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American Journal of Physiology - Gastrointestinal and Liver Physiology logoLink to American Journal of Physiology - Gastrointestinal and Liver Physiology
. 2014 Sep 4;307(8):G863–G870. doi: 10.1152/ajpgi.00457.2013

Rab11, but not Rab4, facilitates cyclic AMP- and tauroursodeoxycholate-induced MRP2 translocation to the plasma membrane

Se Won Park 1, Christopher M Schonhoff 1, Cynthia R L Webster 2, M Sawkat Anwer 1,✉
PMCID: PMC4200318  PMID: 25190474

Abstract

Rab proteins (Ras homologous for brain) play an important role in vesicle trafficking. Rab4 and Rab11 are involved in vesicular trafficking to the plasma membrane from early endosomes and recycling endosomes, respectively. Tauroursodeoxycholate (TUDC) and cAMP increase bile formation, in part, by increasing plasma membrane localization of multidrug resistance-associated protein 2 (MRP2). The goal of the present study was to determine the role of these Rab proteins in the trafficking of MRP2 by testing the hypothesis that Rab11 and/or Rab4 facilitate cAMP- and TUDC-induced MRP2 translocation to the plasma membrane. Studies were conducted in HuH-NTCP cells (HuH7 cells stably transfected with human NTCP), which constitutively express MRP2. HuH-NTCP cells were transfected with Rab11-WT and GDP-locked dominant inactive Rab11-GDP or with Rab4-GDP to study the role of Rab11 and Rab4. A biotinylation method and a GTP overlay assay were used to determine plasma membrane MRP2 and activation of Rab proteins (Rab11 and Rab4), respectively. Cyclic AMP and TUDC increased plasma membrane MRP2 and stimulated Rab11 activity. Plasma membrane translocation of MRP2 by cAMP and TUDC was increased and inhibited in cells transfected with Rab11-WT and Rab11-GDP, respectively. Cyclic AMP (previous study) and TUDC increased Rab4 activity. However, cAMP- and TUDC-induced increases in MRP2 were not inhibited by Rab4-GDP. Taken together, these results suggest that Rab11 is involved in cAMP- and TUDC-induced MRP2 translocation to the plasma membrane.

Keywords: Rab11, Rab4, HuH-NTCP cells

multidrug resistance-associated protein 2 (MRP2), an ATP binding cassette membrane transporter, is present in the canalicular membrane of hepatocytes (2). MRP2 plays an important role in bile formation by mediating secretion of bile acids and organic anions across the canalicular membrane (2). Choleretic agents, such as tauroursodeoxycholate (TUDC) and cAMP, increase bile formation, in part, by increasing plasma membrane localization of MRP2 (5, 32). The mechanisms by which cAMP and TUDC translocate MRP2 to the plasma membrane are incompletely understood, although a role of protein kinase C has been suggested (3). One of the key processes in the translocation of transporters is vesicle trafficking along the microtubules (25), and cAMP stimulates vesicle trafficking in hepatocytes (18). Since Rab proteins are involved in vesicle trafficking (35), it is likely that MRP2 translocation to the plasma membrane may involve Rab proteins.

Rab proteins are a large family of small GTPases, and over 70 Rab proteins have been identified in humans (1). Rab proteins cycle between active GTP bound form, and inactive GDP bound form; active GTP bound Rab protein functions by interacting with a number of effector proteins. The nucleotide exchange from GDP to GTP is enhanced by guanine nucleotide exchange factors (GEFs) and GTP hydrolysis is facilitated by GTPase activating proteins (GAPs) (19). Through this cycling, Rab proteins can act as molecular switches on vesicular trafficking.

Rab proteins represent key regulators of vesicular trafficking including vesicle formation, vesicle transport, vesicle docking and fusion, exocytosis, and endocytosis (35). Rab proteins are localized to a number of different intracellular organelles on both the endocytic and exocytic pathways, suggesting that different Rab proteins are involved in different steps of membrane traffic (46). Among over 70 Rab proteins, Rab1, Rab4, Rab5, Rab7, Rab9, and Rab11 are involved in the vesicular trafficking (28, 47). Rab7 is involved in early endosome-to-late endosome and late endosome-to-lysosome transport (6, 12). Rab9 meditates trafficking from endosome to the trans-Golgi network (41). Rab1a regulates minus-end-directed motility largely by recruiting Kifc1 to early endocytic vesicles (28). Rab5 regulates endocytic vesicular trafficking, and Rab4 facilitates exocytosis (46). Rab11 is involved in trafficking from the recycling endosomes to the plasma membrane (19). Studies so far suggest that Rab4, Rab5, and Rab11 are involved in the trafficking of hepatocellular transporters (34, 40, 44). Among these three Rab proteins, our previous study (48) showed that Rab5 is not involved in cAMP induced NTCP translocation in HuH-NTCP cells. Other studies showed that Rab4 and Rab11 are involved in vesicular trafficking to the plasma membrane from early endosomes and recycling endosomes, respectively (26, 35). There are limited studies suggesting a role for Rab4 and Rab11 in the trafficking of hepatocellular transporters. Thus we focused our present study on the role of Rab11 and Rab4 in MRP2 translocation.

