Pines M, Fukayama S, Costas K, Meurer E, Goldsmith P K, Xu X, Muallem S, Behar V, Chorev M, Rosenblatt M, Tashjian A H, Suva L J
Harvard-Thorndike and Charles A. Dana Laboratories, Department of Medicine, Beth Israel Hospital, Boston, MA, USA.
Bone. 1996 Apr;18(4):381-9. doi: 10.1016/8756-3282(96)00008-7.
We previously reported the preparation and partial characterization of a series of human embryonic kidney cell lines (HEK-293) stably expressing various numbers of the recombinant human (h) parathyroid hormone (PTH)/PTH-related protein (PTHrP) receptor (Rc). Using this expression system we examined ligand (PTH or PTHrP) binding characteristics and cyclic AMP responsiveness. We have now extended these studies to investigate the calcium signal transduction pathways activated by the hPTH/PTHrP Rc. In parental HEK-293 cells, which lack endogenous PTH/PTHrP Rc, incubation with hPTH(1-34) had no effect on cytosolic free Ca2+ concentration [Ca2+]i. In HEK-293 clone C-21, stably expressing approximately 400,000 Rc/cell, PTH stimulated an increase in [Ca2+]i by Ca2+ release from intracellular stores; PTH released Ca2+ exclusively from the IP3 sensitive Ca2+ pool. Unlike previous studies, the ability of PTH to elicit both cAMP responses and [Ca2+]i transients occurred over a wide range of Rc numbers (between 400,000 and 3000 Rc/cell); both responses were always observed at PTH concentrations in the same dose range although the magnitude of the responses decrease with Rc number. Pretreatment of C-21 cells with pertussis toxin for 24 h, which significantly enhanced PTH-stimulated cAMP accumulation, did not modulate PTH-stimulated [Ca2+]i transients. At each PTH concentration tested which resulted in increased cAMP levels, there was also an increase in [Ca2+]i transients. Treatment of C-21 cells with a battery of midregion and C-terminal PTH or PTHrP peptides showed no effect on either [Ca2+]i transients or cAMP accumulation, indicating a lack of functional interactions between these peptides and the form of the hPTH/PTHrP Rc stably expressed in these cells. Immunological analysis of G-protein expression demonstrated the presence of Gs, Gi, and Gq in all parental and transfected cells lines examined. Taken together, these data demonstrate that the hPTH/PTHrP Rc, stably expressed in HEK-293 cells, elicits responses in both the cAMP and IP3-dependent [Ca2+]i pathways and is responsive only to N-terminal PTH/PTHrP peptides.
我们先前报道了一系列稳定表达不同数量重组人（h）甲状旁腺激素（PTH）/PTH相关蛋白（PTHrP）受体（Rc）的人胚肾细胞系（HEK - 293）的制备及部分特性。利用该表达系统，我们检测了配体（PTH或PTHrP）的结合特性及环磷酸腺苷（cAMP）反应性。我们现在扩展了这些研究，以探究由hPTH/PTHrP Rc激活的钙信号转导途径。在缺乏内源性PTH/PTHrP Rc的亲本HEK - 293细胞中，用hPTH(1 - 34)孵育对胞质游离Ca2+浓度[Ca2+]i没有影响。在稳定表达约400,000个Rc/细胞的HEK - 293克隆C - 21中，PTH通过从细胞内储存释放Ca2+刺激[Ca2+]i增加；PTH仅从IP3敏感的Ca2+池中释放Ca2+。与先前的研究不同，PTH引发cAMP反应和[Ca2+]i瞬变的能力在广泛的Rc数量范围内（400,000至3000个Rc/细胞之间）出现；在相同剂量范围内的PTH浓度下总能观察到这两种反应，尽管反应幅度随Rc数量减少。用百日咳毒素预处理C - 21细胞24小时，这显著增强了PTH刺激的cAMP积累，但并未调节PTH刺激的[Ca2+]i瞬变。在每个测试的导致cAMP水平升高的PTH浓度下，[Ca2+]i瞬变也增加。用一系列中部区域和C末端PTH或PTHrP肽处理C - 21细胞，对[Ca2+]i瞬变或cAMP积累均无影响，表明这些肽与在这些细胞中稳定表达的hPTH/PTHrP Rc形式之间缺乏功能相互作用。对G蛋白表达的免疫学分析表明，在所检测的所有亲本和转染细胞系中均存在Gs、Gi和Gq。综上所述，这些数据表明，在HEK - 293细胞中稳定表达的hPTH/PTHrP Rc在cAMP和IP3依赖性[Ca2+]i途径中均引发反应，并且仅对N末端PTH/PTHrP肽有反应。
