Akbar G K, Dasari V R, Sheth S B, Ashby B, Mills D C, Kunapuli S P
Department of Physiology, Temple University School of Medicine, Philadelphia PA 19140, USA.
J Recept Signal Transduct Res. 1996 May-Jul;16(3-4):209-24. doi: 10.3109/10799899609039949.
We have investigated the nature of the nucleotide receptors on human erythro leukemia (HEL) cells, a cell line with some megakaryocytic properties, using a combination of pharmacological, photoaffinity labeling, and molecular biological techniques. Fura-2 loaded HEL cells responded to 2-methylthio ATP, ATP, 2-methylthio ADP, ADP and UTP with an increase in intracellular calcium. 2 Methylthio ADP was the most potent agonist. When external calcium was chelated with EDTA, calcium responses were observed indicating the mobilization of intracellular stores. These responses showed evidence of both homologous and heterologous receptor desensitization. In photoaffinity labeling experiments, beta-[32P]-AzPET-ADP was incorporated into three protein species with mobilities corresponding to M(r) approximately 55 kDa (doublet) and approximately 43 kDa. Labeling of approximately 55 kDa proteins was specifically inhibited by ADP, while that of the approximately 43 kDa was inhibited specifically by UTP. Nucleotide sequence analysis of the positive clones obtained by screening the HEL cell cDNA library with mouse P2U cDNA revealed that the P2U receptor from HEL cells is identical to the previously cloned human P2U receptor. These experiments suggest that the HEL cells contain a P2Y purinoceptor responding to ADP, in addition to a P2U receptor and possibly also a third P2 purinoceptor with a unique agonist profile.
我们运用药理学、光亲和标记及分子生物学技术相结合的方法，研究了人类红白血病（HEL）细胞（一种具有某些巨核细胞特性的细胞系）上核苷酸受体的性质。负载了Fura-2的HEL细胞对2-甲硫基ATP、ATP、2-甲硫基ADP、ADP和UTP产生反应，细胞内钙含量增加。2-甲硫基ADP是最有效的激动剂。当用EDTA螯合细胞外钙时，仍观察到钙反应，这表明细胞内储存钙的动员。这些反应显示出同源和异源受体脱敏的证据。在光亲和标记实验中，β-[³²P]-AzPET-ADP掺入了三种蛋白质，其迁移率分别对应于约55 kDa（双峰）和约43 kDa的分子量。约55 kDa蛋白质的标记被ADP特异性抑制，而约43 kDa蛋白质的标记被UTP特异性抑制。用小鼠P2U cDNA筛选HEL细胞cDNA文库获得的阳性克隆的核苷酸序列分析表明，HEL细胞的P2U受体与先前克隆的人类P2U受体相同。这些实验表明，HEL细胞除了含有对ADP有反应的P2Y嘌呤受体外，还含有一个P2U受体，可能还含有第三个具有独特激动剂谱的P2嘌呤受体。
