Inhibition of Ca2+ signalling by p130, a phospholipase-C-related catalytically inactive protein: Critical role of the p130 pleckstrin homology domain

Hiroshi Takeuchi, Masahiro Oike, Hugh F. Paterson, Victoria Allen, Takashi Kanematsu, Yushi Ito, Christophe Erneux, Matilda Katan, Masato Hirata

Research output: Contribution to journalArticlepeer-review

52 Citations (Scopus)

Abstract

p130 was originally identified as an Ins(1,4,5)P3-binding protein similar to phospholipase C-δ but lacking any phospholipase activity. In the present study we have further analysed the interactions of p130 with inositol compounds in vitro. To determine which of the potential ligands interacts with p130 in cells, we performed an analysis of the cellular localization of this protein, the isolation of a protein-ligand complex from cell lysates and studied the effects of p130 on Ins(1,4,5)P3-mediated Ca2+ signalling by using permeabilized and transiently or stably transfected COS-1 cells (COS-1(p130)). In vitro, p130 bound Ins(1,4,5)P3 with a higher affinity than that for phosphoinositides. When the protein was isolated from COS-1(p130) cells by immunoprecipitation, it was found to be associated with Ins(1,4,5)P3. Localization studies demonstrated the presence of the full-length p130 in the cytoplasm of living cells, not at the plasma membrane. In cell-based assays, p130 had an inhibitory effect on Ca2+ signalling. When fura-2-loaded COS-1(p130) cells were stimulated with bradykinin, epidermal growth factor or ATP, it was found that the agonist-induced increase in free Ca2+ concentration, observed in control cells, was inhibited in COS-1(p130). This inhibition was not accompanied by the decreased production of Ins(1,4,5)P3; the intact p130 pleckstrin homology domain, known to be the ligand-binding site in vitro, was required for this effect in cells. These results suggest that Ins(1,4,5)P3 could be the main p130 ligand in cells and that this binding has the potential to inhibit Ins(1,4,5)P3-mediated Ca2+ signalling.

Original languageEnglish
Pages (from-to)357-368
Number of pages12
JournalBiochemical Journal
Volume349
Issue number1
DOIs
Publication statusPublished - Jul 1 2000

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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