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CLC-5 and KIF3B interact to facilitate CLC-5 plasma membrane expression, endocytosis, and microtubular transport: relevance to pathophysiology of Dent's disease.

Reed AA, Loh NY, Terryn S, Lippiat JD, Partridge C, Galvanovskis J, Williams SE, Jouret F, Wu FT, Courtoy PJ, Nesbit MA, Rorsman P, Devuyst O, Ashcroft FM, Thakker RV - Am. J. Physiol. Renal Physiol. (2009)

Bottom Line: Confocal live cell imaging in kidney cells further demonstrated association of CLC-5 and KIF3B, and transport of CLC-5-containing vesicles along KIF3B microtubules.KIF3B overexpression and underexpression, using siRNA, had reciprocal effects on whole cell chloride current amplitudes, CLC-5 cell surface expression, and endocytosis of albumin and transferrin.Thus, the CLC-5 and KIF3B interaction is important for CLC-5 plasma membrane expression and for facilitating endocytosis and microtubular transport in the kidney.

View Article: PubMed Central - PubMed

Affiliation: Academic Endocrine Unit, Nuffield Department of Medicine, University of Oxford, Oxford Centre for Diabetes, Endocrinology, and Metabolism, Churchill Hospital, Oxford, United Kingdom.

ABSTRACT
Renal tubular reabsorption is important for extracellular fluid homeostasis and much of this occurs via the receptor-mediated endocytic pathway. This pathway is disrupted in Dent's disease, an X-linked renal tubular disorder that is characterized by low-molecular-weight proteinuria, hypercalciuria, nephrolithiasis, and renal failure. Dent's disease is due to mutations of CLC-5, a chloride/proton antiporter, expressed in endosomes and apical membranes of renal tubules. Loss of CLC-5 function alters receptor-mediated endocytosis and trafficking of megalin and cubilin, although the underlying mechanisms remain to be elucidated. Here, we report that CLC-5 interacts with kinesin family member 3B (KIF3B), a heterotrimeric motor protein that facilitates fast anterograde translocation of membranous organelles. Using yeast two-hybrid, glutathione-S-transferase pull-down and coimmunoprecipitation assays, the COOH terminus of CLC-5 and the coiled-coil and globular domains of KIF3B were shown to interact. This was confirmed in vivo by endogenous coimmunoprecipitation of CLC-5 and KIF3B and codistribution with endosomal markers in mouse kidney fractions. Confocal live cell imaging in kidney cells further demonstrated association of CLC-5 and KIF3B, and transport of CLC-5-containing vesicles along KIF3B microtubules. KIF3B overexpression and underexpression, using siRNA, had reciprocal effects on whole cell chloride current amplitudes, CLC-5 cell surface expression, and endocytosis of albumin and transferrin. Clcn5(Y/-) mouse kidneys and isolated proximal tubular polarized cells showed increased KIF3B expression, whose effects on albumin endocytosis were dependent on CLC-5 expression. Thus, the CLC-5 and KIF3B interaction is important for CLC-5 plasma membrane expression and for facilitating endocytosis and microtubular transport in the kidney.

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Schematic representation of a working model for endosomal transport involving CLC-5 and KIF3B interaction in polarized cells. CLC-5 and KIF3B are expressed in polarized renal proximal tubular cells which reabsorb proteins and solutes via the receptor-mediated endocytic pathway that uses megalin and cubilin as the apical plasma membrane (APM) receptors (4, 7, 17). Ligands bind to these receptors at the APM where CLC-5 and interacting proteins Nedd4-2, NHERF2, and cofilin are also present (23, 24, 26). The receptor-ligand complex is internalized in vesicles, containing CLC-5 and CLC-4 (40), which are anterogradely transported along microtubules via the Kinesin-2 complex (17). These vesicles fuse with early endosomes where acidification involves the vacuolar H+-ATPase, with counter ions being provided by CLC-5 (28). In nonpolarized cells, the orientation of the microtubules is reversed (Fig. 3F), and CLC-5 vesicles would be transported to the cell surface.
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Figure 7: Schematic representation of a working model for endosomal transport involving CLC-5 and KIF3B interaction in polarized cells. CLC-5 and KIF3B are expressed in polarized renal proximal tubular cells which reabsorb proteins and solutes via the receptor-mediated endocytic pathway that uses megalin and cubilin as the apical plasma membrane (APM) receptors (4, 7, 17). Ligands bind to these receptors at the APM where CLC-5 and interacting proteins Nedd4-2, NHERF2, and cofilin are also present (23, 24, 26). The receptor-ligand complex is internalized in vesicles, containing CLC-5 and CLC-4 (40), which are anterogradely transported along microtubules via the Kinesin-2 complex (17). These vesicles fuse with early endosomes where acidification involves the vacuolar H+-ATPase, with counter ions being provided by CLC-5 (28). In nonpolarized cells, the orientation of the microtubules is reversed (Fig. 3F), and CLC-5 vesicles would be transported to the cell surface.

