Abou-Samra A B, Goldsmith P K, Xie L Y, Jüppner H, Spiegel A M, Segre G V
Endocrine Unit, Massachusetts General Hospital, Boston 02114.
Endocrinology. 1994 Dec;135(6):2588-94. doi: 10.1210/endo.135.6.7988447.
Recent data have shown that PTH down-regulation of its receptor on opossum kidney (OK) cells is not associated with any change in the steady state level of the PTH/PTH-related peptide (PTHrP) receptor messenger RNA. For analysis of down-regulation of the PTH/PTHrP receptor in OK cells, the present work uses a specific receptor anti-serum, SR-2, that is useful for detection and quantification of PTH/PTHrP receptor immunoreactivity on intact cells bearing the opossum PTH/PTHrP receptor. SR-2 specifically binds to COS-7 cells transiently expressing the opossum PTH/PTHrP receptor complementary DNA (OK-O), to LLCPK1 cells stably expressing the recombinant opossum PTH/PTHrP receptor (AOK cells), and to OK cells expressing endogenous PTH/PTHrP receptors, but not to mock-transfected COS-7 cells or untransfected LLCPK1 cells. SR-2 binding was also linearly correlated with PTH binding in COS-7 cells transfected with different amounts of OK-O plasmid DNA. Treatment with PTH (100 nM) for 4 and 6 h did not significantly down-regulate the PTH/PTHrP receptor immunoreactivity, although PTH binding was decreased to 51% and 49% of control, respectively, and PTH-stimulated cAMP accumulation was decreased to 27% and 28% of control, respectively. Treatment with PTH (100 nM) for 24 and 48 h significantly decreased PTH binding to 51% and 60% of control and decreased PTH/PTHrP receptor immunoreactivity to 68% and 58% of control, respectively. Incubation of OK cells with 0.1 nM to 1 microM PTH for 4 h did not down-regulate the PTH/PTHrP receptor immunoreactivity, although PTH binding was decreased dramatically. Scatchard blot analysis revealed that the binding affinity was decreased by 7-fold in OK cells treated with PTH for 4 h without change in receptor number. Conversely, treatment of OK cells with PTH for 24 h resulted in a parallel decrease in both receptor number and receptor immunoreactivity without any change in receptor binding affinity. Treatment of OK cells with dexamethasone (0.1 nM to 1 microM) had no effect on PTH binding or PTH/PTHrP receptor immunoreactivity. Incubation of OK cells with both dexamethasone (1 microM) and PTH (0.1 nM to 1 microM), however, caused a significantly greater down-regulation of both PTH binding and PTH/PTHrP receptor immunoreactivity than in cells treated with PTH alone. These data indicate that during the first 4 h of exposure of OK cells to PTH, PTH/PTHrP receptors remain on the cell surface but have lowered affinity to bind the ligand and that dexamethasone potentiates the effect of PTH on PTH/PTHrP receptor down-regulation in OK cells.
近期数据显示，甲状旁腺激素（PTH）对负鼠肾（OK）细胞上其受体的下调与甲状旁腺激素/甲状旁腺激素相关肽（PTHrP）受体信使核糖核酸的稳态水平变化无关。为分析OK细胞中PTH/PTHrP受体的下调情况，本研究使用了一种特异性受体抗血清SR-2，它可用于检测和定量表达负鼠PTH/PTHrP受体的完整细胞上的PTH/PTHrP受体免疫反应性。SR-2能特异性结合瞬时表达负鼠PTH/PTHrP受体互补DNA的COS-7细胞（OK-O）、稳定表达重组负鼠PTH/PTHrP受体的LLCPK1细胞（AOK细胞）以及表达内源性PTH/PTHrP受体的OK细胞，但不与mock转染的COS-7细胞或未转染的LLCPK1细胞结合。在转染了不同量OK-O质粒DNA的COS-7细胞中，SR-2结合也与PTH结合呈线性相关。用PTH（100 nM）处理4小时和6小时，虽然PTH结合分别降至对照的51%和49%，PTH刺激的环磷酸腺苷（cAMP）积累分别降至对照的27%和28%，但并未显著下调PTH/PTHrP受体免疫反应性。用PTH（100 nM）处理24小时和48小时，PTH结合分别显著降至对照的51%和60%，PTH/PTHrP受体免疫反应性分别降至对照的68%和58%。用0.1 nM至1 microM的PTH孵育OK细胞4小时，虽然PTH结合显著降低，但并未下调PTH/PTHrP受体免疫反应性。Scatchard印迹分析显示，用PTH处理4小时的OK细胞中，结合亲和力降低了7倍，而受体数量没有变化。相反，用PTH处理OK细胞24小时导致受体数量和受体免疫反应性同时平行下降，而受体结合亲和力没有任何变化。用地塞米松（0.1 nM至1 microM）处理OK细胞对PTH结合或PTH/PTHrP受体免疫反应性没有影响。然而，用1 microM地塞米松和0.1 nM至1 microM的PTH共同孵育OK细胞，与单独用PTH处理的细胞相比，导致PTH结合和PTH/PTHrP受体免疫反应性的下调显著更大。这些数据表明，在OK细胞暴露于PTH的最初4小时内，PTH/PTHrP受体仍留在细胞表面，但与配体结合的亲和力降低，并且地塞米松增强了PTH对OK细胞中PTH/PTHrP受体下调的作用。