Studies in hepatic cells show that Rab4 is present in Ntcp containing vesicles (33) and that cAMP activates Rab4 and Rab4 facilitates cAMP-induced NTCP translocation (34). Rab4 has been shown to regulate transport of the transferrin receptor from the early endosomes to the apical membrane in MDCK cells (27). Thus Rab4 may be involved in apical targeting of hepatocellular transporters. Rab11 is present in vesicles around bile canaliculi (15) and associates with transporters in canalicular membranes of hepatocytes. For example, bile salt export pump (Bsep) cycles between canalicular membrane and Rab11-positive endosomes, and Rab11a is required for bile canalicular formation in WIF-B9 cells (40). However, the role of Rab11 in MRP2 translocation has not been reported. On the basis of these studies, we hypothesize that Rab4 and Rab11 may be involved in the translocation of MRP2 to the plasma membrane by TUDC and cAMP.

The aim of this study was to determine whether Rab11 and/or Rab4 mediate TUDC- and cAMP-stimulated MRP2 translocation to the plasma membrane. Results of our studies with dominant inactive mutants of Rab11 and Rab4 suggest that cAMP- and TUDC-induced MRP2 translocation is facilitated by Rab11 but not by Rab4.

MATERIALS AND METHODS

Materials

TUDC was purchased from Calbiochem (San Diego, CA). 8-Chlorophenylthio cAMP (CPT-cAMP), aprotinin, leupeptin and okadaic acid were purchased from Sigma Chemical (St. Louis, MO). [α-32P]GTP was purchased from Perkin-Elmer (Boston, MA). Polyclonal Rab4 and Rab11 antibodies were obtained from Millipore (Billerica, MA) and Invitrogen (Grand Island, NY), respectively. Rab11-GDP (Rab11S25N, catalog no. 12678) and Rab11-WT (catalog no. 12674) cloned in Clontech C1-pEGFP vector were obtained from Addgene (Cambridge, MA). These constructs have been used (8, 16) and checked for activation status with GTP assay (8). Dominant inactive Rab4-GDP (Rab4S22N) construct was cloned in Clontech C1-CFP vector (26). Expected GTP binding properties of Rab11 and Rab4 constructs were confirmed by the GTP overlay assay. Transfected Rab11-WT but neither Rab11-GDP nor Rab4-GDP did bind GTP. HuH-NTCP cells (HuH-7 cells stably transfected with NTCP), were generously provided by Dr. Gores (Rochester, MN). Monoclonal antibodies of Rab4 and E-cadherin were purchased from BD Transduction Laboratories (Franklin Lakes, NJ). Monoclonal Rab11 antibodies were purchased from BD Transduction Laboratories. MRP2 antibody was obtained from Sigma. Sulfo-NHS-LC-Biotin was purchased from Pierce (Rockford, IL). Streptavidin agarose beads were obtained from EMD Millipore.

Cell Culture

HuH-NTCP cells were grown in Eagle's minimum essential medium supplemented with 10% fetal calf serum, 1.2 g/l G418 100,000 units/l penicillin, 100 mg/l streptomycin, and 25 μg/ml amphotericin B at 37°C with 5% CO2. On the day of the experiment, the medium was changed to serum-free DMEM for 3 h and cells were then treated with or without CPT-cAMP, a cell-permeable analog of cAMP, or TUDC. Other conditions are described in figure legends.

