Characterization of a novel parathyroid hormone (PTH) receptor with specificity for the carboxyl-terminal region of PTH-(1-84)
Carboxyl-terminal fragments of PTH (C-PTH) appear to have biological properties different from those mediated by the amino-terminal portions of PTH and PTH-related peptide (PTHrP). To characterize a C-PTH receptor that may be involved in mediating these functions, we performed RRAs and affinity cross-linking studies with several clonal cell lines. Radiolabeled recombinant [Leu8,18,Tyr34]human PTH-(1-84)[mutPTH-(1-84) and [Tyr34] human PTH-(19-84)[mutPTH-(19-84) showed little or no specific binding to stably expressed recombinant PTH/PTHrP receptors. However, high affinity binding was observed using osteoblast-like and rat parathyroid (PT-r3) cells. The apparent Kd values were 20-30 nM for PTH-(1-84), mutPTH-(1-84), and mutPTH-(19-84), respectively; 400-800 nM for PTH-(39-84); and more than 5000 nM for PTH-(53-84). [Nle8,18,Tyr34]bovine PTH-(1-34)amide [PTH-(1-34)], PTH-(44-68), PTHrP-(37-74), and PTHrP-(109-141) showed no displacement of either radioligand. C-PTH receptor number was increased up to 2-fold by pretreating ROS 17/2.8 cells with increasing doses of PTH-(1-34), PTH-(1-84), or 8-bromo-cAMP, whereas no change was observed in response to dexamethasone or PTH-(39-84). Cross-linking studies using radiolabeled mutPTH-(1-84) or mutPTH-(19-84) revealed specific labeling of two proteins in ROS 17/2.8 cells that were approximately 40 and 90 kilodaltons in size (including the radioligand of approximately 10 kilodaltons). The intensity of affinity labeling of both proteins was dose dependently inhibited by increasing concentrations of unlabeled PTH-(1-84) and several carboxyl-terminal PTH-(1-84) fragments, but not by PTH-(1-34). Similar studies with PT-r3 cells revealed only a single protein band of about 90 kilodaltons. These data indicate that the carboxyl-terminal portion of PTH-(1-84) binds specifically to a unique receptor/binding protein distinct from the previously isolated PTH/PTHrP receptor.
Inomata
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N
Department of Medicine, Massachusetts General Hospital, Boston 02114, USA.
Akiyama
M
M
Kubota
N
N
Jüppner
H
H
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000818:Animals; D002417:Cattle; D003432:Cross-Linking Reagents; D003907:Dexamethasone / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D010006:Osteoblasts / Q000378:metabolism; D012516:Osteosarcoma; D010281:Parathyroid Hormone / Q000378:metabolism* / Q000494:pharmacology; D010446:Peptide Fragments / Q000378:metabolism* / Q000494:pharmacology; D011869:Radioligand Assay; D051381:Rats; D018016:Receptors, Parathyroid Hormone / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D015183:Restriction Mapping; D019379:Teriparatide; D014407:Tumor Cells, Cultured
D003432:Cross-Linking Reagents; D010281:Parathyroid Hormone; D010446:Peptide Fragments; D018016:Receptors, Parathyroid Hormone; D011994:Recombinant Proteins; D019379:Teriparatide; D015124:8-Bromo Cyclic Adenosine Monophosphate; D003907:Dexamethasone