Phospholipase C-beta 1 is regulated by a pertussis toxin-insensitive G-protein.
Regulation of phospholipase C (PLC) by receptors is mediated either through protein tyrosine phosphorylation or by activation of GTP-binding proteins (Gp). For the latter, pertussis toxin (PT)-sensitive and -insensitive pathways have been described, indicating PLC regulation by at least two types of G-proteins. The identity of PLC isoenzymes which are regulated by either type of Gp remains to be determined. Thyrotropin-releasing hormone stimulates a PLC in GH3 cells via a PT-insensitive Gp. Reconstitution methods for the assay of the GH3-cell Gp were developed. Previously, the membrane PLC was found to be reversibly extracted from membranes by high salt and to be activated by guanosine 5'-[gamma-thio]triphosphate (GTP[S]) only when membrane-associated, suggesting that Gp was retained in salt-extracted membranes. In the present work, Gp was cholate-solubilized from PLC-deficient membranes and incorporated into phospholipid vesicles, which were found to confer GTP[S]- and AlF4(-)-stimulated activity on a solubilized membrane PLC. The reconstitution provided a direct assay for the GH3-cell Gp which was shown to be distinct from Gi, Go and Gs proteins by immunodepletion studies. Incorporation of G-protein beta-gamma subunits into phospholipid vesicles with Gp inhibited GTP[S]-stimulated activity in the reconstitution. The results indicated that Gp is a heterotrimeric G-protein with the properties expected for the PT-insensitive GH3-cell Gp protein. PLC-beta 1 was fully purified and shown to be regulated by Gp in the reconstitution. In contrast, PT-sensitive G-proteins failed to affect the activity of PLC-beta 1. The results indicate (1) that a PT-insensitive Gp regulates PLC-beta 1 and (2) that PT-sensitive and -insensitive pathways of PLC regulation employ different PLC isoenzymes as well as different G-proteins.
Martin
T F
TF
Department of Zoology, University of Wisconsin, Madison 53706.
Lewis
J E
JE
Kowalchyk
J A
JA
D000818:Animals; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology*; D002478:Cells, Cultured; D019826:Cholic Acid; D002793:Cholic Acids; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D007527:Isoenzymes / Q000378:metabolism*; D008081:Liposomes; D046911:Macromolecular Substances; D009711:Nucleotides / Q000494:pharmacology; D037342:Pertussis Toxin; D018129:Phosphatidylinositol Phosphates; D010716:Phosphatidylinositols / Q000378:metabolism; D010902:Pituitary Gland / Q000166:cytology; D011233:Precipitin Tests; D010738:Type C Phospholipases / Q000276:immunology / Q000378:metabolism*; D010566:Virulence Factors, Bordetella / Q000494:pharmacology*
D002793:Cholic Acids; D007527:Isoenzymes; D008081:Liposomes; D046911:Macromolecular Substances; D009711:Nucleotides; D018129:Phosphatidylinositol Phosphates; D010716:Phosphatidylinositols; D010566:Virulence Factors, Bordetella; C060980:phosphatidylinositol 3,4-diphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D037342:Pertussis Toxin; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins; D019826:Cholic Acid