Galphaq potentiation of adenylate cyclase type 9 activity through a Ca2+/calmodulin-dependent pathway.
Adenylate cyclase (EC 4.6.1.1) type 9 (AC9) activity has been shown to be inhibited by PMA activation of novel protein kinase C (nPKC) isoforms. In the current study the effect on AC9 activity of activating PKC in physiological relevant manner was examined. Contrary to the anticipated inhibitory effect of activating PKCs through Gq-coupled receptors, activation of transiently expressed Gq-coupled serotonin 5-HT2A or muscarinic M5 receptors resulted in the potentiation of isoproterenol-stimulated cyclic AMP accumulation in HEK293 cells stably expressing AC9 (HEK-AC9). Consistent with Gq-mediated activation of PKC, the addition of the PKC inhibitor bisindolylmaleimide further potentiated isoproterenol-stimulated cyclic AMP accumulation. Expression of a constitutively active mutant of Galphaq in HEK-AC9 cells also produced an enhancement in basal and isoproterenol-stimulated cyclic AMP accumulation. We also examined the role of Galphaq-mediated release of intracellular Ca2+ on the observed potentiation of AC9 activity, by depleting intracellular Ca2+ stores with thapsigargin. In Ca2+-depleted HEK-AC9 cells, activation of transiently expressed M5 receptors resulted in inhibition of isoproterenol-stimulated cyclic AMP accumulation that was blocked by bisindolylmaleimide, indicating that M5 potentiation of AC9 activity requires Ca2+. This prompted us to examine the effects of the calmodulin antagonist W7 and the Ca2+/calmodulin-dependent kinase II (CaMK II) inhibitor KN-93. Pretreating cells with W7 and KN-93 significantly inhibited M5-mediated potentiation of isoproterenol-stimulated cyclic AMP accumulation in HEK-AC9 cells, suggesting that Galphaq potentiation of AC9 activity involves Ca2+/calmodulin and CaMK II. This data provides evidence for Ca2+-mediated potentiation of AC9 activity.
Cumbay
Medhane G
MG
Purdue University, Department of Medicinal Chemistry and Molecular Pharmacology, 575 Stadium Mall Drive, West Lafayette, IN 47907-2051, USA.
Watts
Val J
VJ
D000262:Adenylyl Cyclases / Q000378:metabolism*; D001596:Benzylamines / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D002460:Cell Line; D000242:Cyclic AMP / Q000032:analysis / Q000096:biosynthesis*; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000235:genetics / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D007545:Isoproterenol / Q000494:pharmacology; D008301:Maleimides / Q000494:pharmacology; D009154:Mutation; D011493:Protein Kinase C / Q000378:metabolism; D011976:Receptors, Muscarinic / Q000378:metabolism; D044348:Receptors, Serotonin, 5-HT2 / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D013449:Sulfonamides / Q000494:pharmacology; D019284:Thapsigargin / Q000494:pharmacology; D000096926:Benzenesulfonamides
D000262:Adenylyl Cyclases; D001596:Benzylamines; D002118:Calcium; D002147:Calmodulin; D000242:Cyclic AMP; D004791:Enzyme Inhibitors; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D007211:Indoles; D007545:Isoproterenol; D008301:Maleimides; D011493:Protein Kinase C; D011976:Receptors, Muscarinic; D044348:Receptors, Serotonin, 5-HT2; D013449:Sulfonamides; D019284:Thapsigargin; C072105:KN 93; D000096926:Benzenesulfonamides; C017967:W 7; C413923:adenylate cyclase 9; C088060:bisindolylmaleimide