SFEBES2008 Oral Communications Thyroid (8 abstracts)
Endocytosis involves a CLC-5 and KIF3B interaction: relevance to thyroid and renal tubular function
Anita AC Reed 1 , Nellie Loh 1 , Jonathan Lippiat 2 , Chris Partridge 1 , Juris Galvanovskis 1 , Sian Williams 1 , Francois Jouret 3 , Fiona Wu 1 , Pierre Courtoy 3 , M Andrew Nesbit 1 , Olivier Devuyst 3 , Patrik Rorsman 1 , Frances Ashcroft 1 & Rajesh V Thakker 1
1University of Oxford, Oxford, UK; 2University of Leeds, Leeds, UK; 3University Catholique de Louvain Medical School, Brussels, Belgium.
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
ISSN 1470-3947 (print) | ISSN 1479-6848 (online)
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