Shoichi Fukayama

Inositol 1-,4-,5-trisphosphate-dependent Ca2+ signaling by the recombinant human PTH/PTHrP receptor stably expressed in a human kidney cell line

We previously reported the preparation and partial characterization of a series of human embryonic kidney cell lines (HEK-293) stably expressing various numbers of the recombinant human (h) parathyroid hormone (PTH)/PTH-related protein (PTHrP) receptor (Rc). Using this expression system we examined ligand (PTH or PTHrP) binding characteristics and cyclic AMP responsiveness. We have now extended these studies to investigate the calcium signal transduction pathways activated by the hPTH/PTHrP Rc. In parental HEK-293 cells, which lack endogenous PTH/PTHrP Rc, incubation with hPTH(1-34) had no effect on cytosolic free Ca2+ concentration [Ca2+]i. In HEK-293 clone C-21, stably expressing approximately 400,000 Rc/cell, PTH stimulated an increase in [Ca2+]i by Ca2+ release from intracellular stores; PTH released Ca2+ exclusively from the IP3 sensitive Ca2+ pool. Unlike previous studies, the ability of PTH to elicit both cAMP responses and [Ca2+]i transients occurred over a wide range of Rc numbers (between 400,000 and 3000 Rc/cell); both responses were always observed at PTH concentrations in the same dose range although the magnitude of the responses decrease with Rc number. Pretreatment of C-21 cells with pertussis toxin for 24 h, which significantly enhanced PTH-stimulated cAMP accumulation, did not modulate PTH-stimulated [Ca2+]i transients. At each PTH concentration tested which resulted in increased cAMP levels, there was also an increase in [Ca2+]i transients. Treatment of C-21 cells with a battery of midregion and C-terminal PTH or PTHrP peptides showed no effect on either [Ca2+]i transients or cAMP accumulation, indicating a lack of functional interactions between these peptides and the form of the hPTH/PTHrP Rc stably expressed in these cells. Immunological analysis of G-protein expression demonstrated the presence of Gs, Gi, and Gq in all parental and transfected cells lines examined. Taken together, these data demonstrate that the hPTH/PTHrP Rc, stably expressed in HEK-293 cells, elicits responses in both the cAMP and IP3-dependent [Ca2+]i pathways and is responsive only to N-terminal PTH/PTHrP peptides.

β-Adrenergic Receptor Kinase-1 Acutely Regulates PTH/PTHrP Receptor Signalling in Human Osteoblastlike Cells

To investigate whether G protein-coupled receptor kinases (GRKs) are involved in the regulation of the PTH/PTHrPR, we have established mutant SaOS-2 cells which stably overexpress (> 10-20-fold) a dominant negative form of the beta-adrenergic receptor kinase-1 (beta ARK-1). Acute (< or = 2 h) incubation with hPTH (1-34) induced significantly less (by up to 50%) downregulation of the PTH/PTHrPR in beta ARK-1 mutant SaOS-2 cells than observed in wild-type cells. Pretreatment of wild-type cells with PTH for 2 h induced homologous cAMP desensitisation to a second challenge with PTH, while the effect was blunted by up to 60% in beta ARK-1 mutant cells. We conclude that activation of beta ARK-1 (or a closely related GRK) is a critical component of the acute phase (< or = 2 h) of PTH-induced receptor downregulation and homologous cAMP desensitisation of the PTH/PTHrPR.

Differential regulation of the PTH/PTHrP receptor and its mRNA in human osteoblast-like SaOS-2 cells

Using wild-type SaOS-2 cells and a mutant cAMP-resistant subclone Ca#4A, we have recently shown that homologous down-regulation of PTH/PTHrP receptor (PTH/PTHrPR) is mediated by mechanisms independent of PKA activation, and that homologous down-regulation of PTH/PTHrPR mRNA occurs later than the decline in functional cell surface receptors via a mechanism that does not involve enhanced mRNA degradation or new protein synthesis but does depend on cAMP/PKA. Treatment of SaOS-2 cells with [Arg2]hPTH(1-34) or hPTH(3-34) for 24 h, both of which bind to the human PTH/PTHrPR as well as hPTH(1-34) but fail to stimulate cAMP/PKA efficiently, does not cause down-regulation of PTH/PTHrPR mRNA; however, the same treatment did down-regulate PTH/PTHrPR protein. Incubation with isoproterenol for 24 h, which activates both PKA andβ-adrenergic receptor kinase (βARK) in these cells, induced significant down-regulation of both the PTH/PTHrPR and its mRNA. However, treatment with vasoactive intestinal peptide (VIP) or PGE2 for 24 h, both of which stimulate the cAMP/PKA pathway in these cells, did not down-regulate PTH/PTHrPR mRNA. We conclude that both PKA-dependent and -independent (such asβARK) mechanisms are required for regulation of the PTH/PTHrPR and its mRNA.