Immunoprecipitation of the parathyroid hormone receptor.
An 125I-labeled synthetic analog of bovine parathyroid hormone, [8-norleucine,18-norleucine,34-tyrosine]PTH-(1-34) amide ([Nle]PTH-(1-34)-NH2), purified by high-pressure liquid chromatography (HPLC), was employed to label the parathyroid hormone (PTH) receptor in cell lines derived from PTH target tissues: the ROS 17/2.8 rat osteosarcoma of bone and the CV1 and COS monkey kidney lines. After incubation of the radioligand with intact cultured cells, the hormone was covalently attached to receptors by using either a photoaffinity technique or chemical (affinity) cross-linking. In each case, covalent labeling was specific, as evidenced by a reduction of labeling when excess competing nonradioactive ligand was present. After covalent attachment of radioligand, membranes were prepared from the cells and solubilized in the nonionic detergent Nonidet P-40 or octyl glucoside. The soluble membrane fraction present in the supernatant of a 100,000 X g centrifugation was incubated with IgG prepared from anti-PTH antiserum generated to the amino-terminal region, residues 1-34, of PTH. The IgG-PTH-receptor complex was precipitated with staphylococcal protein A-Sepharose. Analysis of the immunoprecipitate on NaDod-SO4/polyacrylamide gel electrophoresis followed by autoradiography revealed the presence of a doublet of apparent molecular mass 69-70 kDa. Specifically labeled bands of approximate molecular mass 95 and 28 kDa were also observed. The anti-PTH IgG was affinity purified by passage over a PTH-Sepharose column and used to make an immunoaffinity column. The 70- and 28-kDa bands were also observed after labeled solubilized membrane preparations were allowed to bind to this column and then were eluted by using a [Nle]PTH-(1-34)-NH2-containing buffer or acetic acid. These studies suggest that the use of an anti-PTH antiserum that binds receptor-bound hormone is likely to be a useful step in the further physiochemical characterization and purification of the PTH receptor.
D000818:Animals; D001859:Bone Neoplasms / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002522:Chlorocebus aethiops; D002846:Chromatography, Affinity; D007074:Immunoglobulin G; D007668:Kidney / Q000378:metabolism; D008970:Molecular Weight; D012516:Osteosarcoma / Q000378:metabolism; D010281:Parathyroid Hormone / Q000378:metabolism; D051381:Rats; D011956:Receptors, Cell Surface / Q000302:isolation & purification* / Q000378:metabolism; D018016:Receptors, Parathyroid Hormone
D007074:Immunoglobulin G; D010281:Parathyroid Hormone; D011956:Receptors, Cell Surface; D018016:Receptors, Parathyroid Hormone