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

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. Clcn5Y/− 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.

Receptor-mediated endocytosis and endosomal acidification is impaired in proximal tubule epithelial cells of Dent disease patients

“…Proximal tubular cells, which are polarized, facilitate endocytosis of proteins at the apical brush border membrane. The apical endocytosed proteins are then transferred, which is most probably mediated by anterograde microtubule transport, to lysosomes for degradation (3)(4)(5). The anterograde transport is facilitated by orientation of the microtubules such that their minus ends are toward the apical side and their plus ends, which have the microtubule associated protein end-binding protein-1 (EB-1), are extending through the cell to the basolateral side (4, 23).…”

“…Sorted cells were subcloned and treated with CD13-FITC before investigation by flow cytometry (19). DNA sequence analysis of the CLCN5 coding region exons and intron-exon boundaries, and Western blot and RT-PCR analysis of nephron segment specific genes and proteins was performed, as described (4,9). For Z-stack imaging, cells were fixed and coimmunostained with anti-ZO-1 and anti-microtubule associated protein, RB/EB family, member-1 (MAPRE1) (EB-1), and secondary antibodies anti-rabbit Alexa Fluor 488 and anti-mouse Alexa Fluor 594, respectively (4).…”

Impaired Lysosomal Function Underlies Monoclonal Light Chain–Associated Renal Fanconi Syndrome

“…These cells, which keep their differentiation and polarized transport processes, provide a particularly well suited model to investigate receptor-mediated endocytosis of apical ligands and their lysosomal processing. 13,22,23 We show that specific RFS-kLCs accumulate within lysosomes of PT cells, causing marked alterations in cell dynamics and lysosomal function. In turn, these alterations promote dedifferentiation of the cells and defective receptor-mediated endocytosis, sustaining the loss of reabsorptive capacity of the kidney tubule.…”

Thyroid-specific inactivation of KIF3A alters the TSH signaling pathway and leads to hypothyroidism

“…In MEF in the absence of any Pax8 gene alteration, our results clearly indicate that Kif3a inactivation leads to significantly lower β2 adrenergic receptor expression at the cell surface and, as a consequence, to markedly decreased downstream signaling in response to agonist stimulation. The kinesin 2 molecular motor is known to participate to "non intraflagellar" transport in the cell, like retrograde traffic between the Golgi and the endoplasmic reticulum (ER) (Stauber et al 2006), endosome and lysosome transports (Bananis et al 2004;Brown et al 2005), endocytosis and recycling of cell surface receptors like transferrin, cubulin and megalin receptors, or of other proteins like Clc-5, the H + /Cl − exchange transporter (Schonteich et al 2008;Reed et al 2010). The KIF3 molecular motor also plays an important role in the transport of vesicles containing GluR2 and GLUT4 receptors or MT1-MMP and MMP-9 metalloproteinases from the cytosol to the plasma membrane (Imamura et al 2003;Wiesner et al 2010;Hanania et al 2012;Lin et al 2012).…”