Pun K K, Arnaud C D, Nissenson R A
Veterans Administration Medical Center, San Francisco, CA 94121.
J Bone Miner Res. 1988 Aug;3(4):453-60. doi: 10.1002/jbmr.5650030413.
Previous studies have established the presence of parathyroid hormone (PTH)-sensitive adenylate cyclase activity in cultured human skin fibroblasts. The present study was undertaken to identify and quantitate PTH receptors directly in such cells. Human dermal fibroblast cell line CRL 1564 was found to possess specific binding sites for [125I]PTH(1-34). These sites bound PTH selectively; bovine and human PTH(1-34) and PTH(1-84) competed for [125I]PTH(1-34) binding sites, whereas the unrelated peptides calcitonin, insulin, AVP, angiotensin II, and ACTH(1-24) were inactive even at micromolar concentrations. Competitive binding experiments demonstrated the presence of binding site heterogeneity. These data fit a "two-site" model (p less than 0.001) in which one binding component has high affinity (Kd = 2.5 ng/ml = 0.6 nM) and low capacity (10(4) sites/cell) while the other has low affinity (Kd = 5.9 micrograms/ml = 1.5 microM) and high capacity (greater than 10(7) sites/cell). Similar high- and low-affinity [125I]bPTH(1-34) binding sites were seen also in CRL 1564 membranes containing a PTH-responsive adenylate cyclase. The Kd of the high-affinity sites was identical to the concentration of unlabeled bPTH(1-34) (4.2 ng/ml = 1.0 nM) required to half-maximally elevated cyclic AMP in CRL 1564 cells. Affinity labeling of specific PTH binding sites revealed the presence of multiple components with Mrs of 85, 70, 40, 33, and 23 kD on SDS-PAGE. Competition experiments did not disclose structurally discrete high- and low-affinity sites. Thus, structurally homologous PTH receptors in human skin fibroblasts apparently can assume two affinity states: (i) a high-affinity state coupled to adenylate cyclase and (ii) a low-affinity state that may represent uncoupled receptors.
以往的研究已证实在培养的人皮肤成纤维细胞中存在甲状旁腺激素（PTH）敏感的腺苷酸环化酶活性。本研究旨在直接鉴定并定量此类细胞中的PTH受体。发现人真皮成纤维细胞系CRL 1564具有[125I]PTH(1 - 34)的特异性结合位点。这些位点选择性地结合PTH；牛和人PTH(1 - 34)以及PTH(1 - 84)竞争[125I]PTH(1 - 34)结合位点，而不相关的肽降钙素、胰岛素、抗利尿激素（AVP）、血管紧张素II和促肾上腺皮质激素（ACTH(1 - 24)）即使在微摩尔浓度下也无活性。竞争性结合实验证明存在结合位点异质性。这些数据符合“双位点”模型（p < 0.001），其中一个结合成分具有高亲和力（Kd = 2.5 ng/ml = 0.6 nM）和低容量（10(4)个位点/细胞），而另一个具有低亲和力（Kd = 5.9 μg/ml = 1.5 μM）和高容量（大于10(7)个位点/细胞）。在含有PTH反应性腺苷酸环化酶的CRL 1564细胞膜中也观察到类似的高亲和力和低亲和力[125I]bPTH(1 - 34)结合位点。高亲和力位点的Kd与使CRL 1564细胞中环磷酸腺苷（cAMP）升高至最大值一半所需的未标记bPTH(1 - 34)浓度（4.2 ng/ml = 1.0 nM）相同。特异性PTH结合位点的亲和标记显示在十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳（SDS - PAGE）上存在分子量分别为85、70、40、33和23 kD的多种成分。竞争实验未揭示结构上离散的高亲和力和低亲和力位点。因此，人皮肤成纤维细胞中结构同源的PTH受体显然可以呈现两种亲和状态：（i）与腺苷酸环化酶偶联的高亲和力状态和（ii）可能代表未偶联受体的低亲和力状态。
