Processing of pro-islet amyloid polypeptide in the constitutive and regulated secretory pathways of beta cells.
Islet amyloid is a pathologic characteristic of the pancreas in type 2 diabetes comprised mainly of the beta-cell peptide islet amyloid polypeptide (IAPP; amylin). We used a pulse-chase approach to investigate the kinetics of processing and secretion of the IAPP precursor, proIAPP, in beta cells. By only 20 min after synthesis, a COOH-terminally processed proIAPP intermediate (approximately 6 kDa) was already present in beta cells. Formation of this NH2-terminally extended intermediate was not prevented by arresting secretory pathway transport at the trans-Golgi network (TGN) by either brefeldin A or temperature blockade, suggesting that this initial cleavage step occurs in the TGN before entry of (pro)IAPP into granules. Mature IAPP (approximately 4 kDa) was not detected until 60 min of chase, suggesting that NH2-terminal cleavage occurs in granules. Cells chased in low glucose without Ca2+ or with diazoxide, to block regulated release, secreted both proIAPP (approximately 8 kDa) and a partially processed form (approximately 6 kDa) via the constitutive secretory pathway. Stimulation of regulated secretion resulted in secretion primarily of mature IAPP as well as low levels of both unprocessed (approximately 8 kDa) and partially processed (approximately 6 kDa) proIAPP. We conclude that normal processing of proIAPP is a two-step process initiated by cleavage at its COOH terminus (likely by prohormone convertase 1/3 in the TGN) followed by cleavage at its NH2 terminus (by prohormone convertase 2 in granules) to form IAPP. Both proIAPP and its NH2-terminally extended intermediate appear to be normal secretory products of the beta cell that can be released via either the regulated or constitutive secretory pathways.
Marzban
Lucy
L
Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia, Canada V5Z 4H4.
Trigo-Gonzalez
Genny
G
Verchere
C Bruce
CB
D000595:Amino Acid Sequence; D000682:Amyloid / Q000378:metabolism*; D000818:Animals; D020126:Brefeldin A / Q000494:pharmacology; D002461:Cell Line, Transformed; D045683:Furin / Q000737:chemistry; D047468:Immunoprecipitation; D050417:Insulin-Secreting Cells / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D008810:Mice, Inbred C57BL; D008954:Models, Biological; D008969:Molecular Sequence Data; D041681:NIH 3T3 Cells; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D012190:Retroviridae / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D013997:Time Factors; D021601:trans-Golgi Network / Q000378:metabolism
D000682:Amyloid; D010455:Peptides; C422904:pro-islet amyloid polypeptide; D020126:Brefeldin A; D045683:Furin