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.
Native Immunogold Labeling of Cell Surface Proteins and Viral Glycoproteins for Cryo-Electron Microscopy and Cryo-Electron Tomography Applications
