Molecularbiological analysis of the function and localization of membrane proteins.

膜蛋白在顶端或基底侧的适当定位对于溶质在上皮细胞中的生理运输是必需的。在肾囊肿等病理情况下，已观察到膜蛋白定位紊乱。我们研究了膜蛋白之一的钠质子交换蛋白(NHE1)在包被胶原的滤膜上培养的肾小管上皮细胞(LLC-PK1细胞，OK细胞)中的定位。NHE1的反义寡核苷酸仅在LLC-PK1细胞的基底膜上降低固有的NHE1活性。转染人NHE1基因的OK细胞仅在基底膜上检测到NHE1活性，表明NHE1在生理状态下定位于肾脏外膜的基底膜上。在胶原凝胶中培养的MDCK细胞形成囊，但不形成无肝生长因子(HGF)的小管。我们分离了形成无HGF的小管的MDCK细胞(C4)和形成包囊的细胞(C1)。用DNA探针技术检测了9,000多个不同的cDNA在这两种细胞中的差异表达。C1FN表达降低，提示FN在MDCK细胞肾小管形成过程中具有重要意义。

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.