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Influence of the accessory protein SET on M3 muscarinic receptor phosphorylation and G protein coupling

Violaine Simon1, Sukru S Oner, Joelle Cohen-Tannoudji

  • 1University Paris Diderot, Sorbonne Paris Cité, Biologie Fonctionnelle et Adaptative, Centre National de la Recherche Scientifique-Equipe d’Accueil Conventionée 4413, Paris, France.

Molecular Pharmacology
|April 3, 2012
PubMed

Insights

The SET protein inhibits muscarinic acetylcholine receptor 3 (M3-MR) signaling by decreasing receptor dephosphorylation and regulating G protein engagement. SET knockdown enhances M3-MR signaling.

Area of Science:

  • Molecular pharmacology
  • Cellular signaling
  • G protein-coupled receptors

Background:

  • The proto-oncogene SET is an inhibitor of protein phosphatase 2A (PP2A).
  • SET interacts with the M3 muscarinic receptor (M3-MR) and its knockdown augments M3-MR signaling.
  • The precise mechanism by which SET influences M3-MR signaling remains undefined.

Purpose of the Study:

  • To elucidate the mechanism of SET's action on M3-MR signaling.
  • To investigate SET's role in receptor dephosphorylation and G protein coupling.

Main Methods:

  • Small interfering RNA (siRNA) mediated knockdown of SET in Chinese hamster ovary (CHO) cells expressing M3-MR.
  • Assessment of agonist-induced receptor phosphorylation and internalization.
  • Measurement of receptor dephosphorylation kinetics after agonist removal.
  • Competition binding assays to evaluate agonist binding affinity.
  • Glutathione transferase pull-down assays and site-directed mutagenesis to identify SET binding sites and assess G protein coupling.

Main Results:

  • SET knockdown did not affect agonist-induced M3-MR phosphorylation or internalization.
  • SET knockdown increased M3-MR dephosphorylation by approximately 60% after agonist removal.
  • SET knockdown enhanced high-affinity agonist binding to M3-MR.
  • A SET binding site was identified in the M3-MR's third intracellular loop, adjacent to the G protein-binding site.
  • Mutation of this region impaired M3-MR coupling to G protein.

Conclusions:

  • SET inhibits M3-MR signaling by reducing receptor dephosphorylation.
  • SET regulates M3-MR engagement with G proteins.
  • These actions of SET contribute to its inhibitory effect on M3-MR signaling.

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