Nakashima Kohtaro, Takeuchi Seiji, Iwama Shintaro, Kiyota Atsushi, Yasuda Yoshinori, Iwata Naoko, Enomoto Atsushi, Arima Hiroshi, Sugimura Yoshihisa
Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, Nagoya, Aichi 466-8550, Japan.
Department of Pathology, Nagoya University Graduate School of Medicine, Nagoya, Aichi 466-8550, Japan.
Endocr J. 2018 Mar 28;65(3):325-334. doi: 10.1507/endocrj.EJ17-0399. Epub 2018 Jan 23.
The molecular mechanism involved in the exocytosis of arginine vasopressin (AVP) is not fully known. Rabphilin-3A has been suggested as a novel autoantigen in infundibulo-neurohypophysitis (LINH), which leads to central diabetes insipidus through insufficient secretion of AVP. However, the role of rabphilin-3A in the pathogenesis of LINH remains unclear. Thus, the aim of the present study was to identify proteins binding rabphilin-3A in the posterior pituitary. Using glutathione S-transferase (GST)-pulldown assays and proteomic analyses, cullin-associated NEDD8-dissociated protein 1 (CAND1) was identified as a rabphilin-3A-binding protein in the posterior pituitary. Co-immunoprecipitation assays indicated that CAND1 interacted endogenously with rabphilin-3A. In addition, immunohistochemistry experiments showed that CAND1 immunoreactivity was detected mainly in the posterior pituitary, intermediate lobe, and the supraoptic nucleus in the hypothalamus, and less in the anterior lobe, partially co-localizing with rabphilin-3A. Overexpression of CAND1 resulted in deubiquitylation of rabphilin-3A in PC12 cells. Moreover, overexpression of CAND1 in PC12 cells co-transfected with AVP enhanced both basal and KCl-stimulated AVP secretion. The findings indicate that CAND1 inhibits the ubiquitylation of rabphilin-3A and positively regulates AVP secretion. These data shed light on a novel potential mechanism involving rabphilin-3A in AVP secretion, and suggest a new role of CAND1 as a regulator of hormone or neurotransmitter secretion.
精氨酸加压素（AVP）胞吐作用所涉及的分子机制尚未完全明确。Rabphilin-3A被认为是漏斗-神经垂体炎（LINH）中的一种新型自身抗原，LINH通过AVP分泌不足导致中枢性尿崩症。然而，rabphilin-3A在LINH发病机制中的作用仍不清楚。因此，本研究的目的是鉴定垂体后叶中与rabphilin-3A结合的蛋白质。通过谷胱甘肽S-转移酶（GST）下拉试验和蛋白质组学分析，在垂体后叶中鉴定出与rabphilin-3A结合的蛋白质cullin相关的NEDD8解离蛋白1（CAND1）。免疫共沉淀试验表明CAND1与rabphilin-3A在内源性水平上相互作用。此外，免疫组织化学实验表明，CAND1免疫反应性主要在垂体后叶、中间叶和下丘脑视上核中检测到，在前叶中较少，部分与rabphilin-3A共定位。CAND1的过表达导致PC12细胞中rabphilin-3A的去泛素化。此外，在与AVP共转染的PC12细胞中过表达CAND1可增强基础和KCl刺激的AVP分泌。这些发现表明CAND1抑制rabphilin-3A的泛素化并正向调节AVP分泌。这些数据揭示了一种涉及rabphilin-3A在AVP分泌中的新的潜在机制，并提示CAND1作为激素或神经递质分泌调节剂的新作用。
