Expression of a Gαs/Gαi Chimera That Constitutively Activates Cyclic AMP Synthesis

Abstract

A chimeric G α subunit cDNA, referred to as G α s/i(38), was constructed containing the complete 5′-untranslated region of G αs, the first 356 codons of the rat G α s and the last 36 codons and 428 base pairs of the 3′-untranslated region of the rat G α i cDNA. Transient expression of the G αs/i(38) protein in COS cells allowed detection of a chimeric protein which was recognized by antibodies generated against an internal G αs sequence as well as antibodies recognizing the carboxyl terminus of G αi2. Chinese hamster ovary cell clones stably expressing the chimeric G-protein α subunit transcript (G αs/i(38] demonstrated 1.5–2.5-fold constitutively elevated cyclic AMP levels and a 3–4-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 αs. Expression of the rat G αs 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 αs/i(38) transcript were 10–15-fold higher than G αs expressing clones. Adenylyl cyclase activation by guanosine 5′-O-(thiotriphosphate) (GTP γ S) in membranes from clones expressing the G αs/i(38) transcript demonstrated a diminished lag time for maximal activation, indicating an increased relative GDP dissociation rate for the chimeric G α subunit and an increase in total adenylyl cyclase activity relative to wild-type G α s expressing clones. Cholate extracts from membranes of G αs/i(38) expressing clones, when mixed with cyc− S49 membranes, reconstituted an increased GTP γ S-stimulated adenylyl cyclase activity and a diminished lag time for maximal activation compared to cholate extracts prepared from G αs-expressing clones. The G αs/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 αs.