotes

  • ↵Embedded Image The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.

  • The work was supported by the Wellcome Trust [Grant 047600]; the National Institutes of Health National Institute of Mental Health [Grant MH90531]; the National Institutes of Health National Institute of Neurological Disorders and Stroke [Grant NS24821]; Fondation pour la Recherche Médicale; and University Paris 7. S.M.L. was supported by the David R. Bethune/Lederle Laboratories Professorship in Pharmacology and a Research Scholar Award from Yamanouchi Pharmaceutical Company (now Astellas Pharma).

  • Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.

    http://dx.doi.org/10.1124/mol.111.075523.

  • ABBREVIATIONS:

    GPCR
    G protein-coupled receptor
    M3-MR
    M3 muscarinic receptor(s)
    PP2A
    protein phosphatase 2A
    GRK
    G protein-coupled receptor kinase
    siRNA
    small interfering RNA
    CHO
    Chinese hamster ovary
    bp
    base pair
    GST
    glutathione transferase
    PAGE
    polyacrylamide gel electrophoresis
    NMS
    N-methylscopolamine
    GppNHp
    5′-guanylylimidodiphosphate
    M3-i3
    third intracellular loop of M3-MR
    i3 loop
    third intracellular loop.

  • Received September 6, 2011.
  • Accepted March 30, 2012.
  • Copyright © 2012 The American Society for Pharmacology and Experimental Therapeutics
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Molecular Pharmacology: 82 (1)
Molecular Pharmacology
Vol. 82, Issue 1
1 Jul 2012
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Research ArticleArticle

SET and M3 Muscarinic Receptor Regulation

Violaine Simon, Sukru S. Oner, Joelle Cohen-Tannoudji, Andrew B. Tobin and Stephen M. Lanier
Molecular Pharmacology July 1, 2012, 82 (1) 17-26; DOI: https://doi.org/10.1124/mol.111.075523

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Research ArticleArticle

SET and M3 Muscarinic Receptor Regulation

Violaine Simon, Sukru S. Oner, Joelle Cohen-Tannoudji, Andrew B. Tobin and Stephen M. Lanier
Molecular Pharmacology July 1, 2012, 82 (1) 17-26; DOI: https://doi.org/10.1124/mol.111.075523
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