| Distinct intracellular pathways are involved in regulated and constitutive protein secretion from neuronal and endocrine cells, yet the peptide signals and molecular mechanisms responsible for targeting and retention of soluble proteins in secretory granules are incompletely understood. We examined trafficking and the granulogenic role of the neuronal and endocrine peptide precursor VGF that is stored in large dense core vesicles (LDCVs) and undergoes regulated secretion. Deletional analysis and mutagenesis using epitope-tagged VGF identified the sorting sequence as a compact C-terminal alpha-helix and embedded R 564RR566 PC cleavage site; mutation of the R564RR 566 PC site in VGF1-65:GFP:VGF545-617 blocked regulated secretion, while disruption of the alpha-helix had no effect. Mutation of the adjacent H567FHH570 motif, a charged region that might enhance PC cleavage in acidic environments, also blocked regulated release. Inhibition of PC cleavage in PC12 cells using the membrane-permeable synthetic peptide chloromethyl ketone (dec-RVKR-CMK) blocked regulated secretion of VGF. Because secretory granule biogenesis has been shown to depend in part on the granin family proteins chromogranin A (CgA) and chromogranin B (CgB), and is thought to involve interaction of multiple proteins that reside in dense core granules (LDCVs). We proceeded to investigate if VGF, a member of the granin family, was an essential granulogenic factor in Adrenal Chromaffin cells. Genetic ablation of VGF in knockout mice causes depressive behavior and profound abnormalities in energy homeostasis and memory, presumed to result from elimination of the VGF precursor or bioactive VGF-derived peptides, however effects of VGF ablation on LDCV biogenesis have not been previously investigated. We report that targeted germline deletion of the Vgf gene decreases LDCVsize in adrenal medullary chromaffin cells, and alters the relative expression of CgB, but not CgA nor secretogranin II. Phenotypic abnormalities in VGF knockout mice could therefore result, in part, from aberrant peptide, catecholamine, or hormone secretion, in neuronal, endocrine and/or neuroendocrine tissues where VGF loss impacts LDCV biogenesis and potentially function. |
