T K Harden

Role of isoprenoid lipids on the heterotrimeric G protein gamma subunit in determining effector activation.

Post-translational prenylation of heterotrimeric G protein γ subunits is essential for high affinity α-βγ and α-βγ-receptor interactions, suggesting that the prenyl group is an important domain in the βγ dimer. To determine the role of the prenyl modification in the interaction of βγ dimers with effectors, the CAAX (where A indicates alipathic amino acid) motifs in the γ1, γ2, and γ11 subunits were altered to direct modification with different prenyl groups. Six recombinant βγ dimers were overexpressed in baculovirus-infected Sf9 insect cells, purified, and examined for their ability to stimulate three phospholipase C-β isozymes and type II adenylyl cyclase. The native β1γ2 dimer (γ subunit modified with geranylgeranyl) is more potent and effective in activating phospholipase C-β than either the β1γ1 (farnesyl) or the β1γ11 (farnesyl) dimers. However, farnesyl modification of the γ subunit in the β1γ2dimer (β1γ2-L71S) caused a decrement in its ability to activate phospholipase C-β. In contrast, both the β1γ1-S74L (geranylgeranyl) and the β1γ11-S73L (geranylgeranyl) dimers were more active than the native forms. The β1γ2dimer activates type II adenylyl cyclase about 12-fold; however, neither the β1γ1 nor the β1γ11 dimers activate the enzyme. As was the case with phospholipase C-β, the β1γ2-L71S dimer was less able to activate adenylyl cyclase than the native β1γ2dimer. Interestingly, neither the β1γ1-S74Lnor the β1γ11-S73L dimers stimulated adenylyl cyclase. The results suggest that both the amino acid sequence of the γ subunit and its prenyl group play a role in determining the activity of the βγ-effector complex.

PURIFICATION AND G 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 Gαi, Gαo, Gαq, Gαs, and Gβγ 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-βX) that exhibits homology to the PLC-β 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-βX in Sf9 insect cells and have purified a recombinant 150-kDa isoenzyme. PLC-βX catalyzes hydrolysis of phosphatidylinositol(4,5)bisphosphate and phosphatidylinositol(4)monophosphate, and reaction velocity is dependent on Ca2+. Recombinant PLC-βX was activated by both Gαq and Gβγ. PLC-βX exhibited a higher apparent affinity for Gαq than Gβγ, and Gαq was more efficacious than Gβγ at lower concentrations of PLC-βX. Relative to other PLC-β isoenzymes, PLC-βX was less sensitive to stimulation by Gαq than PLC-β1 but similar to PLC-β2 and PLC-βT. PLC-βX was more sensitive to stimulation by Gβγ than PLC-β1 but less sensitive than PLC-β2 and PLC-βT. In contrast PLC-βX was not activated by the pertussis toxin substrate G proteins Gαi1, Gαi2, Gαi3, or Gαo. These results are consistent with the idea that PLC-βX is regulated by α-subunits of the Gq family and by Gβγ and do not support the idea that α-subunits of pertussis toxin-sensitive G proteins are directly involved in regulation of this protein.