Quantification of isozyme-specific activation of phospholipase C-beta2 by Rac GTPases and phospholipase C-epsilon by Rho GTPases in an intact cell assay system.
Phospholipase C (PLC) catalyzes the hydrolysis of PtdIns(4,5)P2, which results in both formation of the second messengers Ins(1,4,5)P3 and diacylglycerol and alteration in the membrane association and/or activity of PtdIns(4,5)P2-binding proteins. The existence of 13 different PLC isozymes suggests multiple mechanisms of regulation of inositol lipid signaling, and the recent realization that Rho-family GTPases directly bind and activate certain PLC isozymes has added to this potential diversity of inositol lipid-related signal transduction. With the goal of delineating a less labor-intensive method for quantification of intracellular inositol phosphate production, we have applied a commercially available yttrium silicate RNA binding resin selective for inositol phosphates to develop a high-throughput inositol phosphate scintillation proximity assay (SPA). We highlight the utility of this assay using COS-7 cells robotically transfected in a 96-well format. This method is readily applied to quantify activation of PLC by receptors and G proteins, and we illustrate here the selective activation of PLC-beta2 by Rac but not by Rho GTPases and the selective activation of PLC-epsilon by Rho but not Rac GTPases.
Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill, USA.
David M
DM
Bourdon
Michele R
MR
Wing
Earl B
EB
Edwards
John
J
Sondek
T Kendall
TK
Harden
D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D004789:Enzyme Activation; D007527:Isoenzymes / Q000378:metabolism*; D010716:Phosphatidylinositols / Q000378:metabolism; D054801:Phosphoinositide Phospholipase C; D054799:Phospholipase C beta; D014162:Transfection / Q000379:methods; D010738:Type C Phospholipases / Q000378:metabolism*; D020744:rac GTP-Binding Proteins / Q000378:metabolism*; D020741:rho GTP-Binding Proteins / Q000378:metabolism*
D007527:Isoenzymes; D010716:Phosphatidylinositols; D010738:Type C Phospholipases; D054801:Phosphoinositide Phospholipase C; D054799:Phospholipase C beta; C423320:phospholipase C epsilon; D020744:rac GTP-Binding Proteins; D020741:rho GTP-Binding Proteins