Cloning of complementary DNAs that encode either of two forms of the α subunit of the guanine nucleotide-binding regulatory protein (G(s)) that stimulates adenylyl cyclase into appropriate plasmid vectors has allowed these proteins to be synthesized in Escherichia coli (Graziano, M. P., Casey, P. J., and Gilman, A. G. (1987) J. Biol. Chem. 262, 11375-11381). A rapid procedure for purification of milligram quantities of these proteins is described. As expressed in E. coli, both forms of G(sα) (apparent molecular weights of 45,000 and 52,000) bind guanosine 5'-(3-O-thio)triphosphate stoichiometrically. The proteins also hydrolyze GTP, although at different rates (i.e. 0.13 · min-1 and 0.34 · min-1 at 20°C for the 45- and the 52-kDa forms, respectively). These rates reflect differences in the rate of dissociation of GDP from the two proteins. Both forms of recombinant G(sα) have essentially the same k(cat) for GTP hydrolysis, ~ 4 · min-1. Recombinant G(sα) interacts functionally with G protein βγ subunits and with β-adrenergic receptors. The proteins can also be ADP-ribosylated stoichiometrically by cholera toxin. This reaction requires the addition of βγ subunits. Both forms of recombinant G(sα) can reconstitute GTP-, isoproterenol + GTP-, guanosine 5-(3-O-thio)triphosphate-, and fluoride-stimulated adenylyl cyclase activity in S49 cyc- membranes to maximal levels, although their specific activities for this reaction are lower than that observed for G(s) purified from rabbit liver. Experiments with purified bovine brain adenylyl cyclase indicate that the affinity of recombinant G(sα) for adenylyl cyclase is 5-10 times lower than that of liver G(s) under these assay conditions; however, the intrinsic capacity of the recombinant protein to activate adenylyl cyclase is normal. These findings suggest that G(sα), when synthesized in E. coli, may fail to undergo a posttranslational modification that is crucial for high affinity interaction of the G protein with adenylyl cyclase.
|Original language||English (US)|
|Number of pages||10|
|Journal||Journal of Biological Chemistry|
|State||Published - 1989|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology