Endocrine Abstracts

Endocytosis involves a CLC-5 and KIF3B interaction: relevance to thyroid and renal tubular function

Anita AC Reed¹, Nellie Loh¹, Jonathan Lippiat², Chris Partridge¹, Juris Galvanovskis¹, Sian Williams¹, Francois Jouret³, Fiona Wu¹, Pierre Courtoy³, M Andrew Nesbit¹, Olivier Devuyst³, Patrik Rorsman¹, Frances Ashcroft¹ & Rajesh V Thakker¹

¹University of Oxford, Oxford, UK; ²University of Leeds, Leeds, UK; ³University Catholique de Louvain Medical School, Brussels, Belgium.

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Endocrine cells utilise endocytosis to scavenge polypeptide hormones, and to generate signals from inactive precursors, such as release of thyroid hormones from thyroglobulin in thyrocytes, and activation of vitamin D after reabsorption of ultrafiltrated pro-vitamin D binding protein complex by renal proximal tubular cells (PTC). We have investigated the role of CLC-5, a chloride/proton antiporter expressed in apical endosomes and the apical plasma membrane of thyrocytes and PTC. The critical role of CLC-5 in these tissues is emphasised by two observations: (i) CLCN5-deficient mice develop goitre with iodine and thyroglobulin accumulation; and (ii) patients with inactivating CLC-5 mutations develop Dent’s disease, an X-linked renal tubular disorder, characterised by low-molecular-weight-proteinuria, hypercalciuria, nephrolithiasis, renal failure and rickets. In order to further elucidate the mechanisms underlying CLC-5 endosomal function, we searched for interacting proteins by performing a yeast two-hybrid screen with the cytoplasmic C-terminal tail of CLC-5 that contains two cystathione-β synthase domains, which are likely to mediate protein–protein interactions. This isolated Kinesin Family Member 3B (KIF3B), a heterotrimeric motor protein for microtubule-based fast anterograde translocation of membranous organelles. Using yeast two-hybrid, GST pull-down and co-immunoprecipitation assays, the C-terminus of CLC-5 and the coiled-coil and globular domains of KIF3B were shown to interact. This was confirmed in vivo by co-immunoprecipitation of endogenous CLC-5 with KIF3B and their co-distribution with endosomal markers in mouse kidneys. Confocal live-cell imaging in kidney cells further demonstrated the association between CLC-5 and KIF3B, and the transport of CLC-5-containing vesicles along KIF3B-labeled microtubules. KIF3B over-expression increased (whilst KIF3B siRNA decreased) whole-cell chloride current amplitudes, CLC-5 cell surface expression, and albumin endocytosis. Thus, our findings have demonstrated that the interaction between CLC-5 and KIF3B is important for CLC-5 apical surface expression, receptor-mediated endocytosis and microtubular transport of CLC-5 containing vesicles.

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Endocrine Abstracts (2008) 15 OC37