Expression of a G alpha s/G alpha i chimera that constitutively activates cyclic AMP synthesis.
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 1.5-2.5-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 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 10-15-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 s/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.
Woon
C W
CW
Department of Biochemistry, University of Massachusetts Medical School, Worcester 01655.
Soparkar
S
S
Heasley
L
L
Johnson
G L
GL
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D000262:Adenylyl Cyclases / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D015152:Blotting, Northern; D002460:Cell Line; D002678:Chimera; D000242:Cyclic AMP / Q000096:biosynthesis*; D004247:DNA / Q000235:genetics; D019204:GTP-Binding Proteins / Q000235:genetics* / Q000502:physiology; D005796:Genes; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D008969:Molecular Sequence Data; D010957:Plasmids; D015183:Restriction Mapping; D013873:Thionucleotides / Q000378:metabolism; D014162:Transfection
D013873:Thionucleotides; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D004247:DNA; D000242:Cyclic AMP; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases; D015056:1-Methyl-3-isobutylxanthine