title: Binding of intact parathyroid hormone to rat osteosarcoma cells: major contribution of binding sites for the carboxyl-terminal region of the hormone.
abstract: Most studies of PTH receptor binding have been carried out with amino-terminal radioligands which only detect binding within that region of the hormone molecule. We studied the binding of electrolytically labeled intact bovine PTH [bPTH-(1-84)], and its amino-terminal fragment, bPTH-(1-34) to intact cloned rat osteosarcoma cells (ROS 17/2.8). We also measured the effects of these hormones on cell cAMP accumulation. Binding equilibrium for the two radioligands was reached by 2 h of incubation at 22 C. However, the cells had higher binding capacity (8-9% or 0.22-0.25 fmol/2 X 10(6) cells) for [125I]bPTH-(1-84) than for [125I]bPTH-(1-34) (4% or 0.11 fmol/2 X 10(6) cells). On the other hand [125I]bPTH-(1-34) bound to ROS cells with higher affinity [dissociation constant (Kd) = 19 nM] than did [125I]bPTH-(1-84) (Kd = 210 nM). Measurements of trichloroacetic acid precipitability and analysis of rebinding of previously incubated radioligand to fresh cells ruled out degradation of the tracer as an explanation for these differences. The maximum cell cAMP response to bPTH-(1-34) (Vmax = 780 +/- 32 pmol/2 X 10(6) cells X 5 min) was reached at 10(-7) M concentration with an affinity [Michaelis-Menten constant (Km)] of 3 nM. On the other hand, the Vmax with intact bPTH-(1-84) was lower (400 +/- 7 pmol/2 X 10(6) cells X 5 min), with a Km of 60 nM). Further studies with the bPTH-(1-84) tracer showed inability of hormonal fragments to compete completely for binding. At a concentration of 3 microM, bPTH-(1-84) reduced tracer binding by 82.5%, compared to 18% by bPTH-(1-34) and 10% by (Nle8,Nle18,Tyr34)bPTH-(1-34)amide, 60% by human PTH (hPTH)-(53-84), and 70% by the combination of bPTH-(1-34) and hPTH-(53-84). hPTH-(53-84) itself did not elicit a cAMP response after 5 min or 1 h of incubation nor did it significantly alter the cAMP response of the cells to bPTH-(1-84). These studies suggest that PTH binds to ROS 17/2.8 cells by sites carboxy-terminal (C-terminal) to position 34, in addition to sites within the amino-terminal portion of the hormone molecule; 72% of the binding of intact hormone to these cells was to the C-terminal 35-84 region of the PTH molecule. The significance of the C-terminal binding sites is presently unclear, but they do not appear to be coupled to adenylate cyclase. Further work is needed to determine the effects of C-terminal PTH fragments on bone cell metabolism.
authors.forename: L G
authors.initials: LG
authors.lastname: Rao
authors.forename: T M
authors.initials: TM
authors.lastname: Murray
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D000242:Cyclic AMP / Q000378:metabolism; D012516:Osteosarcoma / Q000378:metabolism*; D010281:Parathyroid Hormone / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D051381:Rats; D013997:Time Factors
chemical_list: D010281:Parathyroid Hormone; D010446:Peptide Fragments; C035453:parathyroid hormone (53-84); C025076:parathyroid hormone (1-34), bovine; C032505:parathyroid hormone (1-34)amide, 8,18-Nle-34-Tyr-, bovine; D000242:Cyclic AMP