A fusion protein of the human P2Y1 receptor and NTPDase1 exhibits functional activities of the native receptor and ectoenzyme and reduced signaling
A fusion protein of the human P2Y1 receptor and NTPDase1 exhibits functional activities of the native receptor and ectoenzyme and reduced signaling
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DOI：
10.1124/mol.62.3.521
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发表时间：
2002-09-01
影响因子：
3.6
通讯作者：
Boyer, JL
中科院分区：
文献类型：
--
作者：
Alvarado-Castillo, C;Lozano-Zarain, P;Boyer, JL
为了开始解决组成型释放的核苷酸、外切核苷酸酶活性和 P2Y 受体促进的信号转导反应之间的功能相互作用，我们将人 P2Y(1) 受体与外切核苷三磷酸二磷酸水解酶家族的成员 NTPDase1 设计成融合蛋白。由稳定表达野生型 NTPDase1 或 P2Y1 受体-NTPDase1 融合蛋白的中国仓鼠卵巢 (CHO)-K1 细胞制备的膜表现出的核苷酸水解活性比空载体转染细胞膜中测量的活性高 300 倍以上。对于野生型 NTPDase1 和 P2Y1 -NTPDase1 融合蛋白，三磷酸核苷与二磷酸核苷水解的分子比约为 1: 0.4。 P2Y1 -NTPDase1 融合蛋白的稳定表达赋予 CHO-K1 细胞 ADP 和 2MeSADP 促进的 Ca2+ 反应。此外，不可水解激动剂ADPbetaS刺激磷酸肌醇积累的最大能力相似，并且ADPbetaS在融合蛋白中的EC50低于野生型受体。相反，与野生型受体相比，融合蛋白的大量核苷酸水解活性导致ADP激活磷脂酶C的浓度效应曲线向右移动超过50倍。 P2Y(1) 和其他 P2Y 受体的异源表达导致基础磷酸肌醇水平显着增加。鉴于P2Y(1)受体-NTPDase1融合蛋白的高核苷酸酶活性和明显正常的受体信号传导活性，我们定量了稳定表达野生型P2Y(1)受体或融合蛋白的细胞中基础磷酸肌醇的积累。尽管野生型 P2Y(1) 受体表达时肌醇磷酸盐水平显着升高，但表达融合蛋白的细胞中的水平与野生型 CHO-K1 细胞中的水平没有不同。
To begin to address the functional interactions between constitutively released nucleotides, ectonucleotidase activity, and P2Y receptor-promoted signaling responses, we engineered the human P2Y(1) receptor in a fusion protein with a member of the ectonucleoside triphosphate diphosphohydrolase family, NTPDase1. Membranes prepared from Chinese hamster ovary (CHO)-K1 cells stably expressing either wild-type NTPDase1 or the P2Y1 receptor-NTPDase1 fusion protein exhibited nucleotide-hydrolytic activities that were over 300-fold greater than activity measured in membranes from empty vector-transfected cells. The molecular ratio for nucleoside triphosphate versus diphosphate hydrolysis was approximately 1: 0.4 for both the wild-type NTPDase1 and P2Y1 -NTPDase1 fusion protein. Stable expression of the P2Y1 -NTPDase1 fusion protein conferred an ADP and 2MeSADP-promoted Ca2+ response to CHO-K1 cells. Moreover, the maximal capacity of the nonhydrolyzable agonist ADPbetaS to stimulate inositol phosphate accumulation was similar, and the EC50 of ADPbetaS was lower in the fusion protein than the wild-type receptor. In contrast, the substantial nucleotide-hydrolyzing activity of the fusion protein resulted in a greater than 50-fold shift to the right of the concentration-effect curve of ADP for activation of phospholipase C compared with the wild-type receptor. Heterologous expression of the P2Y(1) and other P2Y receptors results in marked increases in basal inositol phosphate levels. Given the high nucleotidase activity and apparently normal receptor signaling activity of the P2Y(1) receptor-NTPDase1 fusion protein, we quantitated basal inositol phosphate accumulation in cells stably expressing either the wild-type P2Y(1) receptor or the fusion protein. Although marked elevation of inositol phosphate levels occurred with wild-type P2Y(1) receptor expression, levels in cells expressing the fusion protein were not different from those in wild-type CHO-K1 cells.