INOSITOL 1-,4-,5-TRISPHOSPHATE-DEPENDENT CA2+ SIGNALING BY THE RECOMBINANT HUMAN PTH PTHRP RECEPTOR STABLY EXPRESSED IN A HUMAN KIDNEY-CELLLINE/

We previously reported the preparation and partial characterization of a series of human embryonic kidney cell lines (HEK-293) stably expres sing 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 inves tigate the calcium signal transduction pathways activated by the hPTH/ PTHrP Rc. In parental HEK-293 cells, which lack endogenous PTH/PTHrP R c, incubation with hPTH(1-34) had no effect on cytosolic free Ca2+ con centration [Ca2+](i). In HEK-293 clone C-21, stably expressing similar to 400,000 Rc/cell, PTH stimulated an increase in [Ca2+](i) by Ca2+ r elease from intracellular stores; PTH released Ca2+ exclusively from t he IP3 sensitive Ca2+ pool, Unlike previous studies, the ability of PT H 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 r esponses were always observed at PTH concentrations in the same dose r ange although the magnitude of the responses decrease with Rc number, Pretreatment of C-21 cells with pertussis toxin for 24 h, which signif icantly enhanced PTH-stimulated cAMP accumulation, did not modulate PT H-stimulated [Ca2+](i) transients, At each PTH concentration tested wh ich resulted in increased cAMP levels, there was also an increase in [ Ca2+](i) transients. Treatment of C-21 cells with a battery of midregi on and C-terminal PTH or PTHrP peptides showed no effect on either [Ca 2+](i) transients or cAMP accumulation, indicating a lack of functiona l interactions between these peptides and the form of the hPTH/PTHrP R c stably expressed in these cells, Immunological analysis of G-protein expression demonstrated the presence of G(s), G(i), and G(q) in all p arental and transfected cell lines examined, Taken together, these dat a demonstrate that the hPTH/PTHrP Rc, stably expressed in HEK-293 cell s, elicits responses in both the cAMP and IP3-dependent [Ca2+](i) path ways and is responsive only to N-terminal PTH/PTHrP peptides.