稳定转染的甲状旁腺激素/甲状旁腺激素相关蛋白受体互补DNA在CHO细胞中的功能表达。

Functional expression of a stably transfected parathyroid hormone/parathyroid hormone related protein receptor complementary DNA in CHO cells.

作者信息

Kaufmann M, Muff R, Born W, Fischer J A

机构信息

Department of Orthopaedic Surgery, University of Zurich, Switzerland.

出版信息

Mol Cell Endocrinol. 1994 Aug;104(1):21-7. doi: 10.1016/0303-7207(94)90047-7.

DOI:10.1016/0303-7207(94)90047-7

PMID:7821704

Abstract

Chinese hamster ovary (CHO) cells were stably transfected with OK-O complementary DNA encoding the parathyroid hormone/parathyroid hormone related protein (PTH/PTHrP) receptor derived from opossum kidney (OK) cells (Jüppner et al., 1991). A subclone of transfected CHO cells, CHO-E2, presented high affinity binding of 125I-labeled [Tyr36]chickenPTHrP(1-36)amide ([125I]chPTHrP(1-36)) (Kd 1.28 +/- 0.10 nM) similar to that of wildtype OK cells (Kd 2.23 +/- 0.16 nM) (P < 0.01). Photoaffinity labeling of the PTH/PTHrP receptors using N-hydroxysuccinimidyl-4-azidobenzoate modified [125I]chPTHrP(1-36) revealed the same specifically labeled 90 kDa protein in CHO-E2 and OK cells. In CHO-cells, chPTHrP(1-36) stimulated cyclic AMP accumulation in dose-dependent fashion (EC50 0.15 +/- 0.04 nM) and raised peak cytosolic free calcium concentration (EC50 2.90 +/- 0.36 nM) independent of extracellular calcium, and stimulated phosphate uptake (EC50 0.21 +/- 0.07 nM). Both, chPTHrP(1-36) and 12-O-tetradecanoylphorbol-13-acetate stimulated phosphate uptake were suppressed by staurosporine. But, Sp-cyclic adenosine-3',5'-monophosphothioate did not affect phosphate uptake in CHO-E2 cells. In conclusion, a PTH/PTHrP receptor stably expressed in CHO cells is linked to stimulation of phosphate uptake. Receptor coupling presumably occurred through the protein kinase C rather than the protein kinase A pathway.

摘要

将编码源自负鼠肾（OK）细胞的甲状旁腺激素/甲状旁腺激素相关蛋白（PTH/PTHrP）受体的OK-O互补DNA稳定转染至中国仓鼠卵巢（CHO）细胞中（于普纳等人，1991年）。转染的CHO细胞亚克隆CHO-E2对125I标记的[酪氨酸36]鸡PTHrP（1-36）酰胺（[125I]chPTHrP（1-36））呈现出高亲和力结合（解离常数Kd为1.28±0.10 nM），与野生型OK细胞相似（Kd为2.23±0.16 nM）（P<0.01）。使用N-羟基琥珀酰亚胺基-4-叠氮苯甲酸修饰的[125I]chPTHrP（1-36）对PTH/PTHrP受体进行光亲和标记，结果显示CHO-E2细胞和OK细胞中特异性标记的90 kDa蛋白相同。在CHO细胞中，chPTHrP（1-36）以剂量依赖方式刺激环磷酸腺苷（cAMP）积累（半数有效浓度EC50为0.15±0.04 nM），并升高胞质游离钙峰值浓度（EC50为2.90±0.36 nM），且不依赖细胞外钙，同时刺激磷酸盐摄取（EC50为0.21±0.07 nM）。chPTHrP（1-36）和12-O-十四烷酰佛波醇-13-乙酸酯刺激的磷酸盐摄取均被星形孢菌素抑制。但是，但Sp-环腺苷-3',5'-单磷酸硫代酸盐不影响CHO-E2细胞中的磷酸盐摄取。总之，稳定表达于CHO细胞中的PTH/PTHrP受体与磷酸盐摄取的刺激有关。受体偶联可能是通过蛋白激酶C而非蛋白激酶A途径发生的。