Cullin-associated NEDD8-dissociated protein a novel interactor of rabphilin-3A, deubiquitylates rabphilin-3A and regulates arginine vasopressin secretion in PC12 cells

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 AVE 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 (CANDI) was identified as a rabphilin-3A-binding protein in the posterior pituitary. Co-immunoprecipitation assays indicated that CANDI interacted endogenously with rabphilin-3A. In addition, immunohistochemistry experiments showed that CANDI 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 CANDI resulted in deubiquitylation of rabphilin-3A in PC12 cells. Moreover, overexpression of CANDI in PC12 cells co-transfected with AVP enhanced both basal and KCI-stimulated AVP secretion. The findings indicate that CANDI 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 CANDI as a regulator of hormone or neurotransmitter secretion.