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 (Jüppner et al., 1991). A subclone of transfected CHO cells, CHO-E2, presented high affinity binding of 125I-labeled [Tyr36]chickenPTHrP(1-36)amide ([125I]chPTHrP(1-36)) (Kd 1.28 +/- 0.10 nM) similar to that of wildtype OK cells (Kd 2.23 +/- 0.16 nM) (P < 0.01). Photoaffinity labeling of the PTH/PTHrP receptors using N-hydroxysuccinimidyl-4-azidobenzoate modified [125I]chPTHrP(1-36) revealed the same specifically labeled 90 kDa protein in CHO-E2 and OK cells. In CHO-cells, chPTHrP(1-36) stimulated cyclic AMP accumulation in dose-dependent fashion (EC50 0.15 +/- 0.04 nM) and raised peak cytosolic free calcium concentration (EC50 2.90 +/- 0.36 nM) independent of extracellular calcium, and stimulated phosphate uptake (EC50 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-E2 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.
Kaufmann
M
M
Department of Orthopaedic Surgery, University of Zurich, Switzerland.
Muff
R
R
Born
W
W
Fischer
J A
JA
D000470:Alkaloids / Q000494:pharmacology; D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000494:pharmacology; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism; D018076:DNA, Complementary / Q000032:analysis* / Q000235:genetics / Q000502:physiology; D007668:Kidney / Q000166:cytology / Q000648:ultrastructure; D009893:Opossums; D044162:Parathyroid Hormone-Related Protein; D010446:Peptide Fragments / Q000378:metabolism / Q000494:pharmacology; D010710:Phosphates / Q000378:metabolism; D011493:Protein Kinase C / Q000502:physiology; D011506:Proteins / Q000378:metabolism / Q000494:pharmacology; D044168:Receptor, Parathyroid Hormone, Type 1; D018016:Receptors, Parathyroid Hormone / Q000032:analysis / Q000235:genetics* / Q000502:physiology; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection
D000470:Alkaloids; D018076:DNA, Complementary; D044162:Parathyroid Hormone-Related Protein; D010446:Peptide Fragments; D010710:Phosphates; D011506:Proteins; D044168:Receptor, Parathyroid Hormone, Type 1; D018016:Receptors, Parathyroid Hormone; C079957:parathyroid hormone-related peptide (1-36); D000242:Cyclic AMP; D011493:Protein Kinase C; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium