Protein kinase C modulates parathyroid hormone- but not prostaglandin E2-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 E2 (PGE2) 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 PGE2-induced cAMP was negligible. Inhibition of the alpha-subunit of the inhibitory guanine nucleotide binding protein (Gi) by pertussis toxin pretreatment also enhanced PTH-mediated cAMP production but had no effect on PGE2-induced cAMP production. These results suggest that although PTH-mediated adenylate cyclase activity is regulated via both the stimulatory (Gs) and inhibitory (Gi) guanine nucleotide binding proteins, only Gs regulates PGE2-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 alpha-subunit of Gi. The alpha-subunit of Gi 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 32P-labeled cells with either PMA or PTH also enhanced incorporation of 32P into the 40 kD band. Using the peptide antisera AS/7 and EC/2, we showed that pertussis toxin-labeled subunits of both Gi1 alpha/Gi2 alpha and Gi3 alpha could be immunoprecipitated, respectively, but immunoprecipitation of membrane proteins after in situ phosphorylation and stimulation with PMA precipitated only Gi2 alpha.(ABSTRACT TRUNCATED AT 250 WORDS)
Koch
H M
HM
Department of Endocrinology and Metabolism, University of Stellenbosch Medical School, Tygerberg, Republic of South Africa.
Muir
H
H
Gelderblom
D
D
Hough
S
S
D037361:Adenylate Cyclase Toxin; D000818:Animals; D000242:Cyclic AMP / Q000378:metabolism*; D015232:Dinoprostone / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D019204:GTP-Binding Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012516:Osteosarcoma / Q000378:metabolism*; D010281:Parathyroid Hormone / Q000494:pharmacology*; D037342:Pertussis Toxin; D010766:Phosphorylation; D011233:Precipitin Tests; D011493:Protein Kinase C / Q000378:metabolism*; D011863:Radioimmunoassay; D051381:Rats; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D037361:Adenylate Cyclase Toxin; D010281:Parathyroid Hormone; D010566:Virulence Factors, Bordetella; D000242:Cyclic AMP; D037342:Pertussis Toxin; D011493:Protein Kinase C; D019204:GTP-Binding Proteins; D015232:Dinoprostone; D013755:Tetradecanoylphorbol Acetate