FUNCTIONAL EXPRESSION OF A STABLY TRANSFECTED PARATHYROID-HORMONE PARATHYROID-HORMONE RELATED PROTEIN-RECEPTOR COMPLEMENTARY-DNA IN CHO CELLS

Chinese hamster ovary (CHO) cells were stably transfected with OK-O complementary DNA encoding the parathyroid hormone/parathyroid hormone related protein (PTH/PTHrP) receptor derived from opossum kidney (OK) cells (Juppner et al., 1991). A subclone of transfected CHO cells, CHO-E(2), presented high affinity binding of I-125-labeled [Tyr(36)]chickenPTHrP(1-36)amide ([I-125]chPTHrP(1-36)) (K-d 1.28 +/- 0.10 nM) similar to that of wildtype OK cells (K-d 2.23 +/- 0.16 nM) (P < 0.01). Photoaffinity labeling of the PTH/PTHrP receptors using N-hydroxysuccinimidyl-4-azidobenzoate modified [I-125]chPTHrP(1-36) revealed the same specifically labeled 90 kDa protein in CHO-E(2) and OK cells. In CHO-cells, chPTHrP(1-36) stimulated cyclic AMP accumulation in dose-dependent fashion (EC(50) 0.15 +/- 0.04 nM) and raised peak cytosolic free calcium concentration (EC(50), 2.90 +/- 0.36 nM) independent of extracellular calcium, and stimulated phosphate uptake (EC(50) 0.21 +/- 0.07 nM). Both, chPTHrP(1-36) and 12-O-tetradecanoylphorbol-13-acetate stimulated phosphate uptake were suppressed by staurosporine. But, Sp-cyclic adenosine-3',5'-monophosphothioate did not affect phosphate uptake in CHO-E(2) cells. In conclusion, a PTH/PTHrP receptor stably expressed in CHO cells is linked to stimulation of phosphate uptake. Receptor coupling presumably occurred through the protein kinase C rather than the protein kinase A pathway.