title: Disruption of a receptor-mediated mechanism for intracellular sorting of proinsulin in familial hyperproinsulinemia.
abstract: 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 site-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 (CPE), previously identified as a prohormone-sorting receptor, is essential for proinsulin sorting. This was demonstrated through short interfering RNA-mediated depletion of CPE and transfection with a dominant negative mutant of CPE in a beta-cell line. Mutant proinsulins found in familial hyperproinsulinemia failed to bind to CPE 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 CPE-mediated sorting of mutant proinsulins to the regulated secretory pathway.
authors.affiliation: Section on Cellular Neurobiology, National Institutes of Health, Bethesda, Maryland 20892-4480, USA.
authors.forename: Savita
authors.initials: S
authors.lastname: Dhanvantari
authors.forename: Fu-Sheng
authors.initials: FS
authors.lastname: Shen
authors.forename: Tiffany
authors.initials: T
authors.lastname: Adams
authors.forename: Christopher R
authors.initials: CR
authors.lastname: Snell
authors.forename: ChunFa
authors.initials: C
authors.lastname: Zhang
authors.forename: Robert B
authors.initials: RB
authors.lastname: Mackin
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Morris
authors.forename: Y Peng
authors.initials: YP
authors.lastname: Loh
mesh_terms: D043423:Carboxypeptidase H / Q000235:genetics / Q000378:metabolism; D005799:Genes, Dominant; D006801:Humans; D006863:Hydrogen-Ion Concentration; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011384:Proinsulin / Q000097:blood* / Q000235:genetics / Q000378:metabolism*; D021382:Protein Sorting Signals; D021381:Protein Transport; D034622:RNA Interference
chemical_list: D021382:Protein Sorting Signals; D011384:Proinsulin; D043423:Carboxypeptidase H