Depletion of carboxypeptidase E, a regulated secretory pathway sorting receptor, causes misrouting and constitutive secretion of proinsulin and proenkephalin, but not chromogranin A.

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.