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Purification from Sf9 cells and characterization of recombinant Gq alpha and G11 alpha. Activation of purified phospholipase C isozymes by G alpha subunits

Hepler, John R. and Kozasa, Tohru and Smrcka, Alan V. and Simon, Melvin I. and Rhee, Sue Goo and Sternweis, Paul C. and Gilman, Alfred G. (1993) Purification from Sf9 cells and characterization of recombinant Gq alpha and G11 alpha. Activation of purified phospholipase C isozymes by G alpha subunits. Journal of Biological Chemistry, 268 (19). pp. 14367-14375. ISSN 0021-9258. http://resolver.caltech.edu/CaltechAUTHORS:HEPjbc93

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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.


Item Type:Article
Related URLs:
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http://www.jbc.org/cgi/content/abstract/268/19/14367PublisherUNSPECIFIED
Additional Information:© 1993 American Society for Biochemistry and Molecular Biology. Received for publication, February 8, 1993. We thank Linda Hannigan for superb technical assistance, Dr. Gabriel Berstein for measuring type 1 muscarinic cholinergic receptor-stimulated GTPγS binding to rGqα and Dr. Elliott Ross for helpful discussions. This work was supported by National Institutes of Health Grants GM34497, GM31954, and GM34236; American Cancer Society Grant BE30; the Perot Family Foundation; the Lucille P. Markey Charitable Trust; the Raymond and Ellen Willie Chair of Molecular Neuropharmacology; National Research Service Awards GM13569 (to J.R.H.) and GM14489 (to A.V.S.); and a Human Frontier Science Program Organization award (to T.K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Funders:
Funding AgencyGrant Number
National Institutes of HealthGM34497
National Institutes of HealthGM31954
National Institutes of HealthGM34236
American Cancer SocietyBE30N
Perot Family FoundationUNSPECIFIED
Lucille P. Markey Charitable TrustUNSPECIFIED
Raymond Willie Chair of Molecular NeuropharmacologyUNSPECIFIED
National Research ServiceGM13569
National Research ServiceGM14489
Human Frontier Science Program OrganizationUNSPECIFIED
Subject Keywords:BINDING REGULATORY PROTEINS; EPIDERMAL GROWTH-FACTOR; ESCHERICHIA-COLI; BETA-GAMMA; IMMUNOLOGICAL IDENTIFICATION; PERTUSSIS TOXIN; BOVINE BRAIN; 2 FORMS; EXPRESSION; RECEPTOR
Record Number:CaltechAUTHORS:HEPjbc93
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:HEPjbc93
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:12030
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:23 Oct 2008 05:28
Last Modified:26 Dec 2012 10:25

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