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 domain 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 process is progressive as pH is reduced from 7.0 to 5.5 and calcium is increased 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 reversible as a function of pH and abolished in the presence of nonionic detergents. Specificity in the homophilic reaction is demonstrated by the exclusion of heterologous proteins (globin, serum albumin, and IgG) from sedimenting complexes. At pH 5.5 in the presence of 20 mM calcium, oligomeric structures (approximately 300 kDa) consistent with tetrameric complexes were observed by gel filtration chromatography and elliptical structures (14-18 nm), frequently arranged in variegated clusters, 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 membrane proteins engage in homotypic binding reactions to establish highly functional membrane (micro)- domains targeted to regulated secretory compartments in polarized epithelial cells.
Freedman
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Charles A. Dana Research Institute, Thorndike Laboratory, Harvard Medical School and Beth Israel Hospital, Boston 02215.
Scheele
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D000818:Animals; D002118:Calcium / Q000378:metabolism; D003594:Cytoplasmic Granules / Q000737:chemistry / Q000648:ultrastructure; D004285:Dogs; D058851:GPI-Linked Proteins; D017261:Glycosylphosphatidylinositols; D006863:Hydrogen-Ion Concentration; D007425:Intracellular Membranes / Q000737:chemistry; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D010179:Pancreas / Q000737:chemistry* / Q000648:ultrastructure; D011485:Protein Binding; D013696:Temperature
C489339:GP2 protein, human; D058851:GPI-Linked Proteins; D017261:Glycosylphosphatidylinositols; D008562:Membrane Glycoproteins; D002118:Calcium