Inomata N, Akiyama M, Kubota N, Jüppner H
Department of Medicine, Massachusetts General Hospital, Boston 02114, USA.
Endocrinology. 1995 Nov;136(11):4732-40. doi: 10.1210/endo.136.11.7588200.
Carboxyl-terminal fragments of PTH (C-PTH) appear to have biological properties different from those mediated by the amino-terminal portions of PTH and PTH-related peptide (PTHrP). To characterize a C-PTH receptor that may be involved in mediating these functions, we performed RRAs and affinity cross-linking studies with several clonal cell lines. Radiolabeled recombinant [Leu8,18,Tyr34]human PTH-(1-84)[mutPTH-(1-84) and [Tyr34] human PTH-(19-84)[mutPTH-(19-84) showed little or no specific binding to stably expressed recombinant PTH/PTHrP receptors. However, high affinity binding was observed using osteoblast-like and rat parathyroid (PT-r3) cells. The apparent Kd values were 20-30 nM for PTH-(1-84), mutPTH-(1-84), and mutPTH-(19-84), respectively; 400-800 nM for PTH-(39-84); and more than 5000 nM for PTH-(53-84). [Nle8,18,Tyr34]bovine PTH-(1-34)amide [PTH-(1-34)], PTH-(44-68), PTHrP-(37-74), and PTHrP-(109-141) showed no displacement of either radioligand. C-PTH receptor number was increased up to 2-fold by pretreating ROS 17/2.8 cells with increasing doses of PTH-(1-34), PTH-(1-84), or 8-bromo-cAMP, whereas no change was observed in response to dexamethasone or PTH-(39-84). Cross-linking studies using radiolabeled mutPTH-(1-84) or mutPTH-(19-84) revealed specific labeling of two proteins in ROS 17/2.8 cells that were approximately 40 and 90 kilodaltons in size (including the radioligand of approximately 10 kilodaltons). The intensity of affinity labeling of both proteins was dose dependently inhibited by increasing concentrations of unlabeled PTH-(1-84) and several carboxyl-terminal PTH-(1-84) fragments, but not by PTH-(1-34). Similar studies with PT-r3 cells revealed only a single protein band of about 90 kilodaltons. These data indicate that the carboxyl-terminal portion of PTH-(1-84) binds specifically to a unique receptor/binding protein distinct from the previously isolated PTH/PTHrP receptor.
甲状旁腺激素（PTH）的羧基末端片段（C-PTH）似乎具有与PTH和甲状旁腺激素相关肽（PTHrP）氨基末端部分介导的生物学特性不同的特性。为了鉴定可能参与介导这些功能的C-PTH受体，我们对几种克隆细胞系进行了放射受体分析（RRAs）和亲和交联研究。放射性标记的重组[Leu8,18,Tyr34]人PTH-（1-84）[mutPTH-（1-84）]和[Tyr34]人PTH-（19-84）[mutPTH-（19-84）]与稳定表达的重组PTH/PTHrP受体几乎没有或没有特异性结合。然而，在成骨细胞样细胞和大鼠甲状旁腺（PT-r3）细胞中观察到高亲和力结合。PTH-（1-84）、mutPTH-（1-84）和mutPTH-（19-84）的表观解离常数（Kd）值分别为20 - 30 nM；PTH-（39-84）为400 - 800 nM；PTH-（53-84）大于5000 nM。[Nle8,18,Tyr34]牛PTH-（1-34）酰胺[PTH-（1-34）]、PTH-（44-68）、PTHrP-（37-74）和PTHrP-（109-141）对两种放射性配体均无置换作用。用递增剂量的PTH-（1-34）、PTH-（1-84）或8-溴-cAMP预处理ROS 17/2.8细胞，C-PTH受体数量增加至2倍，而地塞米松或PTH-（39-84）处理后未观察到变化。使用放射性标记的mutPTH-（1-84）或mutPTH-（19-84）进行的交联研究显示，ROS 17/2.8细胞中有两种蛋白质被特异性标记，大小约为40和90千道尔顿（包括约10千道尔顿的放射性配体）。两种蛋白质的亲和标记强度均受到递增浓度的未标记PTH-（1-84）和几个羧基末端PTH-（1-84）片段的剂量依赖性抑制，但不受PTH-（1-34）的抑制。对PT-r3细胞进行的类似研究仅显示出一条约90千道尔顿的单一蛋白带。这些数据表明，PTH-（1-84）的羧基末端部分与一种独特的受体/结合蛋白特异性结合，该受体/结合蛋白不同于先前分离的PTH/PTHrP受体。
