Role of glycosylation in expression and function of the human parathyroid hormone/parathyroid hormone-related protein receptor.
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.
Bisello
A
A
Harvard-Thorndike Research Laboratory, Department of Medicine, Beth Israel Deaconess Medical Center, Boston, Massachusetts, USA.
Greenberg
Z
Z
Behar
V
V
Rosenblatt
M
M
Suva
L J
LJ
Chorev
M
M
D000345:Affinity Labels / Q000737:chemistry / Q000378:metabolism; D001667:Binding, Competitive; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D003432:Cross-Linking Reagents; D000242:Cyclic AMP / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression / Q000235:genetics; D006031:Glycosylation; D006801:Humans; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / Q000378:metabolism; D010281:Parathyroid Hormone / Q000378:metabolism*; D044162:Parathyroid Hormone-Related Protein; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D018016:Receptors, Parathyroid Hormone / Q000378:metabolism*; D015398:Signal Transduction; D014162:Transfection / Q000235:genetics; D014415:Tunicamycin / Q000494:pharmacology
D000345:Affinity Labels; D003432:Cross-Linking Reagents; C470121:PTHLH protein, human; D010281:Parathyroid Hormone; D044162:Parathyroid Hormone-Related Protein; D010446:Peptide Fragments; D011506:Proteins; D018016:Receptors, Parathyroid Hormone; D014415:Tunicamycin; D000242:Cyclic AMP; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D002118:Calcium