甲胎蛋白受体的性质及其所介导的cAMP-PKA信号转导途径

研究HeLa细胞膜上甲胎蛋白 (alpha fetoprotein ,AFP)受体的存在情况及其介导的信号转导 .先用Na12 5I]标记AFP ;标记的AFP和培养的HeLa细胞结合 ,Scatchard法和受体 配体结合法分析受体数目 ;再用放射免疫结合法分析在百日咳毒素 (pertussistoxin ,PTX)预处理前后AFP对细胞内环腺苷酸 (cAMP)浓度及细胞内蛋白激酶A(proteinkinaseA ,PKA)活性变化的影响 .在HeLa细胞膜表面存在 2种不同解离平衡常数 (Kd)的AFP受体 ,Kd1=5 2pmol L(2 10 0位点 细胞 ) ;Kd2 =2 3nmol L (114 0 0位点 细胞 ) .在AFP(2 0mg L)作用下 ,HeLa细胞内cAMP浓度变化及PKA活性的改变为与对照组比较 ,用PTX预处理前cAMP浓度升高 2 6 7% ,PKA活性增高 10 3 2 % ;用PTX预处理后升高 86 % ,PKA活性增高 2 5 3% .抗甲胎蛋白单克隆抗体可阻断AFP对细胞cAMP浓度和PKA活性的影响 .结果证明 ,在HeLa细胞膜上有 2种不同解离平衡常数的甲胎蛋白受体存在 ,受体有可能通过cAMP PKA途径介导信号转导 .

The Properties and Mediated cAMP-PKA Signal Transduction Pathway of Alpha-fetoprotein Receptor

Alpha-fetoprotein(AFP) can regulate the proliferation of tumor or neoplastic cells. The existence of AFP receptor and its intracellular signaling remain controversial. The goal was to characterize the AFP receptor and its possible mediated signal transduction pathway in HeLa cells. Using Na 125I labeled AFP, i.e. 125I-AFP for receptor binding assay, AFP extracted from human umbilical blood serum was used to act on the cells in vitro. Utilized radioactive immunosorbent assay, cAMP accumulation and protein kinase A(PKA) activity in HeLa cells and the effects of AFP were detected on the concentration and activity in the cells pretreated with pertussis toxin (PTX). The results showed that two subtypes of AFP receptors, with K d1 of 52 pmol/L (2 100 sites per cell ) and K d2 2.3 nmol/L (11 400 sites per cell),were identified in membrane of HeLa cells. AFP (20 mg/L) could significantly increase the concentration of cAMP and the activity of PKA up to 267% and 103.2%,and those pretreated with PTX up to 86% and 25.3%, respectively, compared with control. The monoclonal antibody of AFP could block these effect of AFP. These results showed that there were two subtypes of AFP receptors identified in HeLa cells and it was speculated that the receptor could play its role through the cAMP-protein kinase signal transduction pathway.