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 speci fic G protein-coupled, seven-transmembrane helix-containing receptor. This receptor also binds and is activated by PTH-related protein (PTHr P). The human (h) PTH/PTHrP receptor is a membrane glycoprotein with a n apparent molecular weight of approximately 85 000 which contains fou r 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/PTH rP receptor (HEK-293/C-21). These cells stably express 300000-400000 r eceptors per cell. Inhibition of N-glycosylation with an optimized con centration of tunicamycin yielded completely nonglycosylated hPTH/PTHr P receptor (similar to 60 kDa). This receptor form is fully functional ; it maintains nanomolar binding affinity for PTH- and PTHrP-derived a gonists and antagonists. PTH and PTHrP agonists stimulate cyclic AMP a ccumulation and increases in cytosolic calcium levels. In addition, th e highly potent benzophenone (pBz(2))-containing PTH-derived radioliga nd ,18),Lys(13)(epsilon-pBz(2)),L-2-Nal(23),Tyr(34)(3 -I-125)bPTH(1-34 )NH2 can photoaffinity cross-link specifically to the nonglycosylated receptor. The molecular weight (similar to 60 000) of the band represe nting 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 tre atment 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 lig ands known to interact with the native receptor. The nonglycosylated h PTH/PTHrP receptor remains fully functional with regard to both of its known signal transduction pathways: cAMP-protein kinase A and phospho lipase C-cytosolic calcium.