Analysis and pharmacological targeting of phospholipase C beta interactions with G proteins.
Phosphatidylinositol-specific phospholipase C enzymes (PLC) catalyze hydrolysis of phosphatidylinositol 4,5-bisphosphate generating the second messengers diacylglycerol and inositol 1,4,5-triphosphate. Mammalian phosphoinositide-specific phospholipase C beta (PLCbeta) activity is regulated by the alpha(q) family of G-protein alpha subunits and by Gbetagamma subunits. Regulation of PLCbeta enzymatic activity can be assayed by reconstituting purified G-protein subunits with purified PLCbeta in the presence of phospholipid vesicles containing the substrate phosphatidylinositol 4,5-bisphosphate. This chapter describes methods for expression and purification of PLCbeta and Gbetagamma from insect cells, assay of G-protein-dependent regulation of PLC activity, and assessment of G-protein-PLC direct binding interactions. This combination of functional and direct binding analysis provides a powerful approach to characterizing PLC and G-protein interfaces, identifying inhibitors of this interaction, and potentially uncovering new modes of PLC regulation.
Lehmann
David M
DM
Department of Pharmacology and Physiology, University of Rochester School of Medicine and Dentistry, Rochester, New York, USA.
Yuan
Chujun
C
Smrcka
Alan V
AV
D000818:Animals; D002846:Chromatography, Affinity / Q000379:methods; D019204:GTP-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic; D006493:Heparin; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D007700:Kinetics; D008081:Liposomes; D008322:Mammals; D054799:Phospholipase C beta / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism; D011485:Protein Binding; D021122:Protein Subunits / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012685:Sepharose; D018411:Spodoptera / Q000201:enzymology; D013379:Substrate Specificity
D007527:Isoenzymes; D008081:Liposomes; D010743:Phospholipids; D021122:Protein Subunits; D011994:Recombinant Proteins; D006493:Heparin; D012685:Sepharose; D054799:Phospholipase C beta; D019204:GTP-Binding Proteins