GTP Overlay Assay

Activation of Rab4 or Rab11 was determined by measuring GTP binding to Rab4 or Rab11 in immunoprecipitated Rab4 or Rab11 via a blot overlay assay, as previously described (29). Briefly, cells were incubated in serum-free medium for 3 h and then treated with desired agents as described in the figure legends. Treated cells were washed with ice-cold PBS and then lysed with cell lysis buffer. The soluble fractions were immunoprecipitated by incubation with anti-rabbit polyclonal Rab11 (Invitrogen) or with anti-rabbit polyclonal Rab4 antibody (Millipore) at 4°C overnight, followed by incubation with protein A-Sepharose beads for 2 h. The immunoprecipitated proteins were washed with cell lysis buffer and resolved by SDS-PAGE. After transfer, the blots were incubated in a binding buffer (50 mM sodium phosphate pH 7.5, 10 μM MgCl2, 2 mM DTT, 0.2% Tween 20, 4 μM ATP) for 30 min, followed by incubation for 1 h in the binding buffer containing 1 μCi/ml [α-32P]GTP. Blots were washed and autoradiographed by using Kodak XO-MAT AR films at −80°C to determine GTP-bound Rab4 or Rab11. The bound GTP was stripped followed by immunoblotting for immunoprecipitated Rab4 or Rab11. For immunoblotting, blots were probed with primary antibodies (monoclonal Rab4 or Rab11). After washing, blots were incubated with horseradish peroxidase-linked secondary antibody followed by chemiluminescence detection.

Transfection of Cells with Mutant Isoforms of Rab4 or Rab11

HuH-NTCP cells were transfected with a cyan blue fluorescent protein (CFP)-tagged empty vector or CFP-tagged Rab4-GDP, or an enhanced green fluorescent protein (EGFP)-tagged empty vector (EV), EGFP-tagged Rab11-GDP or EGFP-tagged Rab11-wild-type (WT) by using Lipofectamine 2000 according to the manufacturer's instructions. Briefly, the culture medium was changed to OptiMem containing Lipofectamine and empty vector or mutant constructs of Rab4 or Rab11 and incubated at 37°C for 24 h. Expression of mutant constructs of Rab4 or Rab11 in HuH-NTCP cells was confirmed by immunoblotting with Rab11 and Rab4 antibodies.

Translocation of MRP2

A cell surface biotinylation method was used to quantitate plasma membrane MRP2 (29). Briefly, after various treatments, cells were washed with ice-cold PBS and incubated with sulfo-NHS-LC-Biotin (0.5 mg/ml) at 4°C for 45 min to achieve the selective labeling of cell surface proteins. Cells were washed with 100 μM glycine in PBS (pH 8) three times and then lysed with cell lysis buffer. Biotinylated proteins were isolated with 50 μl of streptavidin-agarose beads followed by immunoblot analysis. To detect the biotinylated plasma membrane MRP2, blots were first incubated with E-cadherin antibody at 4°C overnight and then with MRP2 antibody at room temperature for 2 h. After three washings, blots were incubated with horseradish peroxidase-linked secondary antibodies followed by chemiluminescence detection.

Other Methods

The Lowry method was used to determine cell protein (24). Results were expressed as means ± SE. Data were analyzed by one-way ANOVA-Student-Newman-Keuls method for all pairwise comparison and by paired t-test for comparison between two means. P < 0.05 was considered statistically significant.

RESULTS

Role of Rab11 in cAMP- and TUDC-Induced MRP2 Translocation

We hypothesized that TUDC and cAMP may stimulate MRP2 translocation by activating Rab11. This hypothesis was tested by determining whether 1) TUDC translocates MRP2 to the plasma membrane in HuH-NTCP cells, 2) cAMP and TUDC activate Rab11, and 3) Rab11-GDP inhibits TUDC- and cAMP-induced MRP2 translocation.

TUDC increased MRP2 translocation in HuH-NTCP cells.

Previous studies have shown that TUDC stimulates translocation of vesicles containing Mrp2 to the plasma membrane, and ursodeoxycholic acid enhances transport activity of Mrp2 in rat hepatocytes (5, 10). Cyclic AMP has previously been shown to stimulate Mrp2/MRP2 translocation in rat hepatocytes and HuH-7 cells (29, 32). However, whether TUDC also stimulates translocation of MRP2 in HuH-7 cells has not been determined. Thus, before initiating studies with mutant constructs of Rab11, it was necessary to confirm the expected effect of TUDC on MRP2 translocation in HuH-NTCP cells; this cell line was used to assure cellular entry of TUDC via NTCP.

