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P2Y1 receptor signaling is controlled by interaction with the PDZ scaffold NHERF-2

Sami R Fam1, Maryse Paquet, Amanda M Castleberry

  • 1Department of Pharmacology, Yerkes National Primate Research Center, and Division of Digestive Disease, Emory University School of Medicine, Atlanta, GA 30322, USA.

Proceedings of the National Academy of Sciences of the United States of America
|May 20, 2005
PubMed

Insights

The scaffold protein NHERF-2 specifically binds to P2Y(1) purinergic receptors (P2Y(1)Rs). This interaction regulates intracellular calcium signaling duration, explaining cell-specific P2Y(1)R activity.

Area of Science:

  • Cellular signaling
  • Molecular biology
  • Proteomics

Background:

  • P2Y(1) purinergic receptors (P2Y(1)Rs) are crucial for cellular responses to ATP, but their signaling characteristics, particularly calcium (Ca2+) dynamics, vary significantly across different cell types.
  • The precise molecular mechanisms dictating these cell-specific differences in P2Y(1)R-mediated Ca2+ signaling remain incompletely understood.

Purpose of the Study:

  • To identify specific protein interactions with the P2Y(1)R carboxyl terminus that may modulate its signaling.
  • To investigate the role of identified interacting proteins in regulating P2Y(1)R-mediated Ca2+ signaling duration and characteristics.

Main Methods:

  • Proteomic screening of the P2Y(1)R carboxyl terminus against a library of PDZ (PSD-95/Drosophila Discs large/ZO-1 homology) domains.
  • Co-immunoprecipitation assays to confirm protein-protein interactions in cellular contexts.
  • Calcium imaging studies in glial cells to assess the impact of NHERF-2 on P2Y(1)R-mediated Ca2+ responses.
  • Site-directed mutagenesis to disrupt the P2Y(1)R-NHERF-2 interaction and evaluate functional consequences.

Main Results:

  • A specific interaction was identified between the P2Y(1)R carboxyl terminus and the second PDZ domain of NHERF-2 (Na+/H+ exchanger regulatory factor type 2).
  • P2Y(1)R and NHERF-2 were shown to associate within cells, facilitating the tethering of P2Y(1)R to downstream effectors like phospholipase Cbeta.
  • Coexpression of P2Y(1)R with NHERF-2 in glial cells significantly prolonged P2Y(1)R-mediated Ca2+ signaling.
  • Disruption of the P2Y(1)R-NHERF-2 interaction via point mutations attenuated the duration of P2Y(1)R-mediated Ca2+ responses.

Conclusions:

  • NHERF-2 acts as a critical regulator of P2Y(1)R cellular activity.
  • The interaction between NHERF-2 and P2Y(1)R is a key determinant of the duration and characteristics of P2Y(1)R-mediated Ca2+ signaling.
  • This interaction provides a molecular basis for the observed cell-specific differences in P2Y(1)R signaling pathways.

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