Mentions: Our results establish an interaction between CLC-5 and KIF3B that is important for the plasma membrane expression of CLC-5, microtubular transport of CLC-5 vesicles, and for albumin uptake in polarized and nonpolarized cells. Thus, in polarized epithelial cells such as OK cells, the interaction between CLC-5 and KIF3B results in a reduction in the number of antiporters at the cell surface, as KIF3B is involved in facilitating microtubular transport that removes CLC-5-containing vesicles from the cell surface (Fig. 7). In nonpolarized cells, such as HEK293 cells, the orientation of the microtubules is reversed (Fig. 3F), and the interaction between CLC-5 and KIF3B facilitates transport of CLC-5-containing vesicles to the cell surface. This situation may be analogous to that reported for the interaction between CLC-2 and the microtubule-associated dynein motor complex, which is also involved in regulating CLC-2 cell surface expression through endosomal trafficking (8). However, CLC-2 is ubiquitously expressed, whereas CLC-5 is predominantly expressed in the kidney and extrapolations between these two situations may require cautious interpretation. In the kidney, CLC-5 plays an obligate role in facilitating albumin uptake by the proximal tubule (7, 47), where the cell surface availability of CLC-5 could be predicted to be a rate-limiting step in albumin uptake. Indeed, the marked reduction of albumin uptake by mPTCs of CLC-5-deficient mice (Fig. 6C) is consistent with an important role for CLC-5 in albumin endocytosis. However, the polarized mPTCs from CLC-5-deficient mice are capable of albumin uptake, although at a reduced level. This residual albumin endocytosis is unlikely to be due to compensation by CLC-4, since previous experiments in CLC-5-deficient mPTCs have shown no further reduction in albumin uptake after removal of Cl− ions and inhibition of endosomal acidification (56). This indicates the presence of an alternate CLC-5-independent albumin uptake pathway in mPTCs, whose activity could be altered by KIF3B expression (Fig. 6C). Such an alternative albumin uptake pathway may involve caveolin-1 and caveolin-2, which facilitate albumin transcytosis (29, 38). Moreover, it has been suggested that caveolar transport involves the Kinesin-2 complex (41) and this may provide a possible explanation for the observed reduction in albumin uptake in the mPTCs from the Clcn5Y/− mice (Fig. 6C), although it is important to note that caveolae are not present at the apical surface of renal epithelial cells (53). However, movement of caveolin-1, which is expressed at the apical membrane of renal epithelial cells (36), is disrupted and ceases following treatment with nocodazole (41) and this is similar to our findings of CLC-5 vesicle disruption following nocodazole treatment. In this endocytic process, the Kinesin-2 complex, which consists of KIF3A, KIF3B, and KAP3 that are all expressed in renal epithelial cells, works as a motor for anterograde transport along the microtubules toward their plus ends (17, 21, 60, 61). In renal epithelial cells, such as OK cells, the microtubules are organized longitudinally with the plus end pointing toward the basolateral surface (Fig. 4A) (17, 21). Thus, vesicles that form part of the megalin-cubilin receptor-mediated endocytic pathway (4, 7) and contain CLC-5, via its interaction with KIF3B, will be transported away from the apical membrane and toward the basolateral membrane (Figs. 4A and 7). In our proposed working model for the role of the CLC-5 and KIF3B interaction in endosomal transport, CLC-5 mutations leading to a loss of the CLC-5 and KIF3B interaction will result in a defective microtubular transport of the endocytic vesicles that contain megalin and cubilin, and this in turn will lead to the resorptive abnormalities of solutes and proteins observed in Dent's disease (4, 47).


