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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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Interactions between CLC-5 and KIF3B subunits. A: CLC-5 consists of 18 helices (A to R) with the NH2- and COOH-terminus (C-term) domains being cytoplasmic (membrane boundaries indicated by arrows) (10, 58). B: yeast 2-hybrid using the CLC-5 C-term, which contains 2 CBS domains (11) to screen a renal cDNA library, identified KIF3B as a putative interactor. Interaction between large T and p53 provided a positive control. C: 35S-methionine-labeled CLC-5 C-term was incubated with GST-tagged KIF3B coiled coil (CC)/globular domain (GD). Resin-bound proteins were resolved by SDS-PAGE and detected by autoradiography. Results from 2 independent in vitro translation experiments using 2 different GST-tagged KIF3B clones are shown. The larger band at ∼24.5 kDa represents the 216 amino acid Myc-tagged CLC-5 C-term, whereas the smaller band at ∼18.5 kDa likely represents another translated form of CLC-5 C-term that originated from use of an alternative methionine at codon 583 and hence lacked the NH2 terminal 32 amino acids. This smaller CLC-5 C-term, which was less efficiently translated, had both CBS domains and hence was pulled down by the GST-tagged KIF3B CC/GD. D: endogenous coimmunoprecipitation using wild-type mouse whole kidney extracts and a KIF3B monoclonal antibody (mAb). KIF3B, KIF3A, and KAP3 mAb were used to confirm endogenous coimmunoprecipitation of these proteins which form a trimeric complex. It is important to note that the interaction between CLC-5 and KIF3B is likely to be transient, as endosomes will be released to continue recycling, and that only a small proportion of the total cellular CLC-5 will be present, at a given time point, on the surface of endosomes that are being trafficked anterogradely; these features are consistent with the relatively low amount of KIF3B that is immunopreciptiated by CLC-5. E: coimmunoprecipitation studies using HEK293 cells demonstrated interactions between CLC-5 and KIF3B domains. Lane 1: input; lanes 2, 3, and 4: pull-down with no antibody, anti-Myc, or anti-HA antibodies, respectively. F: summary of KIF3B domains showing presence (+) or absence (−) of interactions with CLC-5. G: coimmunoprecipitation studies using HEK293 cells demonstrated interactions between CLC-4 and KIF3B, and CLC-7 and KIF3B. Lane 1: input; lanes 2, 3, and 4: pull-down with no antibody, anti-Myc, or anti-HA antibodies, respectively.
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Figure 1: Interactions between CLC-5 and KIF3B subunits. A: CLC-5 consists of 18 helices (A to R) with the NH2- and COOH-terminus (C-term) domains being cytoplasmic (membrane boundaries indicated by arrows) (10, 58). B: yeast 2-hybrid using the CLC-5 C-term, which contains 2 CBS domains (11) to screen a renal cDNA library, identified KIF3B as a putative interactor. Interaction between large T and p53 provided a positive control. C: 35S-methionine-labeled CLC-5 C-term was incubated with GST-tagged KIF3B coiled coil (CC)/globular domain (GD). Resin-bound proteins were resolved by SDS-PAGE and detected by autoradiography. Results from 2 independent in vitro translation experiments using 2 different GST-tagged KIF3B clones are shown. The larger band at ∼24.5 kDa represents the 216 amino acid Myc-tagged CLC-5 C-term, whereas the smaller band at ∼18.5 kDa likely represents another translated form of CLC-5 C-term that originated from use of an alternative methionine at codon 583 and hence lacked the NH2 terminal 32 amino acids. This smaller CLC-5 C-term, which was less efficiently translated, had both CBS domains and hence was pulled down by the GST-tagged KIF3B CC/GD. D: endogenous coimmunoprecipitation using wild-type mouse whole kidney extracts and a KIF3B monoclonal antibody (mAb). KIF3B, KIF3A, and KAP3 mAb were used to confirm endogenous coimmunoprecipitation of these proteins which form a trimeric complex. It is important to note that the interaction between CLC-5 and KIF3B is likely to be transient, as endosomes will be released to continue recycling, and that only a small proportion of the total cellular CLC-5 will be present, at a given time point, on the surface of endosomes that are being trafficked anterogradely; these features are consistent with the relatively low amount of KIF3B that is immunopreciptiated by CLC-5. E: coimmunoprecipitation studies using HEK293 cells demonstrated interactions between CLC-5 and KIF3B domains. Lane 1: input; lanes 2, 3, and 4: pull-down with no antibody, anti-Myc, or anti-HA antibodies, respectively. F: summary of KIF3B domains showing presence (+) or absence (−) of interactions with CLC-5. G: coimmunoprecipitation studies using HEK293 cells demonstrated interactions between CLC-4 and KIF3B, and CLC-7 and KIF3B. Lane 1: input; lanes 2, 3, and 4: pull-down with no antibody, anti-Myc, or anti-HA antibodies, respectively.

