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.
Highlights
Members of the G, subfamily of heterotrimeric gua- Heterotrimeric guanine nucleotide-binding regulatory pronine nucleotide-binding proteins (G proteins) activate teins (G proteins)' serve an essential role in cell physiology phospholipase C (PLC)
Recombinant GSal, GL2, Gi-3,GOP(*G), and G, all failed hibited by prior treatment with pertussis toxin, suggesting a to stimulate anyof the isoforms of PLC
When thesesubunitsare expressed together with G protein P and y subunits are reasonable quantities of active, nonaggregated protein generated. Such restrictions complicate the purification of free CY subunits and dictate the use of Recombinant Gq, and Glia uncommon reagents (i.e. &agarose and viruses encoding fl and y)
Summary
Rad); the flow-throughwas collected and passed again over the packed resin. The column was washed at 4 "C with 15 column volumes. RG, was reconstituted with purified recombinant type 1muscarinic cholinergic receptor and brain By in lipid vesicles as described previously [35]; carbachol was vent this problem, rG,, was coexpressed with other Gprotein subunits: p2 (rp2) and 7 2 [1-72] [31] Under these conditions, the majority of the active rG,, was associated with membranes and was not aggregated after extraction with sodium cholate added to stimulate binding of [35S]GTPyS toa determined stoichi- (Fig. 1); most of the G, that remained in the cytosol was ometry. Staining of protein with silver the capacity of GTPyS-activated proteinto stimulate purified nitrate following SDS-polyacrylamide gel electrophoresis was per- brain PLC-pl in reconstitution assays.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
