Identification and characterization of polycystin-2, the PKD2 gene product
Identification and characterization of polycystin-2, the PKD2 gene product
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DOI：
10.1074/jbc.274.40.28557
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发表时间：
1999-10-01
影响因子：
4.8
通讯作者：
Somlo, S
中科院分区：
文献类型：
--
作者：
Cai, ZQ;Maeda, Y;Somlo, S
PKD 2基因是常染色体显性遗传性多囊肾病（ADPKD）的第二个致病基因，它编码的蛋白质多囊蛋白2（polycystin-2）与阳离子通道亚基结构相似，但其功能尚不清楚。我们使用对细胞内NH和COOH末端特异性的多克隆抗血清来鉴定多囊蛋白-2作为类似于110-kDa的整合膜糖蛋白，来自天然组织和培养物中的细胞的多囊蛋白-2对Endo H敏感，这表明持续存在典型的前中Golgis蛋白的高甘露糖寡糖。多囊蛋白-2的免疫荧光细胞染色显示与内质网定位一致的模式。这一发现通过与蛋白质二硫键异构酶的共定位（如通过双间接免疫荧光测定）和与钙连接蛋白在亚细胞分级分离研究中的共分布得到证实。在Gly（821）处或之后截短的多囊蛋白-2翻译产物保留其专属的内质网定位，而在Glu（787）处或之前截短的产物另外运输至质膜。运输至质膜的截短突变体获得Endo H抗性，并且可以在完整细胞中的细胞表面上被生物素化。34个氨基酸的区域Glu（787）-Ser（820）含有两个假定的磷酸化位点，负责多囊蛋白-2的唯一内质网定位，并且是与多囊蛋白-2的尚未鉴定的蛋白质结合伴侣特异性相互作用的位点。全长多囊蛋白-2定位于细胞内膜增加了PKD 2基因产物是细胞内通道复合物亚基的可能性。
PKD2, the second gene for the autosomal dominant polycystic kidney disease (ADPKD), encodes a protein, polycystin-2, with predicted structural similarity to cation channel subunits, However, the function of polycystin-2 remains unknown. We used polyclonal antisera specific for the intracellular NH, and COOH termini to identify polycystin-2 as an similar to 110-kDa integral membrane glycoprotein, Polycystin-2 from both native tissues and cells in culture is sensitive to Endo H suggesting the continued presence of high-mannose oligosaccharides typical of pre-middle Golgis proteins. Immunofluorescent cell staining of polycystin-2 shows a pattern consistent with localization in the endoplasmic reticulum. This finding is confirmed by co-localization with protein-disulfide isomerase as determined by double indirect immunofluorescence and co-distribution with calnexin in subcellular fractionation studies. Polycystin-2 translation products truncated at or after Gly(821) retain their exclusive endoplasmic reticulum localization while products truncated at or before Glu(787) additionally traffic to the plasma membrane. Truncation mutants that traffic to the plasma membrane acquire Endo H resistance and can be biotinylated on the cell surface in intact cells. The 34-amino acid region Glu(787)-Ser(820), containing two putative phosphorylation sites, is responsible for the exclusive endoplasmic reticulum localization of polycystin-2 and is the site of specific interaction with an as yet unidentified protein binding partner for polycystin-2. The localization of full-length polycystin-2 to intracellular membranes raises the possibility that the PKD2 gene product is a subunit of intracellular channel complexes.