Mentions: The role of CLC-5 in endosomal trafficking remains to be established. Loss of CLC-5 function leads to impaired endosomal acidification which may contribute to the observed defects in endosomal trafficking (4). Interestingly, pharmacological inhibition of vacuolar acidification has been reported to decrease endosomal recycling and transfer to lysosomes (1) but not the rate of endocytosis, consistent with a trafficking defect (6, 52). Any possible role of CLC-5 in endosomal transport is likely to involve the CLC-5 COOH terminus (amino acids 551 to 746; Fig. 1A) as this has been shown to be cytoplasmic by an analysis of the three-dimensional structures of homologous bacterial CLCs (10, 58). Moreover, the COOH terminus contains potential binding motifs for regulatory molecules: these include two cystathionine-β-synthase (CBS1 and CBS2) domains, a PY motif (PPLPPY), and a putative PDZ-binding motif (11, 25, 49). Indeed, a number of proteins, including CLC-4, cofilin, WWP2, Nedd4-2, and Na+-H+ exchanger regulatory factor 2 (NHERF2), have been reported to interact with CLC-5.


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)

Interactions between CLC-5 and KIF3B subunits. A: CLC-5 consists of 18 helices (A to R) with the NH2- and COOH-terminus (C-term) domains being cytoplasmic (membrane boundaries indicated by arrows) (10, 58). B: yeast 2-hybrid using the CLC-5 C-term, which contains 2 CBS domains (11) to screen a renal cDNA library, identified KIF3B as a putative interactor. Interaction between large T and p53 provided a positive control. C: 35S-methionine-labeled CLC-5 C-term was incubated with GST-tagged KIF3B coiled coil (CC)/globular domain (GD). Resin-bound proteins were resolved by SDS-PAGE and detected by autoradiography. Results from 2 independent in vitro translation experiments using 2 different GST-tagged KIF3B clones are shown. The larger band at ∼24.5 kDa represents the 216 amino acid Myc-tagged CLC-5 C-term, whereas the smaller band at ∼18.5 kDa likely represents another translated form of CLC-5 C-term that originated from use of an alternative methionine at codon 583 and hence lacked the NH2 terminal 32 amino acids. This smaller CLC-5 C-term, which was less efficiently translated, had both CBS domains and hence was pulled down by the GST-tagged KIF3B CC/GD. D: endogenous coimmunoprecipitation using wild-type mouse whole kidney extracts and a KIF3B monoclonal antibody (mAb). KIF3B, KIF3A, and KAP3 mAb were used to confirm endogenous coimmunoprecipitation of these proteins which form a trimeric complex. It is important to note that the interaction between CLC-5 and KIF3B is likely to be transient, as endosomes will be released to continue recycling, and that only a small proportion of the total cellular CLC-5 will be present, at a given time point, on the surface of endosomes that are being trafficked anterogradely; these features are consistent with the relatively low amount of KIF3B that is immunopreciptiated by CLC-5. E: coimmunoprecipitation studies using HEK293 cells demonstrated interactions between CLC-5 and KIF3B domains. Lane 1: input; lanes 2, 3, and 4: pull-down with no antibody, anti-Myc, or anti-HA antibodies, respectively. F: summary of KIF3B domains showing presence (+) or absence (−) of interactions with CLC-5. G: coimmunoprecipitation studies using HEK293 cells demonstrated interactions between CLC-4 and KIF3B, and CLC-7 and KIF3B. Lane 1: input; lanes 2, 3, and 4: pull-down with no antibody, anti-Myc, or anti-HA antibodies, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Interactions