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 endocyt osis is inhibited at pH 6.0 and progressively activated as the pH is i ncreased to 8.3. Endocytotic activity correlated with the release of G P2, a GPI-linked protein, from the apical plasma membrane. To determin e whether the cleavage of GPI-anchored proteins from the membrane of r at acinar cells was responsible for activation of endocytosis, cells a t 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. Furthe rmore, administration of diacylglycerol or phorbol esters had no effec t on reactivation of endocytosis. PI-PLC did not alter intracellular p H or calcium levels. Two proteins were identified as GPI-linked protei ns on the cell surface. One was GP2, whose release from the apical pla sma membrane correlated with apical endocytosis of horseradish peroxid ase (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 i n nonreducing SDS gels and 54000 in reducing SDS gels. By nondenaturin g gel electrophoresis and staining with naphthylphosphate, this protei n was found to be alkaline phosphatase. In contrast to GP2, alkaline p hosphatase was not endogenously released at pH values of 7.4 or 8.3, c onditions that activate endocytosis of HRP under physiological conditi ons. By electron microscopic evaluation, incubation of cells at pH 6.0 with PI-PLC led to induction of HRP uptake into vesicles at the apica l pole of the cell, a reduction in apical plasma membranes, and a conc omitant contraction of the acinar lumen space. Internalized HRP accumu lated in the Golgi region of the cell. These results suggest that the cleavage of GPI-anchored proteins from the apical plasma membrane acti vates apical endocytosis.