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Co-immunoprecipitation Assay for Studying Functional Interactions Between Receptors and Enzymes
09:40

Co-immunoprecipitation Assay for Studying Functional Interactions Between Receptors and Enzymes

Published on: September 28, 2018

An ITIM-like motif within the CCK2 receptor sequence required for interaction with SHP-2 and the activation of the

Sébastien Vatinel1, Audrey Ferrand, Fréderic Lopez

  • 1INSERM U 531, IFR 31, Institut Louis Bugnard, BP 84225, 31432 Toulouse cedex 4, France.

Biochimica Et Biophysica Acta
|September 12, 2006
PubMed

Insights

This study reveals that the Cholecystokinin 2 receptor (CCK2R) directly interacts with SHP-2 phosphatase, activating the AKT pathway. This finding clarifies a novel role for SHP-2 in G protein-coupled receptor signaling.

Area of Science:

  • Molecular Cell Biology
  • Signal Transduction
  • Gastroenterology

Background:

  • SHP-2 tyrosine phosphatase positively regulates receptor tyrosine kinase (RTK) pathways.
  • G protein-coupled receptors (GPCRs) are rarely linked to SHP-2, and its role in GPCR signaling is poorly understood.
  • The CCK2 receptor (CCK2R) mediates gastrin's mitogenic effects in gastrointestinal cells.

Purpose of the Study:

  • To investigate the role of SHP-2 in CCK2R-mediated signaling.
  • To elucidate the molecular mechanism connecting CCK2R and SHP-2.
  • To determine if SHP-2 activation by CCK2R influences the AKT pathway.

Main Methods:

  • Surface plasmon resonance (SPR) to analyze receptor-phosphatase interaction.
  • Pull-down assays and co-immunoprecipitation to confirm protein interactions.
  • Mutational analysis of the CCK2R C-terminal tail.

Main Results:

  • Identified a direct interaction between SHP-2 and a conserved ITIM motif (tyrosine 438) in the CCK2R C-terminal tail.
  • Confirmed the interaction using SPR, pull-down assays, and co-immunoprecipitation.
  • Demonstrated that CCK2R-SHP-2 interaction and AKT pathway activation are dependent on the ITIM motif and increase upon gastrin stimulation.

Conclusions:

  • The CCK2R directly binds to SHP-2 via its ITIM motif.
  • This interaction is crucial for SHP-2 recruitment and subsequent activation of the AKT pathway.
  • Establishes a novel mechanism for SHP-2 involvement in GPCR signaling, particularly in CCK2R-mediated mitogenesis.

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