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TBC1D9B functions as a GTPase-activating protein for Rab11a in polarized MDCK cells.

Gallo LI, Liao Y, Ruiz WG, Clayton DR, Li M, Liu YJ, Jiang Y, Fukuda M, Apodaca G, Yin XM - Mol. Biol. Cell (2014)

Bottom Line: In contrast, TBC1D9B had no effect on two Rab11a-independent pathways--basolateral recycling of the transferrin receptor or degradation of the epidermal growth factor receptor.Finally, expression of TBC1D9B decreased the amount of active Rab11a in the cell and concomitantly disrupted the interaction between Rab11a and its effector, Sec15A.We conclude that TBC1D9B is a Rab11a GAP that regulates basolateral-to-apical transcytosis in polarized MDCK cells.

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Affiliation: Departments of Medicine, University of Pittsburgh, Pittsburgh, PA 15261.

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TBC1D9B regulates basolateral-to-apical transcytosis of IgA. The fraction of basolaterally internalized [125I]IgA that was transcytosed (A), the fraction of apically internalized [125I]IgA that was recycled (B), the fraction of basolaterally internalized [125I]Tf that was recycled (C), or the fraction of basolaterally internalized [125I]EGF that was degraded (D) was evaluated in filter-grown MDCK cells expressing CFP-TBC1D9B-WT or CFP-TBC1D9B-RYQ/AAA. (E) Top, RT-PCR analysis of MDCK cells expressing scrambled shRNA or specific shRNAs (shRNA1/2) that targeted canine TBC1D9B expression. Bottom, data are quantified. (F) Basolateral-to-apical transcytosis of [125I]IgA was measured in MDCK cells expressing either scrambled shRNA (shRNA Control) or shRNA-1/shRNA-2. In A–F, data were obtained from three independent experiments performed in triplicate, and the means ± SD are shown. Values different from the control, assessed by ANOVA, are indicated (*p < 0.05).
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Figure 8: TBC1D9B regulates basolateral-to-apical transcytosis of IgA. The fraction of basolaterally internalized [125I]IgA that was transcytosed (A), the fraction of apically internalized [125I]IgA that was recycled (B), the fraction of basolaterally internalized [125I]Tf that was recycled (C), or the fraction of basolaterally internalized [125I]EGF that was degraded (D) was evaluated in filter-grown MDCK cells expressing CFP-TBC1D9B-WT or CFP-TBC1D9B-RYQ/AAA. (E) Top, RT-PCR analysis of MDCK cells expressing scrambled shRNA or specific shRNAs (shRNA1/2) that targeted canine TBC1D9B expression. Bottom, data are quantified. (F) Basolateral-to-apical transcytosis of [125I]IgA was measured in MDCK cells expressing either scrambled shRNA (shRNA Control) or shRNA-1/shRNA-2. In A–F, data were obtained from three independent experiments performed in triplicate, and the means ± SD are shown. Values different from the control, assessed by ANOVA, are indicated (*p < 0.05).

Mentions: If TBC1D9B acts as a Rab11a-GAP, we reasoned that increasing its expression should down-regulate Rab11a activity and in doing so alter Rab11a-dependent trafficking events. We first tested the effects of TBC1D9B expression on the efficiency of basolateral-to-apical IgA transcytosis, a known Rab11a-dependent trafficking event (Casanova et al., 1999; Wang et al., 2000b). As expected, expression of TBC1D9B-WT, but not TBC1D9B-RYQ/AAA, significantly slowed the rate of IgA transcytosis, particularly at early time points (Figure 8A). We also tested the effects of TBC1D9B-WT expression on the fraction of IgA that recycles apically, a trafficking event that shows partial dependence on Rab11a (Wang et al., 2000b). Although the effect was not pronounced, there was a small but significant inhibition in the amount of IgA that recycled apically (Figure 8B). We also tested two pathways known to be Rab11a independent, including basolateral recycling of transferrin (Tf) and degradation of basolaterally internalized epidermal growth factor (EGF; Barbieri et al., 2000; Wang et al., 2000b). In both cases, expression of TBC1D9B-WT (or TBC1D9B-RYQ/AAA) had no significant effect (Figure 8, C and D).


