title: Hsp90 Co-localizes with Rab-GDI-1 and regulates agonist-induced amylase release in AR42J cells.
abstract: Rab proteins are small GTPases required for vesicle trafficking through the secretory and endocytic pathways. Rab GDP-dissociation inhibitor (rab-GDI) regulates Rab protein function and localization by maintaining Rab proteins in the GDP-bound conformation. Two isoforms of rab-GDI are present in most mammalian cells: GDI-1 and GDI-2. It has recently been demonstrated that a Heat shock protein 90 (Hsp90) chaperone complex regulates the interactions between Rab proteins and Rab-GDI-1. The AR42J cell line is derived from rat pancreatic exocrine tumor cells and develops an acinar-like phenotype when treated with dexamethasone (Dex). The aim of the present study was to examine the expression of rab-GDI isoforms and Hsp90 in AR42J cells in the presence or absence of Dex. Rab-GDI:Hsp90 interactions were also examined. Both rab-GDI isoforms were detected in AR42J cells by immunoblotting. In Dex-treated cells, quantitative immunoblotting revealed that rab-GDI-1 expression increased by 28%, although this change was not statistically significant. Rab-GDI-2 levels were unaltered by Dex treatment. Approximately 21% rab-GDI-1 was membrane associated, whereas rab-GDI-2 was exclusively cytosolic. Dex treatment did not affect the subcellular distribution of rab-GDI isoforms. Hsp90 was present in the cytosolic and membrane fractions of AR42J cells and co-immunoprecipitated with cytosolic rab-GDI-1. Moreover, density gradient centrifugation of AR42J cell membranes revealed that Hsp90 and rab-GDI-1 co-localize on low- and high-density membrane fractions, including amylase-containing secretory granules. The Hsp90 inhibitor, geldanamycin, inhibited CCK-8-induced amylase release from these cells in a dose-dependent manner. Our results indicate that as AR42J cells differentiate into acinar-like cells, rab-GDI isoform expression and localization is not significantly altered. Moreover, our findings suggest that Hsp90 regulates agonist-induced secretion in exocrine cells by interacting with rab-GDI-1.
authors.affiliation: Hunter College, New York, NY 10010, USA. rraffani@hunter.cuny.edu
authors.forename: Robert
authors.initials: R
authors.lastname: Raffaniello
authors.forename: Daria
authors.initials: D
authors.lastname: Fedorova
authors.forename: Dawn
authors.initials: D
authors.lastname: Ip
authors.forename: Sarwish
authors.initials: S
authors.lastname: Rafiq
mesh_terms: D000681:Amylases / Q000378:metabolism*; D000818:Animals; D016227:Benzoquinones / Q000494:pharmacology; D045744:Cell Line, Tumor; D002499:Centrifugation, Density Gradient; D003907:Dexamethasone / Q000494:pharmacology; D020730:Guanine Nucleotide Dissociation Inhibitors / Q000032:analysis / Q000378:metabolism*; D018841:HSP90 Heat-Shock Proteins / Q000032:analysis / Q000378:metabolism*; D047468:Immunoprecipitation; D047029:Lactams, Macrocyclic / Q000494:pharmacology; D020033:Protein Isoforms / Q000032:analysis / Q000378:metabolism; D051381:Rats; D012844:Sincalide / Q000378:metabolism; D020691:rab GTP-Binding Proteins / Q000032:analysis / Q000378:metabolism*; D062947:rho-Specific Guanine Nucleotide Dissociation Inhibitors
chemical_list: D016227:Benzoquinones; C086796:GDP dissociation inhibitor 1; C521203:Gdi2 protein, rat; D020730:Guanine Nucleotide Dissociation Inhibitors; D018841:HSP90 Heat-Shock Proteins; D047029:Lactams, Macrocyclic; D020033:Protein Isoforms; D062947:rho-Specific Guanine Nucleotide Dissociation Inhibitors; D003907:Dexamethasone; D000681:Amylases; D020691:rab GTP-Binding Proteins; D012844:Sincalide; C001277:geldanamycin