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

p130 was originally identified as an Ins(1,4,5)P-3-binding protein similar to phospholipase C-delta but lacking any phospholipase activity. In the pre sent study we have further analysed the interactions of p130 with inositol compounds in vitro. To determine which of the potential ligands interacts w ith 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)P-3-mediated Ca2+ signalling b y using permeabilized and transiently or stably transfected COS-1 cells (CO S-1(p130)). In vitro, p130 bound Ins(1,4,5)P-3 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) P-3. Localization studies demonstrated the presence of the full-length p130 in the cytoplasm of living cells, not at the plasma membrane. In cell-base d assays, p130 had an inhibitory effect on Ca2+ signalling. When fura-2-loa ded COS-1(p130) cells were stimulated with bradykinin, epidermal growth fac tor or ATP, it was found that the agonist-induced increase in free Ca2+ con centration, observed in control cells, was inhibited in COS-1(p130). This i nhibition was not accompanied by the decreased production of Ins(1,4,5)P-3; the intact p130 pleckstrin homology domain, known to be the ligand-binding site in vitro, was required for this effect in cells. These results sugges t that Ins(1,4,5)P-3 could be the main p130 ligand in cells and that this b inding has the potential to inhibit Ins(1,4,5)P-3-mediated Ca2+ signalling.