CLC-5 and KIF3B interact to facilitate CLC-5 plasma membrane expression, endocytosis, and microtubular transport: relevance to pathophysiology of Dent's disease.

Reed AA, Loh NY, Terryn S, Lippiat JD, Partridge C, Galvanovskis J, Williams SE, Jouret F, Wu FT, Courtoy PJ, Nesbit MA, Rorsman P, Devuyst O, Ashcroft FM, Thakker RV - Am. J. Physiol. Renal Physiol. (2009)

Schematic representation of a working model for endosomal transport involving CLC-5 and KIF3B interaction in polarized cells. CLC-5 and KIF3B are expressed in polarized renal proximal tubular cells which reabsorb proteins and solutes via the receptor-mediated endocytic pathway that uses megalin and cubilin as the apical plasma membrane (APM) receptors (4, 7, 17). Ligands bind to these receptors at the APM where CLC-5 and interacting proteins Nedd4-2, NHERF2, and cofilin are also present (23, 24, 26). The receptor-ligand complex is internalized in vesicles, containing CLC-5 and CLC-4 (40), which are anterogradely transported along microtubules via the Kinesin-2 complex (17). These vesicles fuse with early endosomes where acidification involves the vacuolar H+-ATPase, with counter ions being provided by CLC-5 (28). In nonpolarized cells, the orientation of the microtubules is reversed (Fig. 3F), and CLC-5 vesicles would be transported to the cell surface.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2822520&req=5

Figure 7: Schematic representation of a working model for endosomal transport involving CLC-5 and KIF3B interaction in polarized cells. CLC-5 and KIF3B are expressed in polarized renal proximal tubular cells which reabsorb proteins and solutes via the receptor-mediated endocytic pathway that uses megalin and cubilin as the apical plasma membrane (APM) receptors (4, 7, 17). Ligands bind to these receptors at the APM where CLC-5 and interacting proteins Nedd4-2, NHERF2, and cofilin are also present (23, 24, 26). The receptor-ligand complex is internalized in vesicles, containing CLC-5 and CLC-4 (40), which are anterogradely transported along microtubules via the Kinesin-2 complex (17). These vesicles fuse with early endosomes where acidification involves the vacuolar H+-ATPase, with counter ions being provided by CLC-5 (28). In nonpolarized cells, the orientation of the microtubules is reversed (Fig. 3F), and CLC-5 vesicles would be transported to the cell surface.
Mentions: Our results establish an interaction between CLC-5 and KIF3B that is important for the plasma membrane expression of CLC-5, microtubular transport of CLC-5 vesicles, and for albumin uptake in polarized and nonpolarized cells. Thus, in polarized epithelial cells such as OK cells, the interaction between CLC-5 and KIF3B results in a reduction in the number of antiporters at the cell surface, as KIF3B is involved in facilitating microtubular transport that removes CLC-5-containing vesicles from the cell surface (Fig. 7). In nonpolarized cells, such as HEK293 cells, the orientation of the microtubules is reversed (Fig. 3F), and the interaction between CLC-5 and KIF3B facilitates transport of CLC-5-containing vesicles to the cell surface. This situation may be analogous to that reported for the interaction between CLC-2 and the microtubule-associated dynein motor complex, which is also involved in regulating CLC-2 cell surface expression through endosomal trafficking (8). However, CLC-2 is ubiquitously expressed, whereas CLC-5 is predominantly expressed in the kidney and extrapolations between these two situations may require cautious interpretation. In the kidney, CLC-5 plays an obligate role in facilitating albumin uptake by the proximal tubule (7, 47), where the cell surface availability of CLC-5 could be predicted to be a rate-limiting step in albumin uptake. Indeed, the marked reduction of albumin uptake by mPTCs of CLC-5-deficient mice (Fig. 6C) is consistent with an important role for CLC-5 in albumin endocytosis. However, the polarized mPTCs from CLC-5-deficient mice are capable of albumin uptake, although at a reduced level. This residual albumin endocytosis is unlikely to be due to compensation by CLC-4, since previous experiments in CLC-5-deficient mPTCs have shown no further reduction in albumin uptake after removal of Cl− ions and inhibition of endosomal acidification (56). This indicates the presence of an alternate CLC-5-independent albumin uptake pathway in mPTCs, whose activity could be altered by KIF3B expression (Fig. 6C). Such an alternative albumin uptake pathway may involve caveolin-1 and caveolin-2, which facilitate albumin transcytosis (29, 38). Moreover, it has been suggested that caveolar transport involves the Kinesin-2 complex (41) and this may provide a possible explanation for the observed reduction in albumin uptake in the mPTCs from the Clcn5Y/− mice (Fig. 6C), although it is important to note that caveolae are not present at the apical surface of renal epithelial cells (53). However, movement of caveolin-1, which is expressed at the apical membrane of renal epithelial cells (36), is disrupted and ceases following treatment with nocodazole (41) and this is similar to our findings of CLC-5 vesicle disruption following nocodazole treatment. In this endocytic process, the Kinesin-2 complex, which consists of KIF3A, KIF3B, and KAP3 that are all expressed in renal epithelial cells, works as a motor for anterograde transport along the microtubules toward their plus ends (17, 21, 60, 61). In renal epithelial cells, such as OK cells, the microtubules are organized longitudinally with the plus end pointing toward the basolateral surface (Fig. 4A) (17, 21). Thus, vesicles that form part of the megalin-cubilin receptor-mediated endocytic pathway (4, 7) and contain CLC-5, via its interaction with KIF3B, will be transported away from the apical membrane and toward the basolateral membrane (Figs. 4A and 7). In our proposed working model for the role of the CLC-5 and KIF3B interaction in endosomal transport, CLC-5 mutations leading to a loss of the CLC-5 and KIF3B interaction will result in a defective microtubular transport of the endocytic vesicles that contain megalin and cubilin, and this in turn will lead to the resorptive abnormalities of solutes and proteins observed in Dent's disease (4, 47).

