Role of glycosylation in expression and function of the human parathyroid hormone parathyroid hormone-related protein receptor
Role of glycosylation in expression and function of the human parathyroid hormone parathyroid hormone-related protein receptor
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DOI：
10.1021/bi962111
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发表时间：
1996-12-10
期刊：
影响因子：
2.9
通讯作者：
Chorev, M
中科院分区：
文献类型：
--
作者：
Bisello, A;Greenberg, Z;Chorev, M
甲状旁腺激素（PTH）通过一种特异的G蛋白偶联的、含七跨膜螺旋的受体调节钙代谢。该受体也结合并被PTH相关蛋白（PTHrP）激活。人（h）PTH/PTHrP受体是一种表观分子量约为85000的膜糖蛋白，含有4个推定的N-糖基化位点。为了阐明受体糖基化的功能作用，如果有的话，我们研究了hPTH/PTHrP受体（HEK-293/C-21）转染的人胚肾细胞中的激素结合和信号转导。这些细胞稳定地表达300000-400000个受体/细胞。用优化浓度的衣霉素抑制N-糖基化产生完全非糖基化的hPTH/PTHrP受体（类似于60 kDa）。这种受体形式是完全功能性的;它保持对PTH和PTHrP衍生的激动剂和拮抗剂的纳摩尔结合亲和力。PTH和PTHrP激动剂刺激环AMP积累和细胞溶质钙水平的增加。此外，高效的含二苯甲酮（pBz（2））的PTH衍生的放射性配体[Nle（8，18），Lys（13）（epsilon-pBz（2）），L-2-Nal（23），Tyr（34）（3 -I-125）bPTH（1-34）NH 2可以特异性地与非糖基化受体光亲和交联。代表光交联、非糖基化受体（从衣霉素处理的HEK-293/C-21细胞中获得）的条带的分子量（类似于60 000）与去糖基化光交联受体（通过内切糖苷酶-F/N-糖苷酶-F酶处理获得）的分子量相似。我们的研究结果表明，hPTH/PTHrP受体的糖基化不是必不可少的，其有效的表达在质膜上或已知与天然受体相互作用的配体的结合。非糖基化的hPTH/PTHrP受体在其已知的信号转导途径cAMP-蛋白激酶A和磷脂酶C-胞质钙方面仍然具有完全的功能。
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 85 000 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 (similar to 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 (pBz(2))-containing PTH-derived radioligand [Nle(8,18),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 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.