CLC-5 and KIF3B interact to facilitate CLC-5 plasma membrane expression, endocytosis, and microtubular transport: relevance to pathophysiology of Dent's disease.
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.
Reed
Anita A C
AA
Academic Endocrine Unit, Nuffield Department of Medicine, University of Oxford, Oxford Centre for Diabetes, Endocrinology, and Metabolism, Churchill Hospital, Oxford, United Kingdom.
Loh
Nellie Y
NY
Terryn
Sara
S
Lippiat
Jonathan D
JD
Partridge
Chris
C
Galvanovskis
Juris
J
Williams
Siân E
SE
Jouret
Francois
F
Wu
Fiona T F
FT
Courtoy
Pierre J
PJ
Nesbit
M Andrew
MA
Rorsman
Patrik
P
Devuyst
Olivier
O
Ashcroft
Frances M
FM
Thakker
Rajesh V
RV
D000328:Adult; D000418:Albumins / Q000378:metabolism; D000818:Animals; D019556:COS Cells; D002460:Cell Line; D018118:Chloride Channels / Q000378:metabolism* / Q000502:physiology; D002522:Chlorocebus aethiops; D018076:DNA, Complementary; D015536:Down-Regulation; D004347:Drug Interactions; D004553:Electric Conductivity; D004705:Endocytosis / Q000502:physiology*; D015723:Gene Library; D040181:Genetic Diseases, X-Linked / Q000503:physiopathology; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000378:metabolism*; D007674:Kidney Diseases / Q000503:physiopathology; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000378:metabolism; D016547:Kinesins / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D008870:Microtubules / Q000378:metabolism*; D054730:Protein Interaction Domains and Motifs; D021381:Protein Transport; D020798:Two-Hybrid System Techniques; D015854:Up-Regulation
D000418:Albumins; C097503:CLC-5 chloride channel; D018118:Chloride Channels; D018076:DNA, Complementary; C095899:KIF3B protein, human; C490672:Kif3b protein, mouse; D016547:Kinesins