Protein kinase C modulates parathyroid hormone‐ but not prostaglandin E 2 ‐mediated stimulation of cyclic AMP production via the inhibitory guanine nucleotide binding protein in UMR‐106 osteosarcoma cells

In UMR‐106 osteosarcoma cells we found that PTH activated both the cAMP/protein kinase A and the Ca 2+ ‐dependent phosphoinositide/protein kinase C (PKC) pathways, but prostaglandin E 2 (PGE 2 ) activated only the cAMP pathway. Activation of PKC by the phorbol ester PMA had no effect on cAMP production but enhanced PTH‐stimulated cAMP production by 50% or more; the effect on PGE 2 ‐induced cAMP was negligible. Inhibition of the α‐subunit of the inhibitory guanine nucleotide binding protein (G i ) by pertussis toxin pretreatment also enhanced PTH‐mediated cAMP production but had no effect on PGE 2 ‐induced cAMP production. These results suggest that although PTH‐mediated adenylate cyclase activity is regulated via both the stimulatory (G s ) and inhibitory (G i ) guanine nucleotide binding proteins, only G s regulates PGE 2 ‐mediated adenylate cyclase activity in UMR‐106 cells. Costimulation with pertussis toxin and PMA did not increase PTH‐stimulated cAMP production above that obtained with PMA alone. This implies a similar target of action for pertussis toxin and PMA, that is, the α‐subunit of G i . The α‐subunit of G i was found to be a substrate for in vitro PKC phosphorylation of membrane fractions from UMR‐106 cells, seen as a ±40 kD band on SDS‐PAGE. Stimulation of in situ 32 P‐labeled cells with either PMA or PTH also enhanced incorporation of 32 P into the 40 kD band. Using the peptide antisera AS/7 and EC/2, we showed that pertussis toxin‐labeled subunits of both G i 1α/G i 2α and G i 3α could be immunoprecipitated, respectively, but immuinoprecipitation of membrane proteins after in situ phosphorylation and stimulation with PMA precipitated only G i 2α. We therefore conclude that modulation of adenylate cyclase activity by phorbol esters in UMR‐106 osteosarcoma cells can be ascribed, at least in part, to PKC‐mediated phosphorylation of the α‐subunit of the G i 2 component of the adenylate cyclase regulatory complex.