Bisello A, Greenberg Z, Behar V, Rosenblatt M, Suva L J, Chorev M
Harvard-Thorndike Research Laboratory, Department of Medicine, Beth Israel Deaconess Medical Center, Boston, Massachusetts, USA.
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.
甲状旁腺激素（PTH）通过一种特定的G蛋白偶联的、含七个跨膜螺旋的受体来调节钙代谢。该受体也能结合甲状旁腺激素相关蛋白（PTHrP）并被其激活。人（h）PTH/PTHrP受体是一种膜糖蛋白，表观分子量约为85000，含有四个假定的N-糖基化位点。为了阐明受体糖基化（如果有的话）的功能作用，我们研究了用hPTH/PTHrP受体转染的人胚肾细胞（HEK-293/C-21）中的激素结合和信号转导。这些细胞每个细胞稳定表达300000 - 400000个受体。用优化浓度的衣霉素抑制N-糖基化可产生完全非糖基化的hPTH/PTHrP受体（约60 kDa）。这种受体形式功能完全正常；它对PTH和PTHrP衍生的激动剂和拮抗剂保持纳摩尔级的结合亲和力。PTH和PTHrP激动剂刺激环磷酸腺苷（cAMP）积累并使胞质钙水平升高。此外，含高效二苯甲酮（pBz2）的PTH衍生放射性配体[Nle8,18,Lys13(ε-pBz2),L-2-Nal23,Tyr34 3-125I)]bPTH(1-34)NH2能与非糖基化受体特异性地进行光亲和交联。代表光交联的非糖基化受体的条带（从用衣霉素处理的HEK-293/C-21细胞获得）的分子量（约60000）与去糖基化的光交联受体（通过用内切糖苷酶-F/N-糖苷酶-F进行酶处理获得）相似。我们的研究结果表明，hPTH/PTHrP受体的糖基化对于其在质膜上的有效表达或对于已知与天然受体相互作用的配体的结合并非必不可少。非糖基化的hPTH/PTHrP受体在其已知的两种信号转导途径（cAMP-蛋白激酶A和磷脂酶C-胞质钙）方面仍然功能完全正常。
