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 (G(p)). 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 G(p) remains to be determined. Thyrotropin-releasing hormone stimulates a PLC in GH3 cells via a PT-insensitive G(p). Reconstitution methods for the assay of the GH3-cell G(p) 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 G(p) was retained in salt-extracted membranes. In the present work, G(p) 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 G(p) which was shown to be distinct from G(i), G(o) and G(s) proteins by immunodepletion studies. Incorporation of G-protein beta-gamma subunits into phospholipid vesicles with G(p) inhibited GTP[S]-stimulated activity in the reconstitution. The results indicated that G(p) is a heterotrimeric G-protein with the properties expected for the PT-insensitive GH3-cell G(p) protein. PLC-beta-1 was fully purified and shown to be regulated by G(p) 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 G(p) 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.