Molecularbiological analysis of the function and localization of membrane proteins.

膜蛋白在根尖或基底外侧的适当定位对于上皮内溶质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的紊乱。我们检测了膜蛋白之一钠质子交换剂（NHE1）在胶原包被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lc - pk1细胞的基底膜中降低内在的NHE1活性。转染人NHE1 cDNA的OK细胞仅在基底膜检测到NHE1活性。这些结果表明，在生理情况下，NHE1定位于肾上皮的基底外膜。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。我们分离了不含HGF的形成小管的MDCK细胞（C4）和形成囊肿的细胞（C1）。使用DNA尖端检测了这两种细胞中mRNA的差异表达，其中排列了9000多种不同的cdna。C1中纤维连接蛋白表达降低，说明纤维连接蛋白对MDCK细胞小管形成具有重要意义。

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.