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RGS16 function is regulated by epidermal growth factor receptor-mediated tyrosine phosphorylation

A Derrien1, K M Druey

  • 1Molecular Signal Transduction Section, Laboratory of Allergic Diseases, National Institutes of Health, Rockville, Maryland 20852, USA.

The Journal of Biological Chemistry
|October 17, 2001
PubMed

Insights

Epidermal Growth Factor Receptor (EGFR) phosphorylates Regulator of G protein Signaling 16 (RGS16) on tyrosine residues, enhancing its activity. This EGFR-mediated RGS16 phosphorylation may inhibit Galpha(i)-dependent MAPK activation via a feedback loop.

Area of Science:

  • Cellular signaling pathways
  • Signal transduction mechanisms
  • G protein-coupled receptors

Background:

  • Galpha(i)-coupled receptor stimulation leads to epidermal growth factor receptor (EGFR) phosphorylation and MAPK activation.
  • Regulators of G protein signaling (RGS proteins) attenuate G protein signaling by accelerating Galpha(i) GTP hydrolysis.
  • RGS16 possesses two tyrosine residues (Tyr168 and Tyr177) in its RGS box, predicted sites for phosphorylation.

Purpose of the Study:

  • To investigate the role of RGS16 tyrosine phosphorylation in regulating Galpha(i)-dependent signaling.
  • To determine if EGFR directly phosphorylates RGS16 and how this affects RGS16 function.
  • To elucidate the potential feedback mechanism involving EGFR, RGS16, and MAPK activation.

Main Methods:

  • HEK 293T and COS-7 cells were used to study RGS16 phosphorylation upon receptor stimulation.
  • Mutational analysis (Tyr168Phe, Tyr177Phe) of RGS16 was performed.
  • Co-immunoprecipitation assays were used to assess RGS16-EGFR interaction.
  • In vitro kinase assays with purified EGFR and recombinant RGS16 were conducted.
  • GTPase accelerating protein (GAP) activity assays and measurements of G(i)-mediated MAPK activation and adenylyl cyclase inhibition were performed.

Main Results:

  • RGS16 phosphorylation occurred in response to m2 muscarinic receptor or EGFR stimulation, requiring EGFR kinase activity.
  • EGFR directly phosphorylated RGS16 at Tyr168 in vitro.
  • Phosphorylated RGS16 exhibited enhanced GTPase accelerating (GAP) activity towards Galpha(i).
  • Mutation of Tyr168 abolished RGS16's ability to regulate G(i)-mediated MAPK activation and adenylyl cyclase inhibition, while mutation of Tyr177 had no effect on GAP activity but also abolished signaling regulation.
  • RGS16 co-immunoprecipitated with EGFR, independent of EGFR activation.

Conclusions:

  • Tyrosine phosphorylation, particularly at Tyr168, is a critical regulator of RGS16 function.
  • EGFR enhances RGS16's GAP activity through tyrosine phosphorylation.
  • EGFR may inhibit Galpha(i)-dependent MAPK activation via a feedback loop involving enhanced RGS16 activity.

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