Characterization and agonist-induced down-regulation of parathyroid hormone receptors in clonal rat osteosarcoma cells.-猫眼课题宝

克隆大鼠骨肉瘤细胞中甲状旁腺激素受体的表征和激动剂诱导的下调。

DOI：

10.1210/endo-122-4-1208

发表时间：

1988

期刊：

Endocrinology

影响因子：

4.8

通讯作者：

中科院分区：

医学2区

文献类型：

作者：

I. Yamamoto;C. Shigeno;J. Potts;G. Segre

使用HPLC纯化的、合成的、无硫的、放射性碘标记的牛PTH类似物[Nle 8，Nle 18，Tyr，34]牛PTH-（1-34）酰胺，对两种稳定克隆大鼠骨肉瘤细胞系ROS 17/2和ROS 17/2.8上的PTH受体进行了表征。PTH结合对PTH激动剂和拮抗剂具有特异性，并取决于孵育的时间和温度。在这些完整细胞系统中PTH激动剂和拮抗剂的结合亲和力与犬肾膜中的结合亲和力之间存在极好的相关性。与PTH无关的肽不结合。ROS 17/2和17/2.8都具有单一类别的可饱和的高亲和力PTH结合位点，通过饱和和竞争研究的动力学分析和Scatchard分析，其具有0.8-1.4 nM的解离常数（Kd）。在ROS 17/2和17/2.8中，Bmax分别为每个细胞约36，000和72，000个位点。甲状旁腺素激动剂与ROS 17/2细胞中其受体的结合与其相对生物学效力之间存在密切相关性，所述生物学效力通过刺激从这些细胞制备的质膜中的腺苷酸环化酶来测量。用PTH激动剂长时间处理ROS 17/2和17/2.8细胞导致可用细胞表面结合位点的剂量和时间依赖性减少，而不改变Kd。PTH拮抗剂不调节PTH受体。PTH激动剂对PTH受体的调节依赖于暴露于配体的剂量和时间，剂量范围为10（-8）至10（-11）M。细胞暴露于激动剂（大于或等于10（-8）M）48小时显示受体数量最大程度减少;继续暴露于激动剂（大于或等于10（-8）M）不会进一步减少PTH受体数量，其保持恒定，约为对照值的15%。激动剂诱导的下调发生在小于10（-11）M激动剂的情况下，该浓度小于通过直接配体竞争检测到的最小剂量的10%。用PTH激动剂处理ROS 17/2细胞导致PTH刺激的腺苷酸环化酶的剂量和时间依赖性降低。这种激动剂诱导的脱敏与PTH受体的可用性降低密切相关：在暴露于激动剂（大于10（-8）M）48 h的细胞中最大，并且在细胞继续暴露于激动剂时也不会进一步降低。未来的研究与这些稳定的ROS细胞系应允许详细分析生化机制的同源和异源调节PTH受体和脱敏和敏化的腺苷酸环化酶反应。

PTH receptors on two stable clonal rat osteosarcoma cell lines, ROS 17/2 and ROS 17/2.8, were characterized using an HPLC-purified, synthetic, sulfur-free, radioiodinated analog of bovine PTH, [Nle8,Nle18,Tyr,34]bovine PTH-(1-34)amide. PTH binding is specific for PTH agonists and antagonists and is dependent on the time and temperature of incubation. There is an excellent correlation between binding affinities of PTH agonists and antagonists in these intact cell systems with those in canine renal membranes. Peptides unrelated to PTH do not bind. Both ROS 17/2 and 17/2.8 have a single class of saturable, high affinity PTH binding sites that, by kinetic analysis and Scatchard analysis of saturation and competition studies, has a dissociation constant (Kd) of 0.8-1.4 nM. Bmax is approximately 36,000 and 72,000 sites per cell in ROS 17/2 and 17/2.8, respectively. A close correlation was found between the binding of PTH agonists to their receptors in ROS 17/2 cells with their relative biological potencies as measured by stimulation of adenylate cyclase in plasma membranes prepared from these cells. Prolonged treatment of ROS 17/2 and 17/2.8 cells with PTH agonists results in a dose- and time-dependent decrease of available cell-surface binding sites, without alterations in Kd. PTH antagonists do not regulate PTH receptors. Regulation of PTH receptors by PTH agonists is dependent on the dose and time of exposure to ligand over a dose range of 10(-8) to 10(-11) M. Cells exposed to agonists (greater than or equal to 10(-8) M) for 48 h show maximally decreased receptor number; continued exposure to agonists (greater than or equal to 10(-8) M) does not further decrease PTH receptor number, which remains constant at about 15% of control values. Agonist-induced down-regulation occurs with less than 10(-11) M agonists, a concentration less than 10% of the minimal dose detected by direct ligand competition. Treatment of ROS 17/2 cells with PTH agonists results in a dose- and time-dependent decrease of PTH-stimulated adenylate cyclase. This agonist-induced desensitization correlates closely with the decreased availability of PTH receptors: it is maximal in cells exposed to agonists (greater than 10(-8) M) for 48 h and also does not decrease further with continued exposure of the cells to agonist. Future studies with these stable ROS cell lines should permit detailed analysis of the biochemical mechanisms underlying homologous and heterologous regulation of PTH receptors and desensitization and sensitization of the adenylate cyclase response.