To determine the effect of TUDC on MRP2 translocation, cells were treated with or without TUDC followed by plasma membrane protein biotinylation and determination of the amount of MRP2 in the plasma membrane. A preliminary time-dependent study showed that TUDC increased plasma membrane MRP2 in 10 min, and hence this time point was used for further studies. TUDC significantly increased plasma membrane MRP2 by 1.3-fold (Fig. 1) in HuH-NTCP cells establishing that TUDC also stimulates plasma membrane translocation of MRP2 in a human hepatic cell line.

Fig. 1.

Fig. 1.

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Tauroursodeoxycholate (TUDC) increases plasma membrane multidrug resistance-associated protein 2 (MRP2). HuH-NTCP cells were treated with or without 25 μM TUDC for 10 min. A biotinylation method was used to determine plasma membrane (PM) MRP2. Representative immunoblots of PM MRP2 and E-cadherin (E-cad) (top) and densitometric analysis (bottom) are shown. E-cad served as a loading control for biotinylated proteins. The amount of MRP2 localization in the plasma membrane was expressed as a ratio of PM MRP2 to E-cad. Relative values of MRP2 in the plasma membrane are expressed as means ± SE (n = 6). *Significantly different (P < 0.05) from control values in the absence of TUDC.

TUDC and cAMP increase Rab11 activity.

To determine whether TUDC and cAMP activate Rab11, cells were treated with or without TUDC or CPT-cAMP, followed by GTP overlay assay to determine the effect of these agents on GTP binding ability of Rab11 and hence activation of Rab11. Treatment with either TUDC (Fig. 2A) or cAMP (Fig. 2B) significantly increased GTP binding to Rab11 by 1.2-fold or by 1.5-fold, respectively, indicating that cAMP and TUDC activate Rab11.

Fig. 2.

Fig. 2.

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TUDC and cAMP increase Rab11 activity. HuH-NTCP cells were treated with 25 μM TUDC for 10 min (A) or 100 μM cAMP for 15 min (B). Rab11 activation was determined by GTP overlay assay as described in materials and methods. A representative immunoblot of Rab11-GTP and total Rab11 is shown at top with the densitometric analysis shown at bottom. Rab11 activity (means ± SE, n = 3) was expressed as a ratio of Rab11-GTP to total Rab11 to correct for variations due to loading and immunoprecipitation. Data were analyzed by paired t-test. *Significantly different (P < 0.05) from control values in the absence of TUDC or cAMP.

Rab11 mediates TUDC- and cAMP-stimulated MRP2 translocation.

To determine whether Rab11 is involved in MRP2 translocation by TUDC or cAMP, cells were transfected with EV, Rab11-GDP, or Rab11-WT and then the transfected cells were treated with either TUDC or CPT-cAMP. Expressions of Rab11-WT and Rab11-GDP were confirmed by immunoblotting for Rab11; expression of transfected mutants represented 70–75% of total Rab11 in transfected cells (Figs. 3A and 4A). The expressions of Rab11-WT and Rab11-GDP were not affected by either TUDC or cAMP. As shown in Figs. 3B and 4B, the basal level of MRP2 in the plasma membrane was not affected by either Rab11-GDP or Rab11-WT. Treatment of cells with either TUDC or cAMP significantly increased MRP2 translocation to the plasma membrane in cells transfected with EV or Rab11-WT (Figs. 3B and 4B). The increases in plasma membrane MRP2 by cAMP and TUDC in Rab11-WT transfected cells were 10–15% higher (not statistically significant) than those values in EV-transfected cells. Since Rab11-WT did not affect the basal level of MRP2 in the plasma membrane, the observed increases are due to cAMP and TUDC. However, cAMP and TUDC failed to increase plasma membrane MRP2 in cells transfected with dominant inactive Rab11-GDP. These results suggest that Rab11 activation is necessary for TUDC- and cAMP-mediated MRP2 translocation to the plasma membrane.

Fig. 3.

Fig. 3.

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Rab11 facilitates TUDC-induced MRP2 translocation. HuH-NTCP cells were transfected with empty vector (EV), Rab11-GDP and Rab11-WT followed by treatment with or without 25 μM TUDC for 10 min. A biotinylation method was used to determine plasma membrane MRP2. A: transfection was confirmed with Rab11 antibody (Trans Rab11, transfected Rab11 mutants; Endo Rab11, endogenous Rab11). B: representative immunoblots of PM MRP2 and E-cad (top) and densitometric analysis (bottom) are shown. Relative values of MRP2 in the plasma membrane are expressed as means ± SE (n = 4–15). Data were analyzed by 1-way ANOVA-Student-Newman-Keuls method. *Significantly different (P < 0.05) from respective control values in the absence of TUDC.

