Phosphorylation of G_iα2 attenuates inhibitory adenylyl cyclase in neuroblastoma/glioma hybrid (NG-108-15) cells

Cross-regulation from the stimulatory phospholipase C to the adenylyl cyclase pathways was explored in neuroblastoma-glioma NG-108-15 cells in culture. Activation of protein kinase C by phorbol myristic acid resulted in a markedly attenuated activation of the inhibitory adenylyl cyclase response to δ-opiate agonists and epinephrine but not to the muscarinic agonist carbachol. The ability of okadaic acid to mimic the effects of phorbol myristic acid on the inhibitory response suggested a role for protein phosphorylation. Adenylyl cyclase activity from cells in which protein kinase C had been activated demonstrated a loss in the inhibitory adenylyl cyclase response at the level of the G-protein. Activation of protein kinase C prompted a 2-4-fold increase in phosphorylation of G_iα2 in cells metabolically labeled with [³²P]orthophosphate. The phosphate content of G_iα2 was determined to be ∼0.5 mol/mol subunit in the unstimulated cells and ∼1.5 mol/mol subunit for cells in which protein kinase C was activated. The effects of okadaic acid, 4-α-phorbol, and calphostin C on inhibition of adenylyl cyclase in cells treated with phorbol myristic acid correlate with the effects of these agents on phosphorylation of G_iα2. The time courses for attenuation of inhibitory adenylyl cyclase and that for phosphorylation of G_iα2 were similar in cells challenged with phorbol myristic acid. These data argue for cross-regulation from the stimulatory protein kinase C to inhibitory adenylyl cyclase pathways at the level of G_iα2 via protein phosphorylation.