Characterization and agonist-induced down-regulation of parathyroid hormone receptors in clonal rat osteosarcoma cells.
PTH receptors on two stable clonal rat osteosarcoma cell lines, ROS 17/2 and ROS 17/2.8, were characterized using an HPLC-purified, synthetic, sulfur-free, radioiodinated analog of bovine PTH, [Nle8,Nle18,Tyr,34]bovine PTH-(1-34)amide. PTH binding is specific for PTH agonists and antagonists and is dependent on the time and temperature of incubation. There is an excellent correlation between binding affinities of PTH agonists and antagonists in these intact cell systems with those in canine renal membranes. Peptides unrelated to PTH do not bind. Both ROS 17/2 and 17/2.8 have a single class of saturable, high affinity PTH binding sites that, by kinetic analysis and Scatchard analysis of saturation and competition studies, has a dissociation constant (Kd) of 0.8-1.4 nM. Bmax is approximately 36,000 and 72,000 sites per cell in ROS 17/2 and 17/2.8, respectively. A close correlation was found between the binding of PTH agonists to their receptors in ROS 17/2 cells with their relative biological potencies as measured by stimulation of adenylate cyclase in plasma membranes prepared from these cells. Prolonged treatment of ROS 17/2 and 17/2.8 cells with PTH agonists results in a dose- and time-dependent decrease of available cell-surface binding sites, without alterations in Kd. PTH antagonists do not regulate PTH receptors. Regulation of PTH receptors by PTH agonists is dependent on the dose and time of exposure to ligand over a dose range of 10(-8) to 10(-11) M. Cells exposed to agonists (greater than or equal to 10(-8) M) for 48 h show maximally decreased receptor number; continued exposure to agonists (greater than or equal to 10(-8) M) does not further decrease PTH receptor number, which remains constant at about 15% of control values. Agonist-induced down-regulation occurs with less than 10(-11) M agonists, a concentration less than 10% of the minimal dose detected by direct ligand competition. Treatment of ROS 17/2 cells with PTH agonists results in a dose- and time-dependent decrease of PTH-stimulated adenylate cyclase. This agonist-induced desensitization correlates closely with the decreased availability of PTH receptors: it is maximal in cells exposed to agonists (greater than 10(-8) M) for 48 h and also does not decrease further with continued exposure of the cells to agonist. Future studies with these stable ROS cell lines should permit detailed analysis of the biochemical mechanisms underlying homologous and heterologous regulation of PTH receptors and desensitization and sensitization of the adenylate cyclase response.
Yamamoto
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Department of Medicine, Massachusetts General Hospital, Boston 02114.
Shigeno
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Potts
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Segre
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D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002999:Clone Cells / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D012516:Osteosarcoma / Q000378:metabolism*; D010281:Parathyroid Hormone / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism; D051381:Rats; D011956:Receptors, Cell Surface / Q000502:physiology*; D018016:Receptors, Parathyroid Hormone; D013696:Temperature; D013997:Time Factors; D014407:Tumor Cells, Cultured / Q000378:metabolism
D010281:Parathyroid Hormone; D010446:Peptide Fragments; D011956:Receptors, Cell Surface; D018016:Receptors, Parathyroid Hormone; C032505:parathyroid hormone (1-34)amide, 8,18-Nle-34-Tyr-, bovine; D000262:Adenylyl Cyclases