Fig. 4.

Fig. 4.

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Rab11 facilitates cAMP-induced MRP2 translocation. HuH-NTCP cells were transfected with EV, Rab11-GDP, and Rab11-WT, followed by treatment with or without 100 μM CPT-cAMP for 15 min. A biotinylation method was used to determine plasma membrane MRP2. A: transfection was confirmed with Rab11 antibody. B: representative immunoblots of PM MRP2 and E-cad (top) and densitometric analysis (bottom) are shown. Relative values of MRP2 in the plasma membrane are expressed as means ± SE (n = 6–15). Data were analyzed by 1-way ANOVA-Student-Newman-Keuls method. *Significantly different (P < 0.05) from respective control values in the absence of cAMP.

Role of Rab4 in cAMP- and TUDC-Induced MRP2 Translocation

Cyclic AMP has previously been shown to activate Rab4 in HuH-NTCP cells (34). However, it is not known whether TUDC also activates Rab4. To test the hypothesis that Rab4 may be involved in cAMP and TUDC induced translocation of MRP2, we determined whether TUDC activate Rab4 and whether Rab4-GDP inhibits TUDC- and cAMP-induced MRP2 translocation.

TUDC increases Rab4 activity.

HuH-NTCP cells were treated with or without TUDC (10, 25, and 50 μM) and then GTP overlay assay was used to determine Rab4 activity. In this study, treatment of cells with TUDC significantly increased GTP binding to Rab4 (1.33 ± 0.02, means ± SE, n = 3) at all concentrations tested (Fig. 5), indicating activation of Rab4 by TUDC.

Fig. 5.

Fig. 5.

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TUDC increases Rab4 activity. HuH-NTCP cells were treated with 0, 10, 25, or 50 μM TUDC for 10 min. Rab4 activation was determined by GTP overlay assay as described in materials and methods. A representative immunoblot of Rab4-GTP and total Rab4 is shown in the top with the densitometric analysis shown in the bottom. Rab4 activity (means ± SE, n = 3) was expressed as a ratio of Rab4-GTP to total Rab4 to correct for variations due to loading and immunoprecipitation. Data were analyzed by paired t-test. *,**Significantly different from control values in the absence of TUDC. *P < 0.05 and ** P < 0.01.

Rab4 is not involved in TUDC- and cAMP-induced MRP2 translocation.

To test whether Rab4 mediates TUDC- and cAMP-induced MRP2 translocation, HuH-NTCP cells were transfected with EV or Rab4-GDP followed by treatment with or without TUDC or cAMP. Expressions of GDP-Rab4 were confirmed by immunoblotting with Rab4 antibody and represented 82 ± 2.6% of total Rab4 in transfected cells (Figs. 6A and 7A). TUDC significantly increased the amount of MRP2 in the plasma membrane (Fig. 6B). The basal level of MRP2 in the plasma membrane was not significantly affected by Rab4-GDP. If Rab4 was involved, one would have expected Rab4-GDP to inhibit TUDC-induced MRP2 translocation. However, transfection with Rab4-GDP did not inhibit TUDC-stimulated increases in plasma membrane MRP2, compared with respective controls. Similarly, cAMP significantly increased MRP2 translocation to the plasma membrane in cells transfected with EV and also significantly elevated the amount of MRP2 in the plasma membrane in cells transfected with Rab4-GDP (Fig. 7B). The inability of dominant inactive Rab4-GDP to inhibit increases in plasma membrane MRP2 by cAMP and TUDC suggests that TUDC- and cAMP-induced Rab4 activation does not play a role in MRP2 translocation to the plasma membrane.

Fig. 6.

Fig. 6.

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Rab4 does not mediate TUDC-induced MRP2 translocation. HuH-NTCP cells were transfected with EV or Rab4-GDP followed by treatment with or without 25 μM TUDC for 10 min. A biotinylation method was used to determine PM MRP2. A: transfection was confirmed with Rab4 antibody. B: representative immunoblots of PM MRP2 and E-cad (top) and densitometric analysis (bottom) are shown. The amount of MRP2 localization in the plasma membrane was expressed as a ratio of PM MRP2 to E-cad. Relative values of MRP2 in the plasma membrane are expressed as means ± SE (n = 5). Data were analyzed by paired t-test. *Significantly different (P < 0.05) from respective control values in the absence of TUDC.

