title: Depletion of carboxypeptidase E, a regulated secretory pathway sorting receptor, causes misrouting and constitutive secretion of proinsulin and proenkephalin, but not chromogranin A.
abstract: Previous studies have shown that the prohormone POMC is sorted to the regulated secretory pathway (RSP), at the trans-Golgi network, by binding of a conformation-dependent sorting signal to a sorting receptor, identified as membrane-bound carboxypeptidase E (CPE) (Cool et al., 1997, Cell, 88:73-83). In this study, the role of CPE as a sorting receptor for other RSP proteins that contain sorting signals (proinsulin, proenkephalin, and chromogranin A) was investigated in neuroendocrine cells (Neuro-2a) stably expressing CPE antisense RNA. Whereas these cells were depleted of CPE by greater than 85%, electron microscopy showed that they contain dense core secretory granules identical to wild-type Neuro-2a cells, indicating that CPE is not essential for granulogenesis. Secretion and immunocytochemical studies showed that, in wild-type Neuro-2a cells, endogenous proenkephalin and transfected proinsulin/insulin were localized to punctate secretory granules and were released via the RSP. However, in CPE-depleted cells, these two prohormones were released constitutively and had a Golgi-like distribution but were not localized to punctate secretory granules. In contrast, chromogranin A was present in punctate secretory granules and released via the RSP, in wild-type and CPE-depleted Neuro-2a cells. Thus, the sorting of proinsulin and proenkephalin to the RSP, like POMC, necessitates binding to CPE, and hence, CPE acts as a common sorting receptor for targeting these prohormones to the RSP. In contrast, the sorting signal of chromogranin A does not use CPE as a sorting receptor, suggesting the existence of other sorting receptors for the RSP.
authors.affiliation: Section on Cellular Neurobiology, Laboratory of Developmental Neurobiology, National Institute for Child and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: E
authors.initials: E
authors.lastname: Normant
authors.forename: Y P
authors.initials: YP
authors.lastname: Loh
mesh_terms: D043423:Carboxypeptidase H; D002268:Carboxypeptidases / Q000172:deficiency / Q000235:genetics / Q000502:physiology*; D002460:Cell Line; D053379:Chromogranin A; D002864:Chromogranins / Q000032:analysis / Q000378:metabolism*; D003594:Cytoplasmic Granules / Q000737:chemistry / Q000502:physiology / Q000648:ultrastructure; D004745:Enkephalins / Q000032:analysis / Q000378:metabolism*; D015870:Gene Expression; D006056:Golgi Apparatus / Q000737:chemistry; D006801:Humans; D007150:Immunohistochemistry; D008854:Microscopy, Electron; D009490:Neurosecretory Systems / Q000201:enzymology* / Q000502:physiology / Q000648:ultrastructure; D011384:Proinsulin / Q000032:analysis / Q000378:metabolism*; D011498:Protein Precursors / Q000032:analysis / Q000378:metabolism*; D016372:RNA, Antisense / Q000378:metabolism; D014162:Transfection
chemical_list: C506150:CHGA protein, human; D053379:Chromogranin A; D002864:Chromogranins; D004745:Enkephalins; D011498:Protein Precursors; D016372:RNA, Antisense; C029992:proenkephalin; D011384:Proinsulin; D002268:Carboxypeptidases; D043423:Carboxypeptidase H