Smith D P, Zhang X Y, Frolik C A, Harvey A, Chandrasekhar S, Black E C, Hsiung H M
Endocrine Division, Lilly Research Laboratories, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, IN 46285, USA.
Biochim Biophys Acta. 1996 Jul 17;1307(3):339-47. doi: 10.1016/0167-4781(96)00035-8.
A complementary DNA (cDNA) encoding the receptor for porcine parathyroid hormone/parathyroid hormone-related peptide (PTH/PTHrP) was isolated from a porcine kidney cDNA library. The porcine PTH/PTHrP receptor is a 585 amino acid protein containing seven putative membrane-spanning domains. The porcine PTH/PTHrP receptor has amino acid identity of 95.6%, 80.4%, and 88.7% with human, opossum, and rat PTH/PTHrP receptors, respectively and 53.4% identity to the recently cloned human PTH2 receptor. The receptor cDNA was subsequently cloned into a mammalian cell expression vector (pRC/CMV) which contains a human cytomegalovirus promoter. A human kidney cell line (293), stably transfected with this vector, expressed the receptor at a high level and, when challenged with human PTH(1-34), increased cytoplasmic cAMP and inositol triphosphate production. Radioligand binding studies revealed that the receptor bound both human PTH(1-34), and PTHrP(1-36). Scatchard analyses of three clones showed that the cells harbor a single class of high affinity receptor (Kd = 1-4 nM for human PTH(1-34)) but had varying receptor numbers (10(5)-10(6) receptors/cell). In contrast to PTH(1-34), the [Arg2]PTH(1-34) analog bound to the porcine PTH/PTHrP receptor with low affinity and was a weak agonist for cAMP stimulation with the cloned receptor. These response characteristics differentiate the porcine receptor from the previously cloned rat and opossum PTH/PTHrP receptors.
从猪肾cDNA文库中分离出编码猪甲状旁腺激素/甲状旁腺激素相关肽（PTH/PTHrP）受体的互补DNA（cDNA）。猪PTH/PTHrP受体是一种含585个氨基酸的蛋白质，有7个推定的跨膜结构域。猪PTH/PTHrP受体与人类、负鼠和大鼠的PTH/PTHrP受体的氨基酸同源性分别为95.6%、80.4%和88.7%，与最近克隆的人类PTH2受体的同源性为53.4%。随后将该受体cDNA克隆到一个含有人类巨细胞病毒启动子的哺乳动物细胞表达载体（pRC/CMV）中。用该载体稳定转染的人肾细胞系（293）高水平表达该受体，当用人PTH(1 - 34)刺激时，可增加细胞质中环磷酸腺苷（cAMP）和三磷酸肌醇的产生。放射性配体结合研究表明，该受体能结合人PTH(1 - 34)和PTHrP(1 - 36)。对三个克隆的Scatchard分析表明，这些细胞含有一类高亲和力受体（人PTH(1 - 34)的解离常数Kd = 1 - 4 nM），但受体数量不同（10⁵ - 10⁶个受体/细胞）。与PTH(1 - 34)不同，[Arg2]PTH(1 - 34)类似物与猪PTH/PTHrP受体的结合亲和力较低，并且对于克隆受体刺激cAMP来说是一种弱激动剂。这些反应特性使猪受体与先前克隆的大鼠和负鼠PTH/PTHrP受体区分开来。
