Immunoprecipitation of the parathyroid hormone receptor.

Abstract

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 ADP/ATP carrier is the 32-kilodalton receptor for an NH2-terminally myristylated src peptide but not for pp60src polypeptide.

“…On the basis of this evidence, we have used the MGYsrc peptide as a probe in membrane cross-linking studies to identify potential pp6Qsrc binding proteins. This experimental rationale is based on the ability to use radiolabelled small ligands as probes to identify membrane-bound receptors and has been successfully exploited by other investigators to identify the receptors for, e.g., gamma interferon, erythropoietin, angiotensin II, and parathyroid hormone (21,29,31,39).…”

PTHrP, PTHr, and FGFR3 are involved in the process of endochondral ossification in human osteophytes

“…The antibody against PTHr was characterized by Wright et al (1987) and the antibody against PTHrP by Kramer et al (1991). According to the manufacturer the PTHrP antibody does not reveal crossreactivity with human PTH.…”

Intrauterine occupancy controls expression of the parathyroid hormone-related peptide gene in preterm rat myometrium.

“…Antiserum neutralization experiments were carried out with a rabbit polyclonal antiserum (R13) raised against hPTHRP-(1-74) (20); rabbit nonimmune serum and a rabbit polyclonal anti-bPTH-(1-34) antiserum (JH-42, Meloy Laboratories) were used as controls (29). In these experiments, uterus extracts were incubated for 24 hr at 4°C in phosphate-buffered saline with or without the antisera at a final dilution of 1:50 prior to ROS assay (20).…”