In familial hyperproinsulinemia, specific mutations in the proinsulin gene are linked with a profound increase in circulating plasma proinsulin levels. However, the molecular and cellular basis for this disease remains uncharacterized. Here we investigated how these mutations may disrupt the sorting signal required to target proinsulin to the secretory granules of the regulated secretory pathway, resulting in the unregulated release of proinsulin. Using a combination of molecular modeling and

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... te-directed mutagenesis, we have identified structural molecular motifs in proinsulin that are necessary for correct sorting into secretory granules of endocrine cells. We show that membrane carboxypeptidase E, previously identified as a prohormone sorting receptor, is essential for proinsulin sorting. This was demonstrated through siRNA-mediated depletion of carboxypeptidase E and transfection with a dominant negative mutant of carboxypeptidase E in a β cell line. Mutant proinsulins found in familial hyperproinsulinemia failed to bind to carboxypeptidase E and were not sorted efficiently. These findings provide evidence that the elevation of plasma proinsulin levels found in patients with familial hyperproinsulinemia is caused by the disruption of carboxypeptidase E-mediated sorting of mutant proinsulins to the regulated secretory pathway.