Cleavage of GPI-anchored proteins from the plasma membrane activates apical endocytosis in pancreatic acinar cells

Using rat pancreatic acini, we have recently shown that apical endocytosis is inhibited at pH 6.0 and progressively activated as the pH is increased to 8.3. Endocytotic activity correlated with the release of GP2, a GPI-linked protein, from the apical plasma membrane. To determine whether the cleavage of GPI-anchored proteins from the membrane of rat acinar cells was responsible for activation of endocytosis, cells at pH 6.0 were incubated with PI-specific phospholipase C (PI-PLC). PI-PLC treatment reversed the inhibition of endocytosis observed at pH 6.0. Reactivation of endocytosis correlated with PI-PLC-induced release of GP2 but not cleavage of phospholipids in cellular membranes. Furthermore, administration of diacylglycerol or phorbol esters had no effect on reactivation of endocytosis. PI-PLC did not alter intracellular pH or calcium levels. Two proteins were identified as GPI-linked proteins on the cell surface. One was GP2, whose release from the apical plasma membrane correlated with apical endocytosis of horseradish peroxidase (HRP). The other protein, identified by Western blotting using an antibody directed against a cryptic determinant exposed on GPI-linked proteins after cleavage with PI-PLC, has a molecular weight of 98000 in nonreducing SDS gels and 54000 in reducing SDS gels. By nondenaturing gel electrophoresis and staining with naphthylphosphate, this protein was found to be alkaline phosphatase. In contrast to GP2, alkaline phosphatase was not endogenously released at pH values of 7.4 or 8.3, conditions that activate endocytosis of HRP under physiological conditions. By electron microscopic evaluation, incubation of cells at pH 6.0 with PI-PLC led to induction of HRP uptake into vesicles at the apical pole of the cell, a reduction in apical plasma membranes, and a concomitant contraction of the acinar lumen space. Internalized HRP accumulated in the Golgi region of the cell. These results suggest that the cleavage of GPI-anchored proteins from the apical plasma membrane activates apical endocytosis.