Structural Requirements for Sorting Pro-Vasopressin to the Regulated Secretory Pathway in a Neuronal Cell Line

ABSTRACT

Vasopressin is a peptide hormone normally secreted via the regulated secretory pathway in neuro-endocrine cells. In an effort to determine which region of vasopressin contains sufficient information for sorting, we created five constructs with the cDNA for vasopressin or regions of vasopressin in frame with the gene for green fluorescent protein (GFP). Fluorescence microscopy of Neuro-2a cells expressing the constructs revealed full-length vasopressin-GFP (VP-GFP), neurophysin-GFP (NP-GFP) and arginine-vasopressin/neurophysin-GFP (AN-GFP), were localized to punctate granules in the neurites and accumulated at the tips of neurites, characteristic of regulated secretory granules. These fusion proteins were secreted in a regulated manner as determined by pulse-chase labeling experiments. Two other chimeric proteins, signalpeptide-GFP and AVP-GFP were localized to a perinuclear region, characteristic of the endoplasmic reticulum. Pulse/chase [³⁵S]labeling followed by immunoprecipitation using anti-GFP antibody indicated that these two fusion proteins were constitutively secreted. We conclude that the neurophysin region of pro-vasopressin contains information that is both sufficient and necessary for sorting GFP into the regulated secretory pathway.