Purification from Sf9 cells and characterization of recombinant G(qα) and G(11α). Activation of purified phospholipase C isozymes by G(α) subunits

John R. Hepler, Tohru Kozasa, Alan V. Smrcka, Melvin I. Simon, Sue Goo Rhee, Paul C. Sternweis, Alfred G. Gilman

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234 Scopus citations

Abstract

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 K0.5 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.

Original languageEnglish (US)
Pages (from-to)14367-14375
Number of pages9
JournalJournal of Biological Chemistry
Volume268
Issue number19
StatePublished - Jan 1 1993

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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