title: Purification from Sf9 cells and characterization of recombinant Gq alpha and G11 alpha. Activation of purified phospholipase C isozymes by G alpha subunits.
abstract: Members of the Gq alpha subfamily of heterotrimeric guanine nucleotide-binding proteins (G proteins) activate phospholipase C (PLC). The complementary DNAs (cDNAs) for the G protein alpha subunits Gq alpha and G11 alpha were expressed in insect (Sf9) cells using recombinant baculovirus. Active, nonaggregated, and membrane-associated protein was generated only when the alpha subunit cDNA was expressed together with cDNAs encoding G protein beta and gamma subunits. Recombinant alpha subunits (rGq alpha and rG11 alpha) were purified by three-step procedures, as was a PLC-activating alpha subunit(s) endogenous to Sf9 cells. Guanosine 5'-3-(thio)triphosphate (GTP gamma S) activated rGq alpha and rG11 alpha with an apparent K0.5 of 30 microM; similarly high concentrations of the nucleotide were required to observe [35S]GTP gamma S binding to rGq alpha. Activated rGq alpha and rG11 alpha each stimulated all three isoforms of purified PLC-beta with the rank order of potency PLC-beta 1 = PLC-beta 3 > or = PLC-beta 2; both alpha subunits also stimulated PLC-beta 1 and PLC-beta 3 to a much greater extent (10-fold) than they did PLC-beta 2. In contrast, activated rGq alpha and rG11 alpha failed to stimulate either PLC-delta 1 or PLC-gamma 1. Recombinant Gi alpha 1, Gi alpha 2, Gi alpha 3, Go alpha (A), Gs alpha, and Gz alpha all failed to stimulate any of the isoforms of PLC. The apparent affinities of rGq alpha and rG11 alpha for PLC-beta 1 and their capacities to activate the enzyme were similar to values observed for purified brain Gq alpha/11 alpha. Purified brain beta gamma subunits also stimulated the three isoforms of PLC-beta. The capacities of rGq alpha and rG11 alpha to activate PLC-beta 1 and PLC-beta 3 greatly exceeded those of beta gamma, whereas Gq alpha, G11 alpha and beta gamma were roughly equiefficacious with PLC-beta 2; the alpha subunits were more potent than beta gamma in all cases. The effects of alpha and beta gamma together were nonadditive for both PLC-beta 1 and PLC-beta 2. These results demonstrate that Gq alpha and G11 alpha specifically and selectively stimulate beta isoforms of PLC and confirm the idea that these members of the Gq alpha subfamily of G proteins are physiological regulators of this signaling pathway.
authors.affiliation: Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas 75235.
authors.forename: J R
authors.initials: JR
authors.lastname: Hepler
authors.forename: T
authors.initials: T
authors.lastname: Kozasa
authors.forename: A V
authors.initials: AV
authors.lastname: Smrcka
authors.forename: M I
authors.initials: MI
authors.lastname: Simon
authors.forename: S G
authors.initials: SG
authors.lastname: Rhee
authors.forename: P C
authors.initials: PC
authors.lastname: Sternweis
authors.forename: A G
authors.initials: AG
authors.lastname: Gilman
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004247:DNA; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D005822:Genetic Vectors; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D007527:Isoenzymes / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D009036:Moths; D011994:Recombinant Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D015398:Signal Transduction; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism*
chemical_list: D007527:Isoenzymes; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D004247:DNA; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins