title: The sorting of proglucagon to secretory granules is mediated by carboxypeptidase E and intrinsic sorting signals.
abstract: Proglucagon is expressed in pancreatic alpha cells, intestinal L cells and brainstem neurons. Tissue-specific processing of proglucagon yields the peptide hormones glucagon in the alpha cell and glucagon-like peptide (GLP)-1 and GLP-2 in L cells. Both glucagon and GLP-1 are secreted in response to nutritional status and are critical for regulating glycaemia. The sorting of proglucagon to the dense-core secretory granules of the regulated secretory pathway is essential for the appropriate secretion of glucagon and GLP-1. We examined the roles of carboxypeptidase E (CPE), a prohormone sorting receptor, the processing enzymes PC1/3 and PC2 and putative intrinsic sorting signals in proglucagon sorting. In Neuro 2a cells that lacked CPE, PC1/3 and PC2, proglucagon co-localised with the Golgi marker p115 as determined by quantitative immunofluorescence microscopy. Expression of CPE, but not of PC1/3 or PC2, enhanced proglucagon sorting to granules. siRNA-mediated knockdown of CPE disrupted regulated secretion of glucagon from pancreatic-derived alphaTC1-6 cells, but not of GLP-1 from intestinal cell-derived GLUTag cells. Mutation of the PC cleavage site K70R71, the dibasic R17R18 site within glucagon or the alpha-helix of glucagon, all significantly affected the sub-cellular localisation of proglucagon. Protein modelling revealed that alpha helices corresponding to glucagon, GLP-1 and GLP-2, are arranged within a disordered structure, suggesting some flexibility in the sorting mechanism. We conclude that there are multiple mechanisms for sorting proglucagon to the regulated secretory pathway, including a role for CPE in pancreatic alpha cells, initial cleavage at K70R71 and multiple sorting signals.
authors: Metabolism and Diabetes and Imaging Programs, Lawson Health Research Institute, 268 Grosvenor Street, London, Ontario, Canada.
authors: Rebecca
authors: R
authors: McGirr
authors: Leonardo
authors: L
authors: Guizzetti
authors: Savita
authors: S
authors: Dhanvantari
mesh_terms: D000818:Animals; D043423:Carboxypeptidase H / Q000037:antagonists & inhibitors / Q000187:drug effects / Q000378:metabolism*; D002460:Cell Line; D002478:Cells, Cultured; D019858:Enteroendocrine Cells / Q000378:metabolism / Q000473:pathology; D052216:Glucagon-Like Peptide 1 / Q000378:metabolism; D053767:Glucagon-Like Peptide 2 / Q000378:metabolism; D051379:Mice; D009447:Neuroblastoma / Q000378:metabolism / Q000473:pathology; D010179:Pancreas / Q000378:metabolism / Q000473:pathology; D052336:Proglucagon / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000494:pharmacology; D022142:Secretory Vesicles / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*
chemical_list: D053767:Glucagon-Like Peptide 2; D034741:RNA, Small Interfering; D052336:Proglucagon; D052216:Glucagon-Like Peptide 1; D043423:Carboxypeptidase H