Cyclic adenosine 3',5'-monophosphate (cAMP)-dependent and cAMP-independent regulation of parathyroid hormone receptors on UMR 106-01 osteoblastic osteosarcoma cells.
The osteoblast-like cells, UMR 106-01, express PTH receptors that are coupled to adenylate cyclase. Recently, we reported the isolation of a UMR 106-01 subclone, UMR 4-7, that is stably transfected with a Zn(++)-inducible mutant of the regulatory subunit of protein kinase A. Incubation of UMR 4-7 cells with Zn++ renders the cells unresponsive to cAMP agonists. This subclone, therefore, seemed particularly suitable for studies of PTH receptor regulation. In UMR 106-01 cells, PTH receptors are strikingly down-regulated by pretreatment with 8-Br-cAMP or 3-isobutyl-1-methylxanthine for 2 days. In UMR 4-7 cells, this effect is totally prevented by prior and concurrent treatment with Zn++. Zn++ addition to UMR 106 cells does not modify these responses. Treatment with the PTH agonist [Nle8,18,Tyr34]bovine PTH(1-34)NH2 [(NlePTH(1-34)] also markedly down-regulates PTH receptors in UMR 106 cells, but this effect is only partially inhibited in Zn(++)-induced UMR 4-7 cells. At high doses, the PTH antagonist, [Nle8,18,Tyr34]bovine PTH(3-34)NH2 [NlePTH(3-34)] also (partially) reduces PTH receptor availability. Receptor regulation by NlePTH(3-34) is not blocked in the cAMP-resistant cells, however. Coincubation of submaximal doses of NlePTH(1-34) (1 nM) with NlePTH(3-34) (1 microM) reduces receptor availability more than when the cells are exposed to either ligand alone. This decrease is only partially inhibited in Zn(++)-induced UMR 4-7 cells. In contrast to its additive effect on receptor regulation, NlePTH(3-34) efficiently competes for binding to the PTH receptor in UMR 106-01 cells and antagonizes the stimulatory effects of NlePTH(1-34) on both intracellular cAMP accumulation and gene expression driven by a transiently transfected synthetic cAMP-responsive enhancer. In conclusion, homologous down-regulation of PTH receptors is mediated by activation of both cAMP-dependent (via protein kinase A) and cAMP-independent pathways. PTH activates both pathways, whereas the effect of NlePTH(3-34) appears to be exclusively cAMP-independent. These results give new insights into mechanisms of PTH receptor regulation.
Abou-Samra
A B
AB
Endocrine Unit, Massachusetts General Hospital/Harvard Medical School, Boston 02114.
Zajac
J D
JD
Schiffer-Alberts
D
D
Skurat
R
R
Kearns
A
A
Segre
G V
GV
Bringhurst
F R
FR
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000818:Animals; D002712:Chlorides / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism / Q000502:physiology*; D015536:Down-Regulation / Q000187:drug effects; D004789:Enzyme Activation; D015870:Gene Expression / Q000187:drug effects; D005798:Genes, Bacterial; D010006:Osteoblasts / Q000378:metabolism* / Q000473:pathology; D012516:Osteosarcoma / Q000378:metabolism* / Q000473:pathology; D010281:Parathyroid Hormone / Q000378:metabolism / Q000494:pharmacology; D010446:Peptide Fragments / Q000494:pharmacology; D011494:Protein Kinases / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000378:metabolism*; D018016:Receptors, Parathyroid Hormone; D019379:Teriparatide / Q000031:analogs & derivatives; D014407:Tumor Cells, Cultured; D015032:Zinc / Q000494:pharmacology; D017967:Zinc Compounds
D002712:Chlorides; D010281:Parathyroid Hormone; D010446:Peptide Fragments; D011956:Receptors, Cell Surface; D018016:Receptors, Parathyroid Hormone; D017967:Zinc Compounds; D019379:Teriparatide; D015124:8-Bromo Cyclic Adenosine Monophosphate; C025497:parathyroid hormone (3-34) amide, Nle(8,18)-Nle(34)-; C016837:zinc chloride; C061438:parathyroid hormone (1-34)amide, Nle(8), Nle(18), Tyr(34)-; D000242:Cyclic AMP; D011494:Protein Kinases; D015032:Zinc; D015056:1-Methyl-3-isobutylxanthine