Immunohistochemical evidence for the involvement of protein convertases 5Aand 2 in the processing of pro-neurotensin in rat brain

The neuropeptides/neurotransmitters neurotensin (NT) and neuromedin (NN) ar e synthesized by endoproteolytic cleavage of a common inactive precursor, p ro-NT/NN. In vitro studies have suggested that the prohormone convertases P C5A and PC2 might both be involved in this process. In the present study, w e used dual immunohistochemical techniques to determine whether either one or both of these two convertases were co-localized with pro-NT/NN maturatio n products and could therefore be involved in the physiological processing of this propeptide in rat brain. PC2-immunoreactive neurons were present in all regions immunopositive for NT. All but three regions expressing NT wer e also immunopositive for PC5A. Dual localization of NT with either convert ase revealed that NT was extensively co-localized with both PC5A and PC2, a lbeit with regional differences. These results strongly suggest that PC5A a nd PC2 may play a key role in the maturation of pro-NT/NN in mammalian brai n. The regional variability in NT/PC co-localization patterns may account f or the region-specific maturation profiles previously reported for pro-NT/N N. The high degree of overlap between PC5A and PC2 in most NT-rich areas fu rther suggests that these two convertases may act jointly to process pro-NT /NN. At the subcellular level, PC5A was largely co-localized with the mid-c isternae Golgi marker MG-160. By contrast, PC2 was almost completely exclud ed from MG-160-immunoreactive compartments. These results suggest that PC5A , which is particularly efficient at cleaving the two C-terminal-most dibas ics of pro-NT/NN, may be acting as early as in the Golgi apparatus to relea se NT, whereas PC2, which is considerably more active than PC5A in cleaving the third C-terminal doublet, may be predominantly involved further distal ly along the secretory pathway to release NN. (C) 2000 Wiley-Liss, Inc.