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Cells derived from human giant cell tumors of bone and fibroblasts derived from human neonatal foreskin respond to parathyroid hormone (PTH) by increasing the intracellular and extracellular levels of adenosine cyclic 3',5'-phosphate (cAMP). Using photoaffinity labeling methods, we examined these cells for the presence of a PTH receptor or a binding subunit of a receptor complex. A previously designed biologically active and photolabile radioligand analogue of PTH was reacted with these intact cells. After photolysis, the cells were extracted, and the proteins were denatured, reduced, and separated by electrophoresis on sodium dodecyl sulfate (Na-DodSO4)-polyacrylamide gels followed by autoradiography. A single membrane component, Mr 70 000, was labeled specifically in intact cells cultured from skeletal and dermal tissue. By mixing, in pairs, photolabeled proteins from (a) intact human cells derived from giant cell tumors of bone, (b) intact human fibroblasts, and (c) canine renal cortical membranes, the receptors (or their binding subunits) for PTH were compared directly and found to be identical in terms of molecular size (as determined by the migration position on NaDod-SO4-polyacrylamide gels) across species (dog and human) and target tissue (bone, skin, and kidney). Preincubation of cells cultured from giant cell tumors of bone with PTH resulted in loss of the PTH-induced cAMP response (desensitization). Preincubation with PTH was accompanied by a marked decrease in photoaffinity labeling of the PTH binding component and suggests that the loss of hormone response in cells preincubated with PTH was related to a decrease in the number or availability of PTH receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

An '25I-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) crosslinking. 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 ofa 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-PTHreceptor complex was precipitated with staphylococcal protein A-Sepharose. Analysis of the immunoprecipitate on NaDod-S04/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 immunoaffmity 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 physicochemical characterization and purification of the PTH receptor. was obtained from LKB. Cell culture reagents were obtained from GIBCO.Preparation of 2-51-Labeled [NMe]PTH-(1-34)-NH2. All procedures were performed at room temperature unless otherwise specified. Iodo-Gen (100 ,g) in methylene chloride was used to plate the inside of a 12 x 75 mm test tube according to the manufacturer's instructions. The iodination reaction was started by adding 10 ,ug of [Nle]PTH-(1-34)-NH2 dissolved in 40 ,ul of 8 M urea and 2.0 mCi (1 Ci = 37 GBq) of 1251I. The tube was sealed and the reagents were allowed to react for 15 min with occasional agitation. The reaction was stopped by the addition of0.3 ml ofwater and the mixture was drawn into a syringe containing anion-exchange resin (BioRad AG 1-X8) for removing free 1251. A filter was attached to the syringe and the unbound material was injected onto a 3.9 mm x 15 cm C18 HPLC column (Waters Nova Pak). The flow rate was 1 ml/min of 0.1% trifluoroacetic acid in 70% (vol/vol) water/30% acetonitrile with a linear gradient to 50% acetonitrile over 20 min. Column effluent was monitored ...

The carboxyl-terminal region of human parathyroid hormone (hPTH) was synthesized by the solid phase method. The 32-amino acid fragment, hPTH-(53-84), was prepared for two reasons: 1) to produce antisera directed exclusively against the carboxyl-terminal region of hPTH, which represents the predominant form of the hormone in blood; and 2) to determine whether a portion of the hormone other than the amino-terminal region has any of the biological activity of intact hormone or can bind to PTH receptors. The synthetic fragment was evaluated for heterogeneity by amino acid composition and by several high resolution analytical systems (thin layer electrophoresis, polyacrylamide gel isoelectric focusing, and Edman sequence analysis). Synthetic hPTH-(53-84) lacked PTH-like activity in both in vitro (renal) and in vivo (bone) assays and failed to inhibit the action of native PTH in vitro, indicating lack of receptor binding by the carboxyl-terminal region of the molecule. Because the fragment lacked biological activity, nonchemical evaluation was performed to confirm that the peptide represents the carboxyl terminus of hPTH. Immunological comparison of synthetic hPTH-(53-84) to native hPTH was undertaken using an RIA that employed an antibovine PTH antiserum whose antigenic recognition was limited to the carboxyl-terminal region of the hormone. Synthetic hPTH-(53-84) was more immunoreactive than native hPTH. The immunological findings provide further evidence that the synthetic peptide accurately represents the carboxyl-terminal region of native hPTH.

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