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Molecularbiological analysis of the function and localization of membrane proteins.

膜蛋白功能和定位的分子生物学分析。

基本信息

批准号：
09671151
负责人：
TAKAICHI Kenmei
金额：
$ 1.98万
依托单位：
University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09671151/
关键词：
sodium-proton exchanger NHE1 LLC-PK1 cells OK cells MDCK cells collagen gel DNA tip fibronectin MDCK細胞 differential display フィブロネクチン尿細管 NHE アミロライド BCECF

项目摘要

The appropriate localization of membrane proteins in the apical or basolateral is mandatory for the physiologic transport of the solutes in the epithelia. Derangement of the localization of membrane proteins has been observed in the pathological situations such as renal cysts. We examined the localization of sodium-proton exchanger (NHE1), one of membrane proteins, in the renal tubular epithelial cells (LLC-PK1 cells, OK cells) cultured on the filter membranes coated with collagen. Antisense-Oligo of NHE1 decreased intrinsic NHE1 activity only in the basolateral membranes in LLC-PK1 cells. NHE1 activity was detected only in the basolateral membranes in OK cells transfected with human NHE1 cDNA.These results show that NHE1 is localized in the basolateral membranes in the renal epitelia in the physiologic situations. MDCK cells cultured in the collagen gel form cysts but not tubules without hepatic growth factor (HGF). We isolated the MDCK cells (C4) that form tubules without HGF and cells (C1) that form cysts. The differential expression of mRNA in these two kinds of cells was examined using DNA tip, in which more than 9,000 different cDNAs were arrayed. The expression of fibronectin was decreased in C1, which indicated the significance of fibronectin for tubular formation of MDCK cells.

膜蛋白在顶侧或基底侧的适当定位对于溶质在上皮中的生理运输是必需的。在病理情况下，如肾囊肿，已观察到膜蛋白定位的紊乱。我们研究了钠质子交换器（NHE 1），膜蛋白的一种，在肾小管上皮细胞（LLC-PK 1细胞，OK细胞）培养在过滤膜与胶原蛋白。NHE 1反义寡核苷酸仅在LLC-PK 1细胞的基底外侧膜中降低NHE 1的内在活性。在转染人NHE 1 cDNA的OK细胞中，NHE 1仅在基底外侧膜上有表达，表明在生理状态下，NHE 1定位于肾上皮基底外侧膜上。在胶原凝胶中培养的MDCK细胞在没有肝生长因子（HGF）的情况下形成囊肿，但不形成小管。我们分离了在没有HGF的情况下形成小管的MDCK细胞（C4）和形成囊肿的细胞（C1）。用DNA探针检测了这两种细胞中mRNA的差异表达，其中有9,000多个不同的cDNA。C1细胞中纤维连接蛋白的表达减少，表明纤维连接蛋白在MDCK细胞的肾小管形成中具有重要意义。