Functional expression of a stably transfected parathyroid hormone/parathyroid hormone related protein receptor complementary DNA in CHO cells

Abstract

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 (Jüppner et al., 1991). A subclone of transfected CHO cells, CHO-E 2, presented high affinity binding of 125I-labeled [Tyr 36]chickenPTHrP(1–36)amide ([ 125I]chPTHrP(1–36)) ( K d 1.28 ± 0.10nM) similar to that of wildtype OK cells ( K d 2.23 ± 0.16nM) (P< 0.01). Photoaffmity labeling of the PTH/PTHrP receptors using N-hydroxysuccinimidyl-4-azidobenzoate modified [ 125I]chPTHrP(1–36) revealed the same specifically labeled 90kDa 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±0.04nM) 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 ± 0.07nM). Both, chPTHrP(1–36) and 12-0-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.