Specific binding of parathyroid hormone to living osteoclasts.
We show that osteoclasts bind parathyroid hormone (PTH) in a manner that displays the properties of receptor-dependent hormone binding, that is, saturability, time dependence, temperature dependence, and hormone specificity. Osteoclasts were isolated from the endosteum of 2 to 3 week chick tibiae and maintained in culture for 4-6 days. Bovine PTH-(1-84) was biotinylated with N-hydroxysuccinimidobiotin. Biotinyl-PTH (btPTH, 10(-5)-10(-11) M) was added to the cultured osteoclasts for 2-20 minutes. After rinsing away unbound btPTH, fluorescein isothiocyanate-labeled avidin (FITC-avidin) at a concentration of 66 micrograms/ml was applied. Receptor binding characteristics were assessed: (1) saturation occurred at around 10(-6) M btPTH; (2) competition of excess unlabeled PTH was found, namely, a 10-fold excess abolished fluorescence; (3) specificity was shown by adding other polypeptide hormones (insulin, glucagon, and calcitonin) in 10- to 100-fold excess--no effect on PTH binding was observed; and (4) affinity of btPTH for its binding site was indicated by half-maximal binding approximately equal to 10(-7) M for both osteoclasts and osteoblasts. Biotin (10(-5) M) or FITC-avidin (66 micrograms/ml) alone did not cause fluorescence. The time course of btPTH on the cell exterior was short: at 2 and 5 minutes dots of fluorescence were randomly dispersed over the cell surface, by 10 minutes most of the fluorescence was clustered in one region of the membrane, and by 20 minutes most of the hormone was no longer present on the surface of the cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Agarwala
N
N
Zetachron, Inc., State College, Pennsylvania.
Gay
C V
CV
D001360:Avidin / Q000378:metabolism; D001667:Binding, Competitive / Q000502:physiology; D001710:Biotin; D002469:Cell Separation; D002470:Cell Survival / Q000502:physiology; D004705:Endocytosis / Q000502:physiology; D066298:In Vitro Techniques; D007525:Isoelectric Focusing; D007700:Kinetics; D010010:Osteoclasts / Q000378:metabolism*; D010281:Parathyroid Hormone / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000378:metabolism*; D018016:Receptors, Parathyroid Hormone; D013696:Temperature; D013997:Time Factors
D010281:Parathyroid Hormone; D011956:Receptors, Cell Surface; D018016:Receptors, Parathyroid Hormone; D001360:Avidin; D001710:Biotin