Parathyroid hormone receptors are plasma membrane glycoproteins with asparagine-linked oligosaccharides.

C Shigeno,D P Westerberg,J T Potts,Y Hiraki,G V Segre

doi:10.1016/s0021-9258(18)69006-4

C Shigeno, D P Westerberg + Show 3 more

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https://doi.org/10.1016/s0021-9258(18)69006-4

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Abstract

In the preceding article, we described physicochemical and kinetic properties of parathyroid hormone (PTH) receptors in clonal rat osteosarcoma cells (ROS 17/2.8) using photoaffinity ligand labeling and showed that the physiologically relevant receptor-ligand complex has an apparent Mr = 80,000. In this study, the photoaffinity labeled Mr = 80,000 receptor was localized exclusively on the cell surface plasma membrane and its glycoprotein nature was demonstrated through the use of lectin affinity-chromatography and specific exo- and endoglycosidases. Rinsing ROS cells, preincubated in the dark with 125I-labeled [Nle8, N-epsilon-(4-azido-2-nitrophenyl)Lys13,Nle18,Tyr34]bovine PTH-(1-34)-NH2 (NAP-NlePTH) (4 h, 15 degrees C, equilibrium conditions) with acidic phosphate-buffered saline (pH 2.5, 30 s, 4 degrees C) before photolysis resulted in selective and nearly total disappearance of the labeled Mr = 80,000 receptor. PTH receptor integrity to acid rinsing and photolysis was shown by relabeling the Mr = 80,000 receptor after a second incubation of these cells with 125I-labeled NAP-NlePTH, followed by photolysis. Adsorption of Triton X-100-solubilized, 125I-labeled NAP-NlePTH receptors to wheat germ agglutinin-agarose is nearly complete and highly selective, and elution with N-acetylglucosamine resulted in virtually total recovery of the labeled receptors from the column. The wheat germ agglutinin-retarded PTH receptors show increased electrophoretic mobility upon treatment with neuraminidase which was inhibited by simultaneous addition of 2,3-dehydro-3-desoxy-N-acetylneuraminic acid, a specific neuraminidase inhibitor. Endoglycosidase F treatment of the Mr = 80,000 receptors generated a single, labeled polypeptide with a Mr = 59,000 which migrated as a narrow band. PTH receptors on ROS 17/2.8 cells appear to be monomeric plasma membrane glycoproteins with an apparent Mr of 80,000 which contain a Mr = 59,000 polypeptide backbone and a polymeric arrangement of N-acetylglucosamine with N-acetylneuraminic acid as major terminal sugar residues.

Highlights

From the Endocrine Unit, Massachusetts General Hospital, and the Department of Medicine, Haruard Medical School, Boston, Massachusetts02114
The biological characteristics of the adenylate cyclase-coupled P T H receptors in ROS1712.8 cells have been elucidated in recent years [15,16,17]
Adsorption of Triton X-100-solubilized, labeled NAP-NlePTH receptors to wheat germ agglutinin-agarose is nearly complete and highly selective, tor-ligand complex migrates as a diffuse radioactive band upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in both reducing and nonreducing condiand elution with N-acetylglucosamine resultedin vir- tions [21],suggesting that PTHreceptors mightbe glycoprotually total recoveryof the labeled receptors from the teins [22, 23]

Summary

RESULTS

Polypeptide backbone anda polymeric arrangement of Effect of AcidRinsing on Photoaffinity Labeling of PTH. We investigated the effect of acid treatment after photolysis of ROS 17/2.8 cells incubated with '251-labeled NAP-NlePTH. Photolysis nor was labeling of macromolecules observed when cells weretreated with acidic PBS, butphotolysiswas omitted (Fig. 2, lane 2 ) These results are consistent with our notion that theMI = 80,000 receptor, covalently cross-linked to '"Ilabeled NAP-NlePTH, is localizedexclusively on the cell surface. An apparently identical M , = 80,000 receptor protein was relabeled after the second incubation with the photoligand in the once acid-rinsed and photolyzed cells(lane3 ) ,which, again, was strikingly reduced when the cells wereacid-treated before the second photolysis (lane ). LectinAffinityChromatographyof Triton X-100-solubilized, Photoligand-labeled PTH Receptors-TritonX-100-solubilized lZ5I-labeledNAP-NlePTH-ROS cell plasma membrane complexes werefractionated on WGA-agaroseand LL-Sepharose 4B columns.

Treatment Sequence

In u n

DISCUSSION

Findings

Selective removal of noncovalently bound peptide ligands