between CLC-5 and KIF3B subunits. A: CLC-5 consists of 18 helices (A to R) with the NH2- and COOH-terminus (C-term) domains being cytoplasmic (membrane boundaries indicated by arrows) (10, 58). B: yeast 2-hybrid using the CLC-5 C-term, which contains 2 CBS domains (11) to screen a renal cDNA library, identified KIF3B as a putative interactor. Interaction between large T and p53 provided a positive control. C: 35S-methionine-labeled CLC-5 C-term was incubated with GST-tagged KIF3B coiled coil (CC)/globular domain (GD). Resin-bound proteins were resolved by SDS-PAGE and detected by autoradiography. Results from 2 independent in vitro translation experiments using 2 different GST-tagged KIF3B clones are shown. The larger band at ∼24.5 kDa represents the 216 amino acid Myc-tagged CLC-5 C-term, whereas the smaller band at ∼18.5 kDa likely represents another translated form of CLC-5 C-term that originated from use of an alternative methionine at codon 583 and hence lacked the NH2 terminal 32 amino acids. This smaller CLC-5 C-term, which was less efficiently translated, had both CBS domains and hence was pulled down by the GST-tagged KIF3B CC/GD. D: endogenous coimmunoprecipitation using wild-type mouse whole kidney extracts and a KIF3B monoclonal antibody (mAb). KIF3B, KIF3A, and KAP3 mAb were used to confirm endogenous coimmunoprecipitation of these proteins which form a trimeric complex. It is important to note that the interaction between CLC-5 and KIF3B is likely to be transient, as endosomes will be released to continue recycling, and that only a small proportion of the total cellular CLC-5 will be present, at a given time point, on the surface of endosomes that are being trafficked anterogradely; these features are consistent with the relatively low amount of KIF3B that is immunopreciptiated by CLC-5. E: coimmunoprecipitation studies using HEK293 cells demonstrated interactions between CLC-5 and KIF3B domains. Lane 1: input; lanes 2, 3, and 4: pull-down with no antibody, anti-Myc, or anti-HA antibodies, respectively. F: summary of KIF3B domains showing presence (+) or absence (−) of interactions with CLC-5. G: coimmunoprecipitation studies using HEK293 cells demonstrated interactions between CLC-4 and KIF3B, and CLC-7 and KIF3B. Lane 1: input; lanes 2, 3, and 4: pull-down with no antibody, anti-Myc, or anti-HA antibodies, respectively.
Mentions: The role of CLC-5 in endosomal trafficking remains to be established. Loss of CLC-5 function leads to impaired endosomal acidification which may contribute to the observed defects in endosomal trafficking (4). Interestingly, pharmacological inhibition of vacuolar acidification has been reported to decrease endosomal recycling and transfer to lysosomes (1) but not the rate of endocytosis, consistent with a trafficking defect (6, 52). Any possible role of CLC-5 in endosomal transport is likely to involve the CLC-5 COOH terminus (amino acids 551 to 746; Fig. 1A) as this has been shown to be cytoplasmic by an analysis of the three-dimensional structures of homologous bacterial CLCs (10, 58). Moreover, the COOH terminus contains potential binding motifs for regulatory molecules: these include two cystathionine-β-synthase (CBS1 and CBS2) domains, a PY motif (PPLPPY), and a putative PDZ-binding motif (11, 25, 49). Indeed, a number of proteins, including CLC-4, cofilin, WWP2, Nedd4-2, and Na+-H+ exchanger regulatory factor 2 (NHERF2), have been reported to interact with CLC-5.

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