Fig. 7.

Fig. 7.

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Rab4 does not mediate cAMP-induced MRP2 translocation. HuH-NTCP cells were transfected with EV or Rab4-GDP followed by treatment with or without 100 μM cpt-cAMP for 15 min. A biotinylation method was used to determine PM MRP2. A: transfection was confirmed with Rab4 antibody. B: representative immunoblots of PM MRP2 and E-cad (top) and densitometric analysis (bottom) are shown. The amount of MRP2 localization in the plasma membrane was expressed as a ratio of PM MRP2 to E-cad. Relative values of MRP2 in the plasma membrane are expressed as means ± SE (n = 5). Data were analyzed by paired t-test. *Significantly different (P < 0.05) from respective control values in the absence of cAMP.

DISCUSSION

The aim of this study was to test the hypothesis that Rab11 and Rab4 may mediate TUDC- and cAMP-induced MRP2 translocation to the plasma membrane. Results of the present study suggest that TUDC- and cAMP-induced MRP2 translocation involves Rab11-mediated but not Rab4-mediated exocytosis.

The present study suggests that Rab11 activation is necessary for cAMP- and TUDC-induced translocation of MRP2. This conclusion is supported by results showing that cAMP and TUDC activate Rab11 (Fig. 2) and expression of dominant inactive Rab11-GDP inhibits TUDC- and cAMP-induced increases in plasma membrane MRP2 (Fig. 3B and Fig. 4B). The present study also showed that the basal level of plasma membrane MRP2 was not affected by Rab11-WT. In addition, although cAMP and TUDC significantly increased plasma membrane MRP2 in Rab11-WT transfected cells, this increase was not significantly different from respective values in EV-transfected cells. These results would suggest that, although Rab11 activation is necessary, factors in addition to Rab11 are involved in the translocation of MRP2 to the plasma membrane. The nature of such factors is speculative at this point and may require participation of Rab11-independent proteins involved in vesicle trafficking. In addition, other Rab proteins may also be involved in the translocation of MRP2.

Results of our study are consistent with other studies implicating Rab11 in the trafficking of membrane proteins. Studies have shown that Rab11 plays a role in trafficking from recycling endosomes to the plasma membrane (35), especially in polarized cells (11, 36). Rab11a facilitates transport of Niemann-Pick C1-like protein 1 (NPC1L1) to the plasma membrane in rat hepatoma cell line and Rab11 mediates insulin-stimulated GLUT4 translocation to the plasma membrane in 3T3-L1 adipocytes (9, 45). Expression of dominant negative Rab11 decreased insulin-induced GLUT4 translocation to the plasma membrane in cardiomyocytes and impaired transferrin receptor recycling in CHO and BHK cells (38, 39). In addition, dominant negative Rab25, a member of Rab11 subfamily, inhibits apical recycling of pericentriolar endosomal compartment in MDCK cells (7). Studies have indicated that intracellular fraction of Mrp2 colocalizes with Rab11 and apical recycling involving Rab11 has been described for Bsep (40, 42). Furthermore, Rab11a has been found associated with vesicles containing ABC transporters in hepatocytes (40). Taken together, these studies along with results from the present study would suggest that activation of Rab11 followed by Rab11-mediated exocytosis may be involved in the translocation of canalicular transporters.

The present study also suggests that Rab4 is not involved in TUDC- and cAMP-induced translocation of MRP2. The possibility that Rab4 may be involved in cAMP and TUDC-induced MRP2 translocation was suggested by results that cAMP (34) and TUDC activate Rab4 (Fig. 5) and increase plasma membrane MRP2. If Rab4 activation were involved, one would have expected dominant inactive Rab4-GDP to inhibit cAMP and TUDC-induced increases in plasma membrane MRP2. However, the present study showed that MRP2 translocation by cAMP and TUDC was not inhibited in cells transfected with Rab4-GDP despite that fact that Rab4-GDP represented 82% of total Rab4 in the transfected cells. It may be noted that Rab4-GDP with similar expression level inhibited cAMP-induced plasma membrane translocation of NTCP in HuH-NTCP cells in a previous study (34).

