Baricault L, Fransen J A, Garcia M, Sapin C, Codogno P, Ginsel L A, Trugnan G
Unité de Recherches sur la Neuroendocrinologie et la Biologie Cellulaire Digestives, INSERM U410, Paris, France.
J Cell Sci. 1995 May;108 ( Pt 5):2109-21. doi: 10.1242/jcs.108.5.2109.
The enterocytic differentiation of Caco-2 cells, a human colon adenocarcinoma cell line, is accompanied by the transcriptionally regulated expression of a subset of proteins and their correct sorting towards the cell surface. In the present work we have explored the possibility that post-translational events may interfere with this process by investigating the short term effects of a potent adenylyl cyclase activator, forskolin, on cell surface expression of dipeptidyl peptidase IV. Previous works have shown that this protein is targeted towards the apical domain through either a direct or an indirect route. Domain specific biochemical experiments demonstrate that cell surface expression of neosynthesized dipeptidyl peptidase IV rapidly decreases after a 1 hour forskolin treatment. Both initial basolateral and apical dipeptidyl peptidase IV membrane delivery were altered by forskolin treatment. Decrease of dipeptidyl peptidase IV cell surface expression was not restricted to this protein, since membrane expression of '525' antigen, a basolateral protein and of sucrase-isomaltase, an apically targeted hydrolase, which unlike dipeptidyl peptidase IV mainly follows a direct route to the brush border membrane, also decreases. In addition endocytosis of proteins from the apical and from the basolateral domain was essentially unchanged, suggesting that forskolin's target may be located on the exocytic pathway. Confocal laser scanning microscopy and immuno-electron microscopy studies demonstrate that, within 5 minutes of forskolin treatment, the cell surface proteins studied accumulate in intracellular vesicles which were co-labeled with a polyclonal antibody raised against Lamp-1, a lysosomal membrane marker. Electron microscopy studies show that these vesicles display an autophagic-like morphology. Finally, biochemical experiments indicate that dibutyryl cAMP does not mimick the forskolin effect, thus suggesting that it is a cAMP-independent phenomenon.
人结肠腺癌细胞系Caco-2细胞的肠上皮细胞分化伴随着一组蛋白质的转录调控表达及其向细胞表面的正确分选。在本研究中，我们通过研究强效腺苷酸环化酶激活剂福斯高林对二肽基肽酶IV细胞表面表达的短期影响，探讨了翻译后事件可能干扰这一过程的可能性。先前的研究表明，该蛋白通过直接或间接途径靶向顶端结构域。结构域特异性生化实验表明，新合成的二肽基肽酶IV在福斯高林处理1小时后，其细胞表面表达迅速下降。福斯高林处理改变了二肽基肽酶IV最初向基底外侧和顶端的膜转运。二肽基肽酶IV细胞表面表达的下降并不局限于该蛋白，因为基底外侧蛋白“525”抗原和顶端靶向水解酶蔗糖酶-异麦芽糖酶的膜表达也下降，与二肽基肽酶IV不同，蔗糖酶-异麦芽糖酶主要通过直接途径到达刷状缘膜。此外，从顶端和基底外侧结构域的蛋白质内吞作用基本未改变，这表明福斯高林的作用靶点可能位于外排途径上。共聚焦激光扫描显微镜和免疫电子显微镜研究表明，在福斯高林处理5分钟内，所研究的细胞表面蛋白积聚在细胞内小泡中，这些小泡与针对溶酶体膜标记物Lamp-1的多克隆抗体共同标记。电子显微镜研究表明，这些小泡呈现出自噬样形态。最后，生化实验表明，二丁酰环磷腺苷不能模拟福斯高林的作用，因此表明这是一种不依赖环磷腺苷的现象。
