Hikita C, Takito J, Vijayakumar S, Al-Awqati Q
College of Physicians and Surgeons of Columbia University, New York, New York 10032, USA.
J Biol Chem. 1999 Jun 18;274(25):17671-6. doi: 10.1074/jbc.274.25.17671.
When an intercalated epithelial cell line was seeded at low density and allowed to reach confluence, it located the anion exchanger band 3 in the apical membrane and an H+-ATPase in the basolateral membrane. The same clonal cells seeded at high density targeted these proteins to the reverse location. Furthermore, high density cells had vigorous apical endocytosis, and low density cells had none. The extracellular matrix of high density cells was capable of inducing apical endocytosis and relocation of band 3 to the basolateral membrane in low density cells. A 230-kDa extracellular matrix (ECM) protein termed hensin, when purified to near-homogeneity, was able to reverse the phenotype of the low density cells. Antibodies to hensin prevented this effect, indicating that hensin is necessary for conversion of polarity. We show here that hensin was synthesized by both low density and high density cells. Whereas both phenotypes secreted soluble hensin into their media, only high density cells localized it in their ECM. Analysis of soluble hensin by sucrose density gradients showed that low density cells secreted monomeric hensin, and high density cells secreted higher order multimers. When 35S-labeled monomeric hensin was added to high density cells, they induced its aggregation suggesting that the multimerization was catalyzed by surface events in the high density cells. Soluble monomeric or multimeric hensin did not induce apical endocytosis in low density cells, whereas the more polymerized hensin isolated from insoluble ECM readily induced it. These multimers could be disaggregated by sulfhydryl reagents and by dimethylmaleic anhydride, and treatment of high density ECM by these reagents prevented the induction of endocytosis. These results demonstrate that hensin, like several ECM proteins, needs to be precipitated in the ECM to be functional.
当一种闰管上皮细胞系以低密度接种并使其达到汇合状态时，它会将阴离子交换蛋白带3定位在顶端膜，而将H⁺ -ATP酶定位在基底外侧膜。以高密度接种的相同克隆细胞则将这些蛋白质定位到相反的位置。此外，高密度细胞具有活跃的顶端内吞作用，而低密度细胞则没有。高密度细胞的细胞外基质能够诱导低密度细胞发生顶端内吞作用，并使带3重新定位到基底外侧膜。一种称为hensin的230 kDa细胞外基质（ECM）蛋白，当纯化至接近均一性时，能够逆转低密度细胞的表型。针对hensin的抗体可阻止这种效应，表明hensin是极性转换所必需的。我们在此表明，低密度和高密度细胞均合成hensin。虽然两种表型都将可溶性hensin分泌到培养基中，但只有高密度细胞将其定位在它们的细胞外基质中。通过蔗糖密度梯度分析可溶性hensin表明，低密度细胞分泌单体hensin，而高密度细胞分泌更高阶的多聚体。当将³⁵S标记的单体hensin添加到高密度细胞中时，它们会诱导其聚集，这表明多聚化是由高密度细胞表面事件催化的。可溶性单体或多聚体hensin不会诱导低密度细胞发生顶端内吞作用，而从不溶性细胞外基质中分离出的聚合程度更高的hensin则很容易诱导其发生。这些多聚体可以被巯基试剂和马来酸酐解离，用这些试剂处理高密度细胞外基质可阻止内吞作用的诱导。这些结果表明，hensin与几种细胞外基质蛋白一样，需要在细胞外基质中沉淀才能发挥功能。
