Rab1 1 facilitates cyclic AMP- and tauroursodeoxycholate-induced MRP2 translocation
Se Won Park1, Christopher M. Schonhoff1, Cynthia Webster2, Mohammed S. Anwer1;
1Biomedical Sciences, Tufts Univ Cum-mings School of Veterinary Medicine, North Grafton, MA; 2Clinical Sciences, Tufts Univ Cummings School of Veterinary Medicine, North Grafton, MA
Rab proteins (Ras homologous for brain) play an important role in vesicle trafficking, including endocytosis, vesicle motility and protein trafficking. Rab1 1 regulates vesicle recycling to the plasma membrane and is expressed in apical vesicular populations in discrete epithelial cell populations, including liver. Bile salt exporting pump (Bsep) cycles between canalicular membrane and Rab1 1-positive endosomes, and Rab1 1 is required for bile canalicular formation in WIF-B9 cells (Proc Natl Acad Sci 102: 15087, 2005; Mol Biol Cell 15: 3485, 2004). Both cAMP and tauroursodeoxycholate (TUDC) have been shown to increase plasma membrane Mrp2 in rat hepa-tocytes. However, the role of Rab1 1 in cAMP- and TUDC-induced MRP2 translocation has not been studied. The goal of the present study was to provide further insight into the role of Rab1 1 in the trafficking of MRP2. More specifically, we tested the hypothesis that Rab1 1 facilitates cAMP- and TUDC-induced MRP2 translocation. Studies were conducted in HuH-NTCP cells (HuH7 cells stably transfected with human NTCP), which con-stitutively express MRP2. HuH-NTCP cells were transfected with GFP-tagged wild type Rab1 1 (Rab1 1-WT) or GDP-locked inactive Rab1 1 (Rab1 1-GDP) to study the role of Rab1 1. Overex-pression of mutant constructs of Rab1 1 was confirmed by GFP immunoblots. Cells were treated with either 100 μM cAMP or 25 μM TUDC; these concentrations were shown to increase plasma membrane Mrp2 in hepatocytes. A biotinylation method and a GTP overlay assay were used to determine the plasma membrane MRP2 and the activation of Rab1 1, respectively. Cyclic AMP and TUDC significantly stimulated Rab1 1 activity by 57 +/- 1 1% and 20 +/- 2.3%, respectively (mean +/- SE, n=3), suggesting that Rab1 1 may be involved in MRP2 translocation by cAMP and TUDC. If Rab1 1 is involved, one would expect overexpression of Rab1 1-WT and Rab1 1-GDP to augment and inhibit, respectively, TUDC- and cAMP-induced increases in plasma membrane MRP2. Overexpression of either Rab1 1-WT or Rab1 1-GDP did not significantly affect the basal level of plasma membrane MRP2. In cells transfected with the empty vector, both cAMP and TUDC increased plasma membrane MRP2 by 13 +/- 3% (n=6) and 12 +/- 2% (n=4), respectively. The effect of cAMP and TUDC on plasma membrane MRP2 was further increased to 36 +/- 13% (n=6) and 76 +/-23% (n=4), respectively, in cells transfected with Rab1 1-WT. In contrast, cAMP and TUDC failed to increase plasma membrane MRP2 in cells transfected with Rab1 1-GDP. Taken together, these results suggest that TUDC- and cAMP-induced MRP2 translocation is facilitated by Rab 11.
The following people have nothing to disclose: Se Won Park, Christopher M. Schonhoff, Cynthia Webster, Mohammed S. Anwer