Tomosyn negatively regulates arginine vasopressin secretion in embryonic stem cell-derived neurons

Seiji Takeuchi, Shintaro Iwama, Hiroshi Takagi, Atsushi Kiyota, Kohtaro Nakashima, Hisakazu Izumida, Haruki Fujisawa, Naoko Iwata, Hidetaka Suga, Takashi Watanabe, Kozo Kaibuchi, Yutaka Oiso, Hiroshi Arima, Yoshihisa Sugimura

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Abstract

Arginine vasopressin (AVP) is secreted via exocytosis; however, the precise molecular mechanism underlying the exocytosis of AVP remains to be elucidated. To better understand the mechanisms of AVP secretion, in our study we have identified proteins that bind with a 25 kDa synaptosomal-associated protein (SNAP25). SNAP25 plays a crucial role in exocytosis, in the posterior pituitary. Embryonic stem (ES) cell-derived AVP neurons were established to investigate the functions of the identified proteins. Using glutathione S-transferase (GST)-pulldown assays and proteomic analyses, we identified tomosyn-1 (syntaxinbinding protein 5) as a SNAP25-binding protein in the posterior pituitary. Coimmunoprecipitation assays indicated that tomosyn formed N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes with SNAP25 and syntaxin1. Immunohistochemistry showed that tomosyn localized to the posterior pituitary. Mouse ES cells self-differentiated into AVP neurons (mES-AVP) that expressed tomosyn and two transmembrane SNARE proteins, including SNAP25 and syntaxin1. KCl increased AVP secretion in mES-AVP, and overexpression of tomosyn-1 reduced KCl-stimulated AVP secretion. Downregulation of tomosyn-1 with siRNA increased KCl-stimulated AVP secretion. These results suggested that tomosyn-1 negatively regulated AVP secretion in mES-AVP and further suggest the possibility of using mES-AVP culture systems to evaluate the role of synaptic proteins from AVP neurons.

Original languageEnglish
Article numbere0164544
JournalPloS one
Volume11
Issue number10
DOIs
Publication statusPublished - 10-2016

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All Science Journal Classification (ASJC) codes

  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)
  • General

Cite this

Takeuchi, S., Iwama, S., Takagi, H., Kiyota, A., Nakashima, K., Izumida, H., Fujisawa, H., Iwata, N., Suga, H., Watanabe, T., Kaibuchi, K., Oiso, Y., Arima, H., & Sugimura, Y. (2016). Tomosyn negatively regulates arginine vasopressin secretion in embryonic stem cell-derived neurons. PloS one, 11(10), [e0164544]. https://doi.org/10.1371/journal.pone.0164544