Behar V, Pines M, Nakamoto C, Greenberg Z, Bisello A, Stueckle S M, Bessalle R, Usdin T B, Chorev M, Rosenblatt M, Suva L J
Division of Bone and Mineral Metabolism, Harvard-Thorndike and Charles A. Dana Laboratories, Boston, Massachusetts 02215, USA.
Endocrinology. 1996 Jul;137(7):2748-57. doi: 10.1210/endo.137.7.8770894.
We have generated a series of stably transfected HEK-293 cell lines expressing the newly identified alternate human PTH receptor (hPTH2 receptor). This receptor subtype is selectively activated by N-terminal PTH-(1-34) and not the corresponding N-terminal (1-34) region of the functionally and structurally related hormone, PTH-related protein (PTHrP). A total of 20 distinct clones displaying different levels of PTH-responsive cAMP production were analyzed. None responded to PTHrP-(1-34). One of these clones (BP-16), displaying maximal PTH responsiveness, was chosen for more detailed evaluation. The BP-16 clone (and the parental HEK-293 cell line lacking both the hPTH/PTHrP receptor and the hPTH2 receptor) were examined for PTH binding, PTH-stimulated cAMP accumulation, PTH-stimulated changes in intracellular calcium ([Ca2+]i) levels, and hPTH2 receptor messenger RNA expression. In addition, we studied the photomediated cross-linking of a potent PTH agonist, namely [Nle8,18,Lys13 (epsilon-pBz2), 2-L-Nal23,Tyr34]bPTH(1-34)NH2 (K13), to the hPTH2 receptor on BP-16 cells. Photoaffinity cross-linking identified an approximately 90-kDa cell membrane component that was specifically competed by PTH-(1-34) and other receptor-interacting ligands. PTH-(1-34) and K13 are potent stimulators of both cAMP accumulation and increases in (Ca2+]i levels, and both bind to the hPTH2 receptor with high affinity (apparent Kd, 2.8 +/- 0.9 x 10(-8) and 8.5 +/- 1.7 x 10(-8) M, respectively). There was no apparent binding, cAMP-stimulating activity, or [Ca 2+]i signaling observed, nor was specific competition vs. binding of a PTH-(1-34) radioligand ([125I]PTH) with PTHrP-(1-34)NH2 found. PTHrP-(1-34) failed to inhibit cross-linking of the hPTH2 receptor by radiolabeled K13 ([125I]K13). However, effective competition vs. [125I]PTH and [125I]K13 binding and [125I]K13 cross-linking were observed with the potent PTH/PTHrP receptor antagonists, PTHrP-(7-34)NH2 and PTH-(7-34)NH2. PTHrP-(7-34)NH2 was shown to be a partial agonist that weakly stimulates both cAMP accumulation and increases in [Ca 2+]i levels in BP-16 cells. These data suggest that the hPTH2 receptor is distinct from the hPTH/PTHrP receptor in the structural features it requires for ligand binding in the family of PTH and PTHrP peptides.
我们构建了一系列稳定转染的HEK - 293细胞系，这些细胞系表达新鉴定出的人甲状旁腺激素替代受体（hPTH2受体）。该受体亚型可被N端甲状旁腺激素 - (1 - 34)选择性激活，而不能被功能和结构相关激素甲状旁腺激素相关蛋白（PTHrP）的相应N端(1 - 34)区域激活。总共分析了20个表现出不同水平甲状旁腺激素反应性cAMP产生的不同克隆。没有一个对PTHrP - (1 - 34)有反应。选择其中一个表现出最大甲状旁腺激素反应性的克隆（BP - 16）进行更详细的评估。对BP - 16克隆（以及既缺乏hPTH/PTHrP受体又缺乏hPTH2受体的亲本HEK - 293细胞系）进行了甲状旁腺激素结合、甲状旁腺激素刺激的cAMP积累、甲状旁腺激素刺激的细胞内钙（[Ca2 + ]i）水平变化以及hPTH2受体信使核糖核酸表达的检测。此外，我们研究了一种强效甲状旁腺激素激动剂，即[Nle8,18,Lys13(ε - pBz2),2 - L - Nal23,Tyr34]bPTH(1 - 34)NH2（K13）与BP - 16细胞上hPTH2受体的光介导交联。光亲和交联鉴定出一种约90 kDa的细胞膜成分，该成分可被甲状旁腺激素 - (1 - 34)和其他与受体相互作用的配体特异性竞争。甲状旁腺激素 - (1 - 34)和K13都是cAMP积累和[Ca2 + ]i水平升高的强效刺激剂，并且两者都以高亲和力结合hPTH2受体（表观Kd分别为2.8 ± 0.9×10 - 8和8.5 ± 1.7×10 - 8 M）。未观察到明显的结合、cAMP刺激活性或[Ca2 + ]i信号传导，也未发现PTHrP - (1 - 34)NH2与甲状旁腺激素 - (1 - 34)放射性配体（[125I]PTH）结合的特异性竞争。PTHrP - (1 - 34)未能抑制放射性标记的K13（[125I]K13）对hPTH2受体的交联。然而，用强效PTH/PTHrP受体拮抗剂PTHrP - (7 - 34)NH2和PTH - (7 - 34)NH2观察到了对[125I]PTH和[125I]K13结合以及[125I]K13交联的有效竞争。PTHrP - (7 - 34)NH2被证明是一种部分激动剂，可在BP - 16细胞中微弱刺激cAMP积累和[Ca2 + ]i水平升高。这些数据表明，在甲状旁腺激素和甲状旁腺激素相关蛋白肽家族中，hPTH2受体在配体结合所需的结构特征上与hPTH/PTHrP受体不同。
