Cyclic Adenosine 3′,5′-Monophosphate (cAMP)-Dependent and cAMP-Independent Regulation of Parathyroid Hormone Receptors on UMR 106–01 Osteoblastic Osteosarcoma Cells

Abstract

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.

Protein kinase A-dependent inhibition of alkaline phosphatase release by SaOS-2 human osteoblastic cells: studies in new mutant cell lines that express a cyclic AMP-resistant phenotype.

“…It is of interest that the characteristics of PTHreceptor binding in the PKA-deficient mutant cells are indistinguishable from normal SaOS-2 cells, which we have also found in REV mutant UMR 4-7 rat osteosarcoma cells (Abou-Samra et al, 1991). These findings suggest that PTHreceptor expression may not be strongly influenced by ambient PKA activity in unstimulated osteoblastic cells.…”

Individual and combined effects of calciotropic hormones and growth factors on mineral metabolism in embryonic chick tibiae

“…The purpose of the present study was to investigate possible interactions of PTH with other calciotropic hormones and to correIate major effects of PTH on bone metabolism to domains known to be selective for the cAMP and PKC messenger system (1,2,27). Our results with PTH(1-34) showed a time-and dose-dependent induction of calcium mobilization and inhibition of ALP activity.…”

Heterotrimeric G proteins in the control of parathyroid hormone actions

“…In addition to Gs, the PTH1R couples to G q/11 -dependent activation of phospholipase C (PLC) (Abou-Samra, et al 1992; Abou-Samra et al 1991; Bringhurst et al 1993). Upon activation, G q/11 leads to the generation of the second messengers inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) through the action of phospholipase C on the membrane phospholipids (Bourne et al 1991; Cabrera-Vera et al 2003; Syrovatkina et al 2016).…”

“…PTH1R couples to multiple different G proteins, including G s and G q/11 (Abou-Samra, et al 1991; Bringhurst, et al 1993) (Figure 1). The amino terminal portion of PTH can also bind and activate another, closely related G protein-coupled receptor, termed PTH2R (Usdin, et al 1995); however, it is now known that the latter receptor is primarily for the actions of the neuropeptide tuberoinfundibular peptide of 39 residues (TIP39) (Usdin, et al 1999).…”