RESUMO
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.
Assuntos
Carboxipeptidase H/metabolismo , Proglucagon/metabolismo , Vesículas Secretórias/metabolismo , Transdução de Sinais/fisiologia , Animais , Carboxipeptidase H/antagonistas & inibidores , Carboxipeptidase H/efeitos dos fármacos , Linhagem Celular , Células Cultivadas , Células Enteroendócrinas/metabolismo , Células Enteroendócrinas/patologia , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Peptídeo 2 Semelhante ao Glucagon/metabolismo , Camundongos , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Pâncreas/metabolismo , Pâncreas/patologia , RNA Interferente Pequeno/farmacologiaRESUMO
It is discovered that chronic consumption of ethanol induced decrease of carboxypeptidase H activity in striatum by 27%; increase of carboxypeptidase M activity in hippocampus by 67% and decrease in cerebral hemispheres by 34%; phenylmethylsulfonyl fluoride-inhibited carboxypeptidase activity increase in hypothalamus by 141%, in striatum by 60% and in optic and lamina quadrigemina by 34%. The role of basic carboxypeptidases in mechanisms of ethanol influence on the peptidergic systems are discussed.