Role of glycosylation in expression and function of the human parathyroid hormone/parathyroid hormone-related protein receptor

Biochemistry. 1996 Dec 10;35(49):15890-5. doi: 10.1021/bi962111+.


Parathyroid hormone (PTH) regulates calcium metabolism through a specific G protein-coupled, seven-transmembrane helix-containing receptor. This receptor also binds and is activated by PTH-related protein (PTHrP). The human (h) PTH/PTHrP receptor is a membrane glycoprotein with an apparent molecular weight of approximately 85000 which contains four putative N-glycosylation sites. To elucidate the functional role of receptor glycosylation, if any, we studied hormone binding and signal transduction in human embryonic kidney cells transfected with hPTH/PTHrP receptor (HEK-293/C-21). These cells stably express 300000-400000 receptors per cell. Inhibition of N-glycosylation with an optimized concentration of tunicamycin yielded completely nonglycosylated hPTH/PTHrP receptor (approximately 60 kDa). This receptor form is fully functional; it maintains nanomolar binding affinity for PTH- and PTHrP-derived agonists and antagonists. PTH and PTHrP agonists stimulate cyclic AMP accumulation and increases in cytosolic calcium levels. In addition, the highly potent benzophenone (pBz2)-containing PTH-derived radioligand [Nle8,18,Lys13(epsilon-pBz2),L-2-Nal23,Tyr34 3-125I)]bPTH(1-34)NH2 can photoaffinity cross-link specifically to the nonglycosylated receptor. The molecular weight (approximately 60000) of the band representing the photo-cross-linked, nonglycosylated receptor (obtained from the tunicamycin-treated HEK-293/C-21 cells) was similar to that of the deglycosylated photo-cross-linked receptor (obtained by enzymatic treatment with Endoglycosidase-F/N-glycosidase-F). Our findings indicate that glycosylation of the hPTH/PTHrP receptor is not essential for its effective expression on the plasma membrane or for the binding of ligands known to interact with the native receptor. The nonglycosylated hPTH/PTHrP receptor remains fully functional with regard to both of its known signal transduction pathways: cAMP-protein kinase A and phospholipase C-cytosolic calcium.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Affinity Labels / chemistry
  • Affinity Labels / metabolism
  • Binding, Competitive
  • Calcium / metabolism
  • Cells, Cultured
  • Cross-Linking Reagents
  • Cyclic AMP / metabolism
  • Electrophoresis, Polyacrylamide Gel
  • Gene Expression / genetics
  • Glycosylation
  • Humans
  • Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / metabolism
  • Parathyroid Hormone / metabolism*
  • Parathyroid Hormone-Related Protein
  • Peptide Fragments / chemistry
  • Peptide Fragments / metabolism
  • Protein Binding
  • Proteins / metabolism*
  • Receptors, Parathyroid Hormone / metabolism*
  • Signal Transduction
  • Transfection / genetics
  • Tunicamycin / pharmacology


  • Affinity Labels
  • Cross-Linking Reagents
  • PTHLH protein, human
  • Parathyroid Hormone
  • Parathyroid Hormone-Related Protein
  • Peptide Fragments
  • Proteins
  • Receptors, Parathyroid Hormone
  • Tunicamycin
  • Cyclic AMP
  • Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase
  • Calcium