Nguyen Hang Thi Thu, Charrier-Hisamuddin Laetitia, Dalmasso Guillaume, Hiol Abel, Sitaraman Shanthi, Merlin Didier
Dept. of Medicine, Division of Digestive Diseases, Emory Univ. School of Medicine, 615 Michael St., Atlanta, GA 30322, USA.
Am J Physiol Gastrointest Liver Physiol. 2007 Dec;293(6):G1155-65. doi: 10.1152/ajpgi.00334.2007. Epub 2007 Oct 11.
The transporter PepT1, apically expressed in intestinal epithelial cells, is responsible for the uptake of di/tripeptides. PepT1 is also expressed in nonpolarized immune cells. Here we investigated the localization of PepT1 in lipid rafts in small intestinal brush border membranes (BBMs) and polarized and nonpolarized cells, as well as functional consequences of the association of PepT1 with lipid rafts. Immunoblot analysis showed the presence of PepT1 in low-density fractions isolated from mouse intestinal BBMs, polarized intestinal Caco2-BBE cells, and nonpolarized Jurkat cells by solubilization in ice-cold 0.5% Triton X-100 and sucrose gradient fractionation. PepT1 colocalized with lipid raft markers GM1 and N-aminopeptidase in intestinal BBMs and Caco2-BBE cell membranes. Disruption of lipid rafts with methyl-beta-cyclodextrin (MbetaCD) shifted PepT1 from low- to high-density fractions. Remarkably, we found that MbetaCD treatment increased PepT1 transport activity in polarized intestinal epithelia but decreased that in intestinal BBM vesicles and nonpolarized immune cells. Mutational analysis showed that phenylalanine 293, phenylalanine 297, and threonine 281 in transmembrane segment 7 of the human di/tripeptide transporter, hPepT1, are important for the targeting to lipid rafts and transport activity of hPepT1. In conclusion, the association of PepT1 with lipid rafts differently modulates its transport activity in polarized and nonpolarized cells.
转运体PepT1在肠道上皮细胞顶端表达，负责二肽/三肽的摄取。PepT1也在非极化免疫细胞中表达。在此，我们研究了PepT1在小肠刷状缘膜（BBM）、极化和非极化细胞的脂筏中的定位，以及PepT1与脂筏结合的功能后果。免疫印迹分析表明，通过在冰冷的0.5% Triton X-100中溶解并进行蔗糖梯度分级分离，从小鼠肠道BBM、极化的肠道Caco2-BBE细胞和非极化的Jurkat细胞中分离得到的低密度组分中存在PepT1。PepT1在肠道BBM和Caco2-BBE细胞膜中与脂筏标记物GM1和N-氨肽酶共定位。用甲基-β-环糊精（MβCD）破坏脂筏会使PepT1从低密度组分转移到高密度组分。值得注意的是，我们发现MβCD处理增加了极化肠道上皮细胞中PepT1的转运活性，但降低了肠道BBM囊泡和非极化免疫细胞中的转运活性。突变分析表明，人二肽/三肽转运体hPepT1跨膜区段7中的苯丙氨酸293、苯丙氨酸297和苏氨酸281对hPepT1靶向脂筏和转运活性很重要。总之，PepT1与脂筏的结合以不同方式调节其在极化和非极化细胞中的转运活性。