The observed different role of Rab4 and Rab11 in MRP2 translocation may be related to known role of Rab4 and Rab11 in vesicular trafficking (35). Although both Rab4 and Rab11 are involved in outward recycling, their site of action is slightly different. Transporting internalized proteins back to the plasma membrane can occur directly from the early endosome (fast cycle) or via the recycling endosome (slow cycle) (19). It has been suggested that Rab4 is involved in fast recycling from early endosomes, whereas Rab11 regulates slow recycling from recycling endosomes (19), and Rab4 and Rab11 coordinately regulate the recycling of angiotensin II type I receptor (AT1R) (23). During the early recycling stage, internalized AT1Rs were mainly associated with Rab4 and during the late recycling stage with Rab11 (23). Rab11 and Rab4 have been shown to be associated with different population of endocytic vesicles in hepatocyte. For example, Rab11 resides in apical recycling endosome and mediates BSEP translocation to the apical membrane in hepatocytes (40). In addition, Rab11 is not involved in the basolateral recycling of transferrin in polarized Madin-Darby canine kidney cells (43). Rab4 is associated with hepatocyte-derived early endocytic vesicles, colocalizes with NTCP containing vesicles, and facilitates cAMP-induced NTCP translocation (4, 33, 34). In addition, trafficking of proteins to the right membrane is regulated at multiple steps by Rab effector proteins, which include motors, tethering factors, and docking complexes (17, 37). Myosin Vb and Rab11-FIP2 are required for Rab11-mediated NPC1L1 translocation in rat hepatocytes (9) and kinesin is needed for Rab4-mediated GLUT4 translocation in adipocytes (20). These studies highlight the differences between Rab11- and Rab4-mediated vesicle trafficking. Since Rab11 and not Rab4 is involved, it is likely that cAMP and TUDC stimulate translocation of MRP2 to the plasma membrane by activating Rab11-mediated recycling from recycling endosomes and not from early endosomes. Since Rab11 is associated with pericanalicular vesicles and MRP2 is a canalicular membrane protein, it can be speculated that Rab11 regulates vesicular trafficking to the canalicular membrane.

The mechanism by which cAMP and TUDC activate Rab4 and Rab11 is not known. One possibility may be that cAMP and TUDC stimulate GEF to facilitate the conversion of GDP to GTP or inhibits GAP. Phosphorylation has also been shown to increase the affinity of a few members of Rab proteins for GTP (13, 14). For example, phosphorylated Rab4, which is increased during mitosis, is in GTP-bound form (14), and PKC-mediated phosphorylation of Rab6 increased the affinity for GTP (13). In addition, phosphorylation of Rab11 by PKC may be involved in transferring trafficking in HeLa cells (30). Both TUDC and cAMP have been shown to activate protein kinase C (3). Therefore, it is also possible that the activation of Rab4 and Rab11 by TUDC and cAMP involves PKC-mediated phosphorylation.

The mechanism by which Rab11 facilitates TUDC- and cAMP-induced MRP2 translocation is also unclear. Rab proteins mediate vesicular trafficking by interacting with a wide variety of effector proteins (17, 19). Rab effector proteins respond to a specific Rab protein and mediate their downstream effects (17). For example, Rab11 binds to RIP11, one of the Rab11 effector proteins, and this Rab11/RIP11 complex regulates trafficking of subapical endosomes to the apical membrane in rat brain (31). It has also been reported that Rab proteins recruit motor proteins leading to the regulation of vesicle trafficking including vesicle budding, fusion, and transport (21). Previous studies showed that myosin Vb, an actin-associated motor protein, binds to Rab11 (22) and recruitment of myosin Vb and Rab11a are required for bile canalicular formation in WIF-B9 cells (40). Thus it is possible that TUDC and cAMP promote the binding of Rab11 to myosin Vb to facilitate translocation of MRP2 containing vesicles to the plasma membrane.

In summary, the present study for the first time showed that TUDC- and cAMP-induced MRP2 translocation to the plasma membrane is mediated via activation of Rab11. In addition, activation of Rab4 is not involved in the translocation of MRP2 by TUDC and cAMP.

GRANTS

This study was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK90010.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

S.W.P. and M.S.A. conception and design of research; S.W.P. performed experiments; S.W.P. analyzed data; S.W.P., C.R.W., and M.S.A. interpreted results of experiments; S.W.P. prepared figures; S.W.P. drafted manuscript; S.W.P., C.M.S., and M.S.A. edited and revised manuscript; M.S.A. approved final version of manuscript.

ACKNOWLEDGMENTS

We thank Holly Jameson and Ariel Hobson for excellent technical assistance.

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