Bottom Line: Confocal live cell imaging in kidney cells further demonstrated association of CLC-5 and KIF3B, and transport of CLC-5-containing vesicles along KIF3B microtubules.KIF3B overexpression and underexpression, using siRNA, had reciprocal effects on whole cell chloride current amplitudes, CLC-5 cell surface expression, and endocytosis of albumin and transferrin.Thus, the CLC-5 and KIF3B interaction is important for CLC-5 plasma membrane expression and for facilitating endocytosis and microtubular transport in the kidney.

View Article: PubMed Central - PubMed

Affiliation: Academic Endocrine Unit, Nuffield Department of Medicine, University of Oxford, Oxford Centre for Diabetes, Endocrinology, and Metabolism, Churchill Hospital, Oxford, United Kingdom.

ABSTRACT
Renal tubular reabsorption is important for extracellular fluid homeostasis and much of this occurs via the receptor-mediated endocytic pathway. This pathway is disrupted in Dent's disease, an X-linked renal tubular disorder that is characterized by low-molecular-weight proteinuria, hypercalciuria, nephrolithiasis, and renal failure. Dent's disease is due to mutations of CLC-5, a chloride/proton antiporter, expressed in endosomes and apical membranes of renal tubules. Loss of CLC-5 function alters receptor-mediated endocytosis and trafficking of megalin and cubilin, although the underlying mechanisms remain to be elucidated. Here, we report that CLC-5 interacts with kinesin family member 3B (KIF3B), a heterotrimeric motor protein that facilitates fast anterograde translocation of membranous organelles. Using yeast two-hybrid, glutathione-S-transferase pull-down and coimmunoprecipitation assays, the COOH terminus of CLC-5 and the coiled-coil and globular domains of KIF3B were shown to interact. This was confirmed in vivo by endogenous coimmunoprecipitation of CLC-5 and KIF3B and codistribution with endosomal markers in mouse kidney fractions. Confocal live cell imaging in kidney cells further demonstrated association of CLC-5 and KIF3B, and transport of CLC-5-containing vesicles along KIF3B microtubules. KIF3B overexpression and underexpression, using siRNA, had reciprocal effects on whole cell chloride current amplitudes, CLC-5 cell surface expression, and endocytosis of albumin and transferrin. Clcn5(Y/-) mouse kidneys and isolated proximal tubular polarized cells showed increased KIF3B expression, whose effects on albumin endocytosis were dependent on CLC-5 expression. Thus, the CLC-5 and KIF3B interaction is important for CLC-5 plasma membrane expression and for facilitating endocytosis and microtubular transport in the kidney.

Show MeSH
Related in: MedlinePlus