Generation and characterization of human kidney cell lines stably expressing recombinant human PTH/PTHrP receptor: lack of interaction with a C-terminal human PTH peptide.
Generation and characterization of human kidney cell lines stably expressing recombinant human PTH/PTHrP receptor: lack of interaction with a C-terminal human PTH peptide.
复制标题
稳定表达重组人 PTH/PTHrP 受体的人肾细胞系的生成和表征：缺乏与 C 端人 PTH 肽的相互作用。
作者：
M. Pines;A. E. Adams;S. Stueckle;R. Bessalle;V. Rashti;M. Chorev;M. Rosenblatt;L. Suva
甲状旁腺激素（PTH）通过与主要在骨和肾中表达的膜结合G蛋白偶联受体结合来发挥其生物学作用。在这项研究中，我们描述了一组细胞系的生产和表征，来自人胚肾细胞系（HEK-293），其中每一个稳定表达不同量的重组人甲状旁腺激素/甲状旁腺相关蛋白（PTHrP）受体（Rc）。分析了总共52个显示不同水平的PTH响应性cAMP产生的不同克隆;选择三个克隆进行更详细的评价。检查这些克隆（和缺乏受体的亲本细胞系）的PTH结合、PTH刺激的环AMP积累和PTH/PTHrP Rc mRNA表达。受体阳性克隆显示与受体数量/细胞和受体mRNA水平相关的PTH反应谱。在表达最高量Rc（> 400，000 Rc/细胞）的细胞中检查C-末端hPTH-（52-84）肽与稳定表达的人受体的相互作用。hPTH-（52-84）与放射性标记的PTH-（1-34）无直接结合或特异性竞争。然而，观察到bPTH-（1 - 34）、hPTH-（1-84）和hPTHrP-（1-34）与放射性标记的PTH-（1-34）的竞争。这些数据表明hPTH-（52-84）不与人PTH/PTHrP Rc的唯一已知形式相互作用。因此，报告的PTH-（52-84）在其他系统中的影响必须通过替代（尚未确定）机制。在人靶细胞背景中表达不同量的人PTH/PTHrP Rc应有助于表征Rc的配体结合性质和生理信号转导机制。
Parathyroid hormone (PTH) exerts its biological action by binding to membrane-bound, G-protein coupled receptors expressed predominantly in bone and kidney. In this study, we describe the production and characterization of a panel of cell lines, derived from a human embryonic kidney cell line (HEK-293), each of which stably express different amounts of the recombinant human PTH/parathyroid hormone-related protein (PTHrP) receptor (Rc). A total of 52 distinct clones displaying different levels of PTH-responsive cAMP production were analyzed; three clones were chosen for more detailed evaluation. These clones (and the receptor-lacking parental cell line) were examined for PTH binding, PTH-stimulated cyclic AMP accumulation and PTH/PTHrP Rc mRNA expression. Receptor-positive clones display a spectrum of PTH-responsiveness that correlates with receptor number/cell and level of receptor mRNA present. The interaction of a C-terminal hPTH-(52-84) peptide with the stably expressed human receptor was examined in cells expressing the highest amount of Rc (> 400,000 Rc/cell). There was no direct binding of hPTH-(52-84) or specific competition versus radiolabeled PTH-(1-34). However, competition versus radiolabeled PTH-(1-34) was observed with bPTH-(1-34), hPTH-(1-84) and hPTHrP-(1-34). These data suggest that hPTH-(52-84) does not interact with the only known form of the human PTH/PTHrP Rc. Therefore, the reported effects of PTH-(52-84) in other systems must be via an alternate (as yet unidentified) mechanism(s). The expression of various amounts of the human PTH/PTHrP Rc in a human target cell background should facilitate characterization of the ligand-binding properties and physiological signal transduction mechanism of the Rc.