Purification and gr protein subunit regulation of a phospholipase C-beta from Xenopus laevis oocytes

Xenopus oocytes exhibit both pertussis toxin-sensitive and -insensitive inositol lipid signaling responses to G protein-coupled receptor activation. The G protein subunits G alpha(i), G alpha(o), G alpha(q), G alpha(s), and G(beta gamma) all have been proposed to function as activators of phospholipase C in oocytes. Ma et al. (Ma, H.-W., Blitzer, R, D., Healy, E. C., Premont, R. T., Landau, E. M., and Iyengar, R. J. Biol. Chem. 268, 19915-19918) cloned a Xenopus phospholipase C (PLC-beta X) that exhibits homology to the PLC-beta class of isoenzymes. Although this enzyme was proposed to function as a signaling protein in the pertussis toxin sensitive inositol Lipid signaling pathway of oocytes, its regulation by G protein subunits has not been directly assessed. As such we have utilized baculovirus-promoted overexpression of PLC-beta X in Sf9 insect cells and have purified a recombinant 150-kDa isoenzyme. PLC-beta X catalyzes hydrolysis of phosphatidylinositol(4,5)bisphosphate and phosphatidylinositol(4)monophosphate, and reaction velocity is de pendent on Ca2+. Recombinant PLC-beta X was activated by both G alpha(q), and G(beta gamma). PLC-beta X exhibited a higher apparent affinity for G alpha(q) than G(beta gamma), and G alpha(q) was more efficacious than G(beta gamma) at lower concentrations of PLC-beta X Relative to other PLC-beta isoenzymes, PLC-beta X was less sensitive to stimulation by Ga-q than PLC-beta 1 but similar to PLC-beta 2 and PLC-beta T, PLC beta X was more sensitive to stimulation by G(beta gamma) than PLC-beta 1 but less sensitive than PLC-beta 2 and PLC-beta T. in contrast PLC-beta X was not activated by the pertussis toxin substrate G proteins G alpha(i1), G alpha(i2), G alpha(i3), or G alpha(o). These results are consistent with the idea that PLC-PX is regulated by alpha-subunits of the G(q) family and by G(beta gamma) and do not support the idea that alpha-subunits of pertussis toxin-sensitive G proteins are directly involved in regulation of this protein.