Abstract

Nine membrane-bound members of the mammalian adenylate cyclase family have been identified. The least characterized and most divergent in sequence of the nine adenylate cyclase isoforms is AC9. Stimulation by Gα_s and inhibition by Ca²⁺/calcineurin are two modes of regulation that have been reported for AC9. We explored the possibility of additional modes of regulation of human AC9. We now report that quinpirole activation of the inhibitory G protein-coupled D_2L dopamine receptor inhibits Gα_s stimulation of AC9 by approximately 50%. The effects of quinpirole were reversed by the D₂ antagonist spiperone and by pertussis toxin pretreatment. We also report the first evidence for regulation of AC9 by protein kinase C (PKC). Specifically, phorbol ester activation of PKC significantly attenuated (∼50%) Gα_s-stimulated AC9 activity. The effect of PKC activation on AC9 was reversed by the PKC inhibitor bisindolylmaleimide. Gα_s-stimulated cyclic accumulation was reduced more by simultaneous addition of both quinpirole and phorbol 12-myristate 13-acetate than by either drug alone. Additional studies investigated the role of glycosylation on AC9 activity. The results show that blocking glycosylation of AC9 significantly attenuates Gα_s stimulation. In contrast, the ability of PKC and Gα_i/o to negatively regulate AC9 did not seem to be affected by the glycosylation state of AC9. These observations demonstrate the diverse regulatory features of AC9 and the ability of AC9 to integrate multiple signals.