Expression-level dependent activation of recombinant human parathyroid hormone/parathyroid hormone-related peptide receptor: effect of human parathyroid hormone (1-34), (1-31), and (28-48).
A stable recombinant chinese hamster ovary (CHO) cell model system expressing the human type-1 receptor for parathyroid hormone and parathyroid hormone-related peptide (hPTH-R) was established for the analysis of human PTH (hPTH) variants. The cell lines showed receptor expression in the range from 10(5) to I.9 x 10(6) receptors per cell. The affinity of the receptors for hPTH-(1-34) was independent of the receptor number per cell (Kd approximately = 8 nmol/1). The induction of cAMP by hPTH-(1-34) is maximal in clones expressing >2x10(5) receptors per cell and Ca++ signals were maximal in cell lines expressing >1.4x10(6) receptors per cell. Second messenger specific inhibitors demonstrated that PTH-induced increases in intracellular cAMP and Ca++ are independent and Ca++ ions are derived from intracellular stores. The cAMP-specific receptor activator hPTH-(1-31) showed also an increase in intracellular Ca++. Even in cell lines expressing more than 10(6) receptors per cell the Ca++/PKC specific activator hPTH-(28-48) did not activate hPTH-Rs. Based on these results, synthesis of further derivatives of PTH is required to identify pathway-specific ligands for the type-1 hPTH-R.
Tonn
O
O
Roche Diagnostics GmbH, Pharma Research, Penzberg, Germany.
Kriegbaum
S
S
Braitmaier
A
A
Schäfer
W
W
Esswein
A
A
Dony
C
C
Kaluza
K
K
Honold
K
K
D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D006224:Cricetinae; D016048:Dideoxyadenosine / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D004963:Estrenes / Q000494:pharmacology; D006801:Humans; D010281:Parathyroid Hormone / Q000494:pharmacology*; D010446:Peptide Fragments / Q000494:pharmacology*; D011493:Protein Kinase C / Q000502:physiology; D011760:Pyrrolidinones / Q000494:pharmacology; D044168:Receptor, Parathyroid Hormone, Type 1; D018016:Receptors, Parathyroid Hormone / Q000187:drug effects*; D011994:Recombinant Proteins / Q000187:drug effects
D004963:Estrenes; D010281:Parathyroid Hormone; D010446:Peptide Fragments; D011760:Pyrrolidinones; D044168:Receptor, Parathyroid Hormone, Type 1; D018016:Receptors, Parathyroid Hormone; D011994:Recombinant Proteins; C101312:parathyroid hormone (1-31); C069678:parathyroid hormone (28-48); C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; D016048:Dideoxyadenosine; D004533:Egtazic Acid; D011493:Protein Kinase C; D002118:Calcium