AN EFFICIENT CELLULAR-SYSTEM FOR MUTATIONAL ANALYSIS OF PROHORMONE PROCESSING

A novel system for heterologous expression of prohormones based on tra nsient transfection of the HIT beta-cell line was established using hu man progastrin as a model. Progastrin was expressed at high levels com pared to other gene transfer systems in endocrine cells, and the proce ssing pattern was similar to that of normal antral gastrin cells. Thus , gastrin was partially tyrosine O-sulfated and carboxyamidated. Cell extracts contained mainly gastrin-17 and gastrin-34 and the correspond ing glycine-extended forms. In contrast, the media contained more inco mpletely processed gastrin forms, This suggests that gastrin was direc ted to the regulated secretory pathway but that some progastrin produc ts were constitutively secreted. Glucose increased both the level of g astrin gene expression and maturation to carboxyamidated peptides, ind icating that glucose influences the activity of the amidation enzyme c omplex, peptidylglycine alpha-amidating mono-oxygenase (PAM), in insul in cells. Mutational analysis of tyrosine sulfation of gastrin demonst rated that substitution of the uncharged residue carboxy-terminal to t he tyrosine with an acidic residue does not increase sulfation in cont rast to previous results, where the amino-terminal residue was replace d with an acidic residue, The mutant peptides displayed sulfation-depe ndent processing, supporting our recent suggestion that tyrosine sulfa tion increases the proteolytic processing of prohormones.