Inhibition of Ca(2+) 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 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)P(3)-mediated Ca(2+) signalling by using permeabilized and transiently or stably transfected COS-1 cells (COS-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-based assays, p130 had an inhibitory effect on Ca(2+) 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 Ca(2+) 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)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 suggest that Ins(1,4,5)P(3) could be the main p130 ligand in cells and that this binding has the potential to inhibit Ins(1,4,5)P(3)-mediated Ca(2+) signalling.
Takeuchi
H
H
Department of Biochemistry, Faculty of Dentistry, Kyushu University and Kyushu University Station for Collaborative Research, Fukuoka 812-8582, Japan.
Oike
M
M
Paterson
H F
HF
Allen
V
V
Kanematsu
T
T
Ito
Y
Y
Erneux
C
C
Katan
M
M
Hirata
M
M
D016475:3T3 Cells; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001798:Blood Proteins / Q000737:chemistry; D001920:Bradykinin / Q000494:pharmacology; D019556:COS Cells; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000737:chemistry; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism*; D002384:Catalysis; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004285:Dogs; D004305:Dose-Response Relationship, Drug; D004815:Epidermal Growth Factor / Q000494:pharmacology; D049452:Green Fluorescent Proteins; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D053496:Inositol 1,4,5-Trisphosphate Receptors; D008024:Ligands; D008081:Liposomes / Q000378:metabolism; D008164:Luminescent Proteins / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D010750:Phosphoproteins / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction; D013997:Time Factors; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism*
D001798:Blood Proteins; D015220:Calcium Channels; D002352:Carrier Proteins; D053496:Inositol 1,4,5-Trisphosphate Receptors; D008024:Ligands; D008081:Liposomes; D008164:Luminescent Proteins; D010750:Phosphoproteins; C430714:Plcl1 protein, rat; D018160:Receptors, Cytoplasmic and Nuclear; D011993:Recombinant Fusion Proteins; C039077:platelet protein P47; D049452:Green Fluorescent Proteins; D004815:Epidermal Growth Factor; D015544:Inositol 1,4,5-Trisphosphate; D000255:Adenosine Triphosphate; D010738:Type C Phospholipases; D001920:Bradykinin; D002118:Calcium