EGFP-tagged vasopressin precursor protein sorting into large dense core vesicles and secretion from PC12 cells

Bing Jun Zhang, Mitsuo Yamashita, Ray Fields, Kiyoshi Kusano, Harold Gainer

Research output: Contribution to journalArticlepeer-review

12 Citations (Scopus)

Abstract

1. Hypothalamic magnocellular neurons synthesize, store, and secrete large quantities of the neuropeptides, vasopressin (VP) and oxytocin (OT), which are synthesized as protein precursors also containing proteins called neurophysins. These protein precursors are sorted through the regulated secretory pathway (RSP), packaged into large dense core vesicles LDCVs, and their peptide products are secreted from nerve terminals in the posterior pituitary. 2. It has been hypothesized that this efficient packaging is dependent on the interaction of the peptide with neurophysin in a complex that forms the granule core. To test this, PC12 cells were transfected with vasopressin precursor DNA constructs that either contained or deleted the neurophysin moiety and tagged with enhanced green fluorescent protein (EGFP) as reporters. The intracellular routing and secretion of the EGFP-tagged VP precursor proteins were studied by in differentiated PC12 cells by fluorescence microscopy, electron microscopic immunocytochemistry, and fluorescent imaging techniques. 3. The data showed that only when the neurophysin was present in the VP precursor construct did the fluorescent fusion protein become routed to the RSP and get efficiently packaged into LDCVs and secreted. These data are consistent with the view that routing of the precursor to LDCVs requires the amino acids that encode the intravesicular chaperone, neurophysin.

Original languageEnglish
Pages (from-to)581-605
Number of pages25
JournalCellular and Molecular Neurobiology
Volume25
Issue number3-4
DOIs
Publication statusPublished - 2005 Jun

Keywords

  • Exocytosis
  • Green fluorescent protein
  • Neurophysin
  • Regulated secretory pathway
  • Vasopressin

ASJC Scopus subject areas

  • Cellular and Molecular Neuroscience
  • Cell Biology

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