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首页> 外文期刊>American Journal of Physiology >Caveolin-1 directly interacts with UT-A1 urea transporter: the role of caveolae/lipid rafts in UT-A1 regulation at the cell membrane.
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Caveolin-1 directly interacts with UT-A1 urea transporter: the role of caveolae/lipid rafts in UT-A1 regulation at the cell membrane.

机译：Caveolin-1直接与UT-A1尿素转运蛋白相互作用：Caveolae / Lipid Rafts在细胞膜的UT-A1调节中的作用。

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摘要

The cell plasma membrane contains specialized microdomains called lipid rafts which contain high amounts of sphingolipids and cholesterol. Lipid rafts are involved in a number of membrane protein functions. The urea transporter UT-A1, located in the kidney inner medullary collecting duct (IMCD), is important for urine concentrating ability. In this study, we investigated the possible role of lipid rafts in UT-A1 membrane regulation. Using sucrose gradient cell fractionation, we demonstrated that UT-A1 is concentrated in the caveolae-rich fraction both in stably expressing UT-A1 HEK293 cells and in freshly isolated kidney IMCD suspensions. In these gradients, UT-A1 at the cell plasma membrane is codistributed with caveolin-1, a major component of caveolae. The colocalization of UT-A1 in lipid rafts/caveolae was further confirmed in isolated caveolae from UT-A1-HEK293 cells. The direct association of UT-A1 and caveolin-1 was identified by immunoprecipitation and GST pull-down assay. Examination of internalized UT-A1 in pEGFP-UT-A1 transfected HEK293 cells fluorescent overlap with labeled cholera toxin subunit B, a marker of the caveolae-mediated endocytosis pathway. Disruption of lipid rafts by methyl-beta-cyclodextrin or knocking down caveolin-1 by small-interference RNA resulted in UT-A1 cell membrane accumulation. Functionally, overexpression of caveolin-1 in oocytes decreased UT-A1 urea transport activity and UT-A1 cell surface expression. Our results indicate that lipid rafts/caveolae participate in UT-A1 membrane regulation and this effect is mediated via a direct interaction of caveolin-1 with UT-A1.
机译：细胞血浆膜含有含有大量鞘脂和胆固醇的脂质筏的专用微滴。脂筏参与了许多膜蛋白功能。位于肾内髓质收集管道（IMCD）中的尿素转运蛋白质UT-A1对于尿液浓缩能力是重要的。在这项研究中，我们研究了脂肪筏在UT-A1膜调节中的可能作用。使用蔗糖梯度细胞分级，我们证明了UT-A1在稳定表达UT-A1 HEK293细胞中并在新鲜分离的肾IMCD悬浮液中浓缩。在这些梯度中，细胞血浆膜的UT-A1与Caveolae的主要成分用Caveolin-1编译。来自UT-A1-HEK293细胞的分离的Caveolae中进一步证实了脂质筏/ caveolae中的UT-A1的分致化。通过免疫沉淀和GST下拉测定鉴定UT-A1和Caveolin-1的直接关系。在PEGFP-UT-A1转染的HEK293细胞中的内化UT-A1检测荧光重叠与标记的霍乱毒素亚基B，Caveolae介导的内吞作用的标志物。通过小干扰RNA通过甲基-β-环糊精或敲下Caveolin -1的脂质筏的破坏导致UT-A1细胞膜积累。在功能上，卵母细胞中Caveolin-1的过表达降低了UT-A1尿素传输活性和UT-A1细胞表面表达。我们的结果表明，脂质筏/ Caveolae参与UT-A1膜调节，并且通过Caveolin-1与UT-A1的直接相互作用介导该效果。

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