Prohormone convertases 1 and 2 process ProPACAP and generate matured, bioactive PACAP38 and PACAP27 in transfected rat pituitary GH(4)C(1) cells

Pituitary adenylate cyclase-activating polypetide (PACAP) exists in two ami dated forms, PACAP38 and PACAP27, which are expressed in the magnocellular and parvocellular neurons of the paraventricular nucleus (PVN) and the magn ocellular neurons of the supraoptic nucleus (SON) of the hypothalamus. The prohormone convertases PC1 and PC2, subtilisin-like PCs of the Kex2 family, are expressed in neuroendocrine cells. Immunocytochemistry and in situ hyb ridization of PC1 and PC2 in the hypothalamus have shown that PC1 and PC2 a re also present in the PVN and SON. Therefore, it is possible that the prec ursor of PACAP is processed by PC1 and/or PC2 in the hypothalamic nuclei an d then converted to its mature forms. To test th is hypothesis, rat pituita ry GH(4)C(1) cells were supertransfected with human PACAP cDNA and either r at PC1 or PC2 cDNA. The acid extracts of these cells were analyzed by rever sed-phase HPLC for proPACAP, PACAP38 and/or PACAP27 radioimmunoassays using three antibodies with different recognition sites, and then bioassayed for the ability to stimulate adenylate cyclase. The cells transfected with PAC AP cDNA alone yielded PACAP-like immunoreactivity (PACAP-Ii) corresponding to molecular weights between 15 and 20 kDa without PACAP bioactivity. Cotra nsfection of these cells with PC1 or PC2 generated PACAP-Ii, which coeluted with synthetic PACAP38 and PACAP27, respectively. Western blot also reveal ed 4.5- and 3.0-kDa PACAP-Ii bands, which correspond to the molecular weigh ts of PACAP38 and PACAP27, respectively. The HPLC fractions containing PACA P-Ii, which were coeluted with synthetic PACAP38 and PACAP27, showed marked bioactivities. These findings suggest that the precursor of PACAP expresse d in the PVN and SON of the hypothalamus could be efficiently processed by PC1 and PC2, and then converted to mature, bioactive PACAP38 and PACAP27.