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Purification of heterotrimeric G protein alpha subunits by GST-Ric-8 association: primary characterization of purified G alpha(olf).

ABSTRACT: Ric-8A and Ric-8B are nonreceptor G protein guanine nucleotide exchange factors that collectively bind the four subfamilies of G protein ? subunits. Co-expression of G? subunits with Ric-8A or Ric-8B in HEK293 cells or insect cells greatly promoted G? protein expression. We exploited these characteristics of Ric-8 proteins to develop a simplified method for recombinant G protein ? subunit purification that was applicable to all G? subunit classes. The method allowed production of the olfactory adenylyl cyclase stimulatory protein G?(olf) for the first time and unprecedented yield of G?(q) and G?(13). G? subunits were co-expressed with GST-tagged Ric-8A or Ric-8B in insect cells. GST-Ric-8·G? complexes were isolated from whole cell detergent lysates with glutathione-Sepharose. G? subunits were dissociated from GST-Ric-8 with GDP-AlF(4)(-) (GTP mimicry) and found to be >80% pure, bind guanosine 5'-[?-thio]triphosphate (GTP?S), and stimulate appropriate G protein effector enzymes. A primary characterization of G?(olf) showed that it binds GTP?S at a rate marginally slower than G?(s short) and directly activates adenylyl cyclase isoforms 3, 5, and 6 with less efficacy than G?(s short).

SUBMITTER: Chan P

PROVIDER: S-EPMC3024758 | biostudies-literature | 2011 Jan

REPOSITORIES: biostudies-literature

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Purification of heterotrimeric G protein alpha subunits by GST-Ric-8 association: primary characterization of purified G alpha(olf).

Chan PuiYee P Gabay Meital M Wright Forrest A FA Kan Wei W Oner Sukru S SS Lanier Stephen M SM Smrcka Alan V AV Blumer Joe B JB Tall Gregory G GG

The Journal of biological chemistry 20101129 4

Ric-8A and Ric-8B are nonreceptor G protein guanine nucleotide exchange factors that collectively bind the four subfamilies of G protein α subunits. Co-expression of Gα subunits with Ric-8A or Ric-8B in HEK293 cells or insect cells greatly promoted Gα protein expression. We exploited these characteristics of Ric-8 proteins to develop a simplified method for recombinant G protein α subunit purification that was applicable to all Gα subunit classes. The method allowed production of the olfactory a ...[more]

PMID: 21115479

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Project description:The heterotrimeric G-protein alpha subunit has long been considered a bimodal, GTP-hydrolyzing switch controlling the duration of signal transduction by seven-transmembrane domain (7TM) cell-surface receptors. In 1996, we and others identified a superfamily of "regulator of G-protein signaling" (RGS) proteins that accelerate the rate of GTP hydrolysis by Galpha subunits (dubbed GTPase-accelerating protein or "GAP" activity). This discovery resolved the paradox between the rapid physiological timing seen for 7TM receptor signal transduction in vivo and the slow rates of GTP hydrolysis exhibited by purified Galpha subunits in vitro. Here, we review more recent discoveries that have highlighted newly-appreciated roles for RGS proteins beyond mere negative regulators of 7TM signaling. These new roles include the RGS-box-containing, RhoA-specific guanine nucleotide exchange factors (RGS-RhoGEFs) that serve as Galpha effectors to couple 7TM and semaphorin receptor signaling to RhoA activation, the potential for RGS12 to serve as a nexus for signaling from tyrosine kinases and G-proteins of both the Galpha and Ras-superfamilies, the potential for R7-subfamily RGS proteins to couple Galpha subunits to 7TM receptors in the absence of conventional Gbetagamma dimers, and the potential for the conjoint 7TM/RGS-box Arabidopsis protein AtRGS1 to serve as a ligand-operated GAP for the plant Galpha AtGPA1. Moreover, we review the discovery of novel biochemical activities that also impinge on the guanine nucleotide binding and hydrolysis cycle of Galpha subunits: namely, the guanine nucleotide dissociation inhibitor (GDI) activity of the GoLoco motif-containing proteins and the 7TM receptor-independent guanine nucleotide exchange factor (GEF) activity of Ric8/synembryn. Discovery of these novel GAP, GDI, and GEF activities have helped to illuminate a new role for Galpha subunit GDP/GTP cycling required for microtubule force generation and mitotic spindle function in chromosomal segregation.

Dataset Information

Purification of heterotrimeric G protein alpha subunits by GST-Ric-8 association: primary characterization of purified G alpha(olf).

Publications

Purification of heterotrimeric G protein alpha subunits by GST-Ric-8 association: primary characterization of purified G alpha(olf).

Similar Datasets

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