EXPRESSION OF A G-ALPHA-S/G-ALPHA-I CHIMERA THAT CONSTITUTIVELY ACTIVATES CYCLIC-AMP SYNTHESIS

  • 5 April 1989
    • journal article
    • research article
    • Vol. 264 (10), 5687-5693
Abstract
A chimeric G.alpha. subunit cDNA, referred to as G.alpha.s/i(38), was constructed containing the complete 5''-untranslated region of G.alpha.s, the first 356 codons of the rat G.ALPHA.s and the last 36 codons and 428 base pairs of the 3''-untranslated region of the rat G.alpha.i cDNA. Transient expression of the G.alpha.s/i(38) protein in CoS cells allowed detection of a chimeric protein which was recognized by antibodies generated against an internal G.alpha.s sequence as well as antibodies recognizing the carboxyl terminus of G.alpha.i2. Chinese hamster ovary cell clones stably expressing the chimeric G-protein .alpha. subunit transcript (G.alpha.s/i(38)) demonstrated 1.5- to 2.5-fold constitutively elevated cyclic AMP levels and a 3- to 4-fold increase in the activity ratio of cyclic AMP-dependent protein kinase, although expression of the chimeric polypeptide could not be demonstrated presumably because of low expression of the mutant .alpha.s. Expression of the rat G.alpha.s transcript yielded clones that were similar to wild-type Chinese hamster ovary cells in regard to cyclic AMP levels and protein kinase activity. In the presence of methyl isobutylxanthine, a cyclic AMP phosphodiesterase inhibitor, cyclic AMP levels in clones expressing the G.alpha.s/i(38) transcript were 10- to 15-fold higher than G.alpha.s expressing clones. Adenylyl cyclase activation by guanosine 5''-O-(thiotriphosphate) (GTP.gamma.S) in membranes from clones expressing the G.alpha.a/i(38) transcript demonstrated a diminished lag time for maximal activation, indicating an increased relative GDP dissociation rate for the chimeric G.alpha. subunit and an increase in total adenylyl cyclase activity relative to wild-type G.alpha.s expressing clones. Cholate extracts from membranes of G.alpha.s/i(38) expressing clones, when mixed with cyc- S49 membranes, reconstituted an increased GTP.gamma.S-stimulated adenylyl cyclase activity and a diminished lag time for maximal activation compared to cholate extracts prepared from G.alpha.s-expressing clones. The G.alpha.s/i(38) construct confers a dominant constitutive activation of adenylyl cyclase when expressed in cells in the presence of a background of wild-type G.alpha.s.