Arginine vasopressin (AVP) is secreted via exocytosis; however, the precise molecular mechanism underlying the exocytosis of AVP is unknown. To better understand the mechanisms of AVP secretion, here we study to identify proteins that bind with a 25 kDa synaptosomal-associated protein (SNAP25), a protein that 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 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 reduced KCl-stimulated AVP secretion. Downregulation of tomosyn with siRNA increased KCl-stimulated AVP secretion. In addition, pituitary adenylate cyclase-activating polypeptide (PACAP) increased the PKA-catalysed phosphorylation of tomosyn and thus increased AVP secretion. These results suggest that tomosyn negatively regulates AVP secretion and that phosphorylation of tomosyn by PKA is involved in tomosyn-regulated AVP secretion in mES-AVP.An important next step would be to screen for gene mutations in tomosyn in patients with idiopathic CDI. We anticipate our methods of mES-AVP culturing to provide a more sophisticated in vitro model of secretion of AVP, including application to studies of CDI-specific human samples and induced pluripotent stem cells.
18 - 21 May 2019
European Society of Endocrinology