REVERSIBLE PH-INDUCED HOMOPHILIC BINDING OF GP2, A GLYCOSYL-PHOSPHATIDYLINOSITOL-ANCHORED PROTEIN IN PANCREATIC ZYMOGEN GRANULE MEMBRANES

GP2, the major zymogen granule membrane (ZGM) protein in the pancreas, is linked to the lumenal leaflet of the lipid bilayer via a glycosyl- phosphatidylinositol (GPI) moiety. We demonstrate that the peptide dom ain of GP2 (pGP2, approximately 75 kDa), purified from pancreatic ZGMs after phospholipase C cleavage, shows pH- and calcium-dependent self- association into sedimenting complexes. This homophilic binding proces s is progressive as pH is reduced from 7.0 to 5.5 and calcium is incre ased from 0 to 10-20 mm. This self-association reaction is temperature -dependent, optimal between 20 and 37-degrees-C, progressively reduced below 20-degrees-C, and eliminated at 10-degrees-C. The reaction is r eversible as a function of pH and abolished in the presence of nonioni c detergents. Specificity in the homophilic reaction is demonstrated b y the exclusion of heterologous proteins (globin, serum albumin, and I gG) from sedimenting complexes. At pH 5.5 in the presence of 20 mm cal cium, oligomeric structures (approximately 300 kDa) consistent with te trameric complexes were observed by gel filtration chromatography and elliptical structures (14-18 nm), frequently arranged in variegated cl usters, were observed in the electron microscope by negative staining techniques. The pH-and calcium-dependent self-association observed for GP2 may represent an important mechanism by which GPI-anchored membra ne proteins engage in homotypic binding reactions to establish highly functional membrane (micro)domains targeted to regulated secretory com partments in polarized epithelial cells.