Purification from Sf9 cells and characterization of recombinant G_qα and Q_11α: Activation of purified phospholipase C isozymes by G_α subunits

Members of the G_qα subfamily of heterotrimeric guanine nucleotide-binding proteins (G proteins) activate phospholipase C (PLC). The complementary DNAs (cDNAs) for the G protein α subunits G_qα and G_11α were expressed in insect (Sf9) cells using recombinant baculovirus. Active, nonaggregated, and membrane-associated protein was generated only when the α subunit cDNA was expressed together with cDNAs encoding G protein β and γ subunits. Recombinant α subunits (rG_qα and rG_11α) were purified by three-step procedures, as was a PLC-activating α subunit(s) endogenous to Sf9 cells. Guanosine 5′-3-(thio)triphosphate (GTPγS) activated rG_qα and rG_11α with an apparent K_0.5 of 30 μM; similarly high concentrations of the nucleotide were required to observe [³⁵S]GTPγS binding to rG_qα. Activated rG_qα and rG_11α each stimulated all three isoforms of purified PLC-β with the rank order of potency PLC-β1 = PLC-β3 ≥ PLC-β2; both α subunits also stimulated PLC-β1 and PLC-β3 to a much greater extent (10-fold) than they did PLC-β2. In contrast, activated rG_qα and rG_11α failed to stimulate either PLC-δ1 or PLC-γ1. Recombinant G_iα1, G_iα2, G_iα3, G_oα(A), G_sα, and G_zα all failed to stimulate any of the isoforms of PLC. The apparent affinities of rG_qα and rG_11α for PLC-β1 and their capacities to activate the enzyme were similar to values observed for purified brain G_qα/11α. Purified brain βγ subunits also stimulated the three isoforms of PLC-β. The capacities of rG_qα and rG_11α to activate PLC-β1 and PLC-β3 greatly exceeded those of βγ, whereas G_qα, G_11α and βγ were roughly equiefficacious with PLC-β2; the α subunits were more potent than βγ in all cases. The effects of α and βγ together were nonadditive for both PLC-β1 and PLC-β2. These results demonstrate that G_qα and G_11α specifically and selectively stimulate β isoforms of PLC and confirm the idea that these members of the G_qα subfamily of G proteins are physiological regulators of this signaling pathway.