title: Tomosyn Negatively Regulates Arginine Vasopressin Secretion in Embryonic Stem Cell-Derived Neurons.
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 (syntaxin-binding 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.
authors.affiliation: Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, Nagoya, Japan.
authors.forename: Seiji
authors.initials: S
authors.lastname: Takeuchi
authors.affiliation: Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, Nagoya, Japan.
authors.forename: Shintaro
authors.initials: S
authors.lastname: Iwama
authors.affiliation: Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, Nagoya, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Takagi
authors.affiliation: Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, Nagoya, Japan.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Kiyota
authors.affiliation: Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, Nagoya, Japan.
authors.forename: Kohtaro
authors.initials: K
authors.lastname: Nakashima
authors.affiliation: Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, Nagoya, Japan.
authors.forename: Hisakazu
authors.initials: H
authors.lastname: Izumida
authors.affiliation: Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, Nagoya, Japan.
authors.forename: Haruki
authors.initials: H
authors.lastname: Fujisawa
authors.affiliation: Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, Nagoya, Japan.
authors.forename: Naoko
authors.initials: N
authors.lastname: Iwata
authors.affiliation: Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, Nagoya, Japan.
authors.forename: Hidetaka
authors.initials: H
authors.lastname: Suga
authors.affiliation: Department of Cell Pharmacology, Nagoya University Graduate School of Medicine, Nagoya, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Watanabe
authors.affiliation: Department of Cell Pharmacology, Nagoya University Graduate School of Medicine, Nagoya, Japan.
authors.forename: Kozo
authors.initials: K
authors.lastname: Kaibuchi
authors.affiliation: Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, Nagoya, Japan.
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Oiso
authors.affiliation: Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, Nagoya, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Arima
authors.affiliation: Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, Nagoya, Japan.
authors.forename: Yoshihisa
authors.initials: Y
authors.lastname: Sugimura
mesh_terms: D000818:Animals; D001127:Arginine Vasopressin / Q000378:metabolism*; D002460:Cell Line; D008297:Male; D051379:Mice; D000066450:Mouse Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000032:analysis / Q000378:metabolism*; D055495:Neurogenesis; D009474:Neurons / Q000166:cytology* / Q000378:metabolism; D010904:Pituitary Gland, Posterior / Q000378:metabolism / Q000648:ultrastructure; D011485:Protein Binding; D050765:Qa-SNARE Proteins / Q000378:metabolism; D050683:R-SNARE Proteins / Q000032:analysis / Q000378:metabolism*; D017207:Rats, Sprague-Dawley; D050825:Synaptosomal-Associated Protein 25 / Q000378:metabolism
chemical_list: D009419:Nerve Tissue Proteins; D050765:Qa-SNARE Proteins; D050683:R-SNARE Proteins; C484769:Stxbp5 protein, rat; D050825:Synaptosomal-Associated Protein 25; C112618:tomosyn protein, mouse; D001127:Arginine Vasopressin