Distribution of neuroendocrine regulatory peptide-1 and -2, and proteolytic processing of their precursor VGF protein in the rat.

Neuroendocrine regulatory peptide (NERP)-1 and NERP-2 are biologically active peptides recently discovered by peptidomic analysis. NERPs are processed out from the 594-residue VGF protein which contains many prohormone convertase cleavage motifs. VGF-deficient mice exhibit a hypermetabolic and infertile phenotype, for which VGF protein-derived peptides including NERPs are presumably responsible. To provide a solid basis for elucidating physiological roles of NERPs, we investigated rat VGF protein processing by chromatographic and mass spectrometric analysis, and immunoblotting, using antibodies against NERPs and the VGF protein C-terminus (VGF-C). Cellular and tissue distribution of immunoreactive (ir) NERPs were also analyzed in the rat. Both ir-NERP-1 and ir-NERP-2, which occur abundantly in the CNS and pituitary, moderately in the gastrointestinal (GI) tract, were mainly localized in neuronal structures. Major endogenous forms of ir-NERPs in the brain and GI tract were identified as NERP-1, NERP-2, and big NERP-2 (NERP-1 + NERP-2), with NERP-1 and big NERP-2 being predominant. Regarding ir-VGF-C peptides, VGF[588-617], VGF[556-617], and VGF[509-617] were found to be major forms. Immunoblotting with the NERP-2 and VGF-C antibodies revealed processing intermediates of 10-37 kDa. Taken together, we deduce that VGF protein is primarily cleaved at 10 sites through the processing pathway common to the brain and GI tract.