RAPID SEQUESTRATION OF DPP IV CD26 AND OTHER CELL-SURFACE PROTEINS INAN AUTOPHAGIC-LIKE COMPARTMENT IN CACO-2 CELLS TREATED WITH FORSKOLIN/

The enterocytic differentiation of Caco-2 cells, a human colon adenoca rcinoma cell line, is accompanied by the transcriptionally regulated e xpression of a subset of proteins and their correct sorting towards th e cell surface. In the present work we have explored the possibility t hat posttranslational events may interfere with this process by invest igating the short term effects of a potent adenylyl cyclase activator, forskolin, on cell surface expression of dipeptidyl peptidase IV. Pre vious works have shown that this protein is targeted towards the apica l domain through either a direct or an indirect route. Domain specific biochemical experiments demonstrate that cell surface expression of n eosynthesized dipeptidyl peptidase IV rapidly decreases after a 1 hour forskolin treatment. Both initial basolateral and apical dipeptidyl p eptidase IV membrane delivery were altered by forskolin treatment. Dec rease of dipeptidyl peptidase IV cell surface expression was not restr icted to this protein, since membrane expression of '525' antigen, a b asolateral protein and of sucrase-isomaltase, an apically targeted hyd rolase, which unlike dipeptidyl peptidase IV mainly follows a direct r oute to the brush border membrane, also decreases. In addition endocyt osis of proteins from the apical and from the basolateral domain was e ssentially unchanged, suggesting that forskolin's target may be locate d on the exocytic pathway. Confocal laser scanning microscopy and immu ne-electron microscopy studies demonstrate that, within 5 minutes of f orskolin treatment, the cell surface proteins studied accumulate in in tracellular vesicles which were co-labeled with a polyclonal antibody raised against Lamp-1, a lysosomal membrane marker. Electron microscop y studies show that these vesicles display an autophagic-like morpholo gy. Finally, biochemical experiments indicate that dibutyryl cAMP does not mimick the forskolin effect, thus suggesting that it is a cAMP-in dependent phenomenon.