TBC1D9B functions as a GTPase-activating protein for Rab11a in polarized MDCK cells.

Gallo LI, Liao Y, Ruiz WG, Clayton DR, Li M, Liu YJ, Jiang Y, Fukuda M, Apodaca G, Yin XM - Mol. Biol. Cell (2014)

TBC1D9B regulates basolateral-to-apical transcytosis of IgA. The fraction of basolaterally internalized [125I]IgA that was transcytosed (A), the fraction of apically internalized [125I]IgA that was recycled (B), the fraction of basolaterally internalized [125I]Tf that was recycled (C), or the fraction of basolaterally internalized [125I]EGF that was degraded (D) was evaluated in filter-grown MDCK cells expressing CFP-TBC1D9B-WT or CFP-TBC1D9B-RYQ/AAA. (E) Top, RT-PCR analysis of MDCK cells expressing scrambled shRNA or specific shRNAs (shRNA1/2) that targeted canine TBC1D9B expression. Bottom, data are quantified. (F) Basolateral-to-apical transcytosis of [125I]IgA was measured in MDCK cells expressing either scrambled shRNA (shRNA Control) or shRNA-1/shRNA-2. In A–F, data were obtained from three independent experiments performed in triplicate, and the means ± SD are shown. Values different from the control, assessed by ANOVA, are indicated (*p < 0.05).
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Figure 8: TBC1D9B regulates basolateral-to-apical transcytosis of IgA. The fraction of basolaterally internalized [125I]IgA that was transcytosed (A), the fraction of apically internalized [125I]IgA that was recycled (B), the fraction of basolaterally internalized [125I]Tf that was recycled (C), or the fraction of basolaterally internalized [125I]EGF that was degraded (D) was evaluated in filter-grown MDCK cells expressing CFP-TBC1D9B-WT or CFP-TBC1D9B-RYQ/AAA. (E) Top, RT-PCR analysis of MDCK cells expressing scrambled shRNA or specific shRNAs (shRNA1/2) that targeted canine TBC1D9B expression. Bottom, data are quantified. (F) Basolateral-to-apical transcytosis of [125I]IgA was measured in MDCK cells expressing either scrambled shRNA (shRNA Control) or shRNA-1/shRNA-2. In A–F, data were obtained from three independent experiments performed in triplicate, and the means ± SD are shown. Values different from the control, assessed by ANOVA, are indicated (*p < 0.05).
Mentions: If TBC1D9B acts as a Rab11a-GAP, we reasoned that increasing its expression should down-regulate Rab11a activity and in doing so alter Rab11a-dependent trafficking events. We first tested the effects of TBC1D9B expression on the efficiency of basolateral-to-apical IgA transcytosis, a known Rab11a-dependent trafficking event (Casanova et al., 1999; Wang et al., 2000b). As expected, expression of TBC1D9B-WT, but not TBC1D9B-RYQ/AAA, significantly slowed the rate of IgA transcytosis, particularly at early time points (Figure 8A). We also tested the effects of TBC1D9B-WT expression on the fraction of IgA that recycles apically, a trafficking event that shows partial dependence on Rab11a (Wang et al., 2000b). Although the effect was not pronounced, there was a small but significant inhibition in the amount of IgA that recycled apically (Figure 8B). We also tested two pathways known to be Rab11a independent, including basolateral recycling of transferrin (Tf) and degradation of basolaterally internalized epidermal growth factor (EGF; Barbieri et al., 2000; Wang et al., 2000b). In both cases, expression of TBC1D9B-WT (or TBC1D9B-RYQ/AAA) had no significant effect (Figure 8, C and D).

Bottom Line: In contrast, TBC1D9B had no effect on two Rab11a-independent pathways--basolateral recycling of the transferrin receptor or degradation of the epidermal growth factor receptor.Finally, expression of TBC1D9B decreased the amount of active Rab11a in the cell and concomitantly disrupted the interaction between Rab11a and its effector, Sec15A.We conclude that TBC1D9B is a Rab11a GAP that regulates basolateral-to-apical transcytosis in polarized MDCK cells.

View Article: PubMed Central - PubMed

Affiliation: Departments of Medicine, University of Pittsburgh, Pittsburgh, PA 15261.

Show MeSH
Related in: MedlinePlus