Abstract
Mutations in the gene encoding polycystin-2 (PC2) result in autosomal dominant polycystic kidney disease and defects in left-right asymmetry during embryogenesis. PC2 is a TRP-type Ca(2+)-permeable non-selective cation channel, which is expressed in kidney and other organs. PC2 is present and functional in microtubule-containing primary cilia of renal epithelial cells. However, no information is yet available as to whether PC2 interacts with microtubules. Here, we assessed the role of microtubular dynamics in regulating PC2 channel function in primary cilia. Isolated ciliary membranes from LLC-PK1 epithelial cells were reconstituted in a lipid bilayer system. The acute addition of the microtubular disrupter colchicine (15 mum) rapidly abolished, whereas the addition of the microtubular stabilizer paclitaxel (taxol, 15 mum) increased ciliary PC2 channel activity. The further addition of alpha-tubulin plus GTP also stimulated PC2 channel activity in ciliary membranes. However, alpha-tubulin and GTP had no effect on in vitro translated PC2. Using the yeast two-hybrid assay, we found that PC2 interacts with the microtubule-dependent motor kinesin-2 subunit KIF3A, a protein involved in polycystic kidney disease. The interaction occurred through the carboxyl termini domain of both proteins, which was further confirmed by in vitro glutathione S-transferase pull-down and dot blot overlay assays. Co-immunoprecipitation experiments showed that PC2 and KIF3A are in the same complex in native HEK293, Madin-Darby canine kidney cells (MDCK), and LLC-PK1 cells. Immunofluorescent staining also showed substantial PC2 and KIF3A co-localization in primary cilia of renal epithelial cells. The data indicate that microtubular organization regulates PC2 function, which may explain, among others, the regulatory role of PC2 in the sensory function of primary cilia.
Highlights
Recent observations support the idea that autosomal dominant polycystic kidney disease cyst formation implicates specific structural/functional abnormalities in primary cilia of renal epithelial cells [8, 9]
Tubulin itself was incapable of regulating the isolated PC2 channel, suggesting that immediate anchoring proteins mediate this interaction. In search for such molecular linkage, we identified a physical interaction between PC2 and KIF3A and observed their colocalization in primary cilia of renal epithelial cells
Microtubular Regulation of PC2 Function from Primary Cilia— To assess a functional interaction between PC2 and microtubular structures, we explored the primary cilium of renal epithelial cells, where functional PC2 has recently been observed
Summary
Recent observations support the idea that autosomal dominant polycystic kidney disease cyst formation implicates specific structural/functional abnormalities in primary cilia of renal epithelial cells [8, 9]. Various forms of cystic kidney disease not directly linked to mutations in the PKD genes have been associated with microtubular-associated ciliary proteins [8, 13,14,15,16]. Fibrocystin, encoded by PKHD1, whose mutations lead to autosomal recessive polycystic kidney disease, is present in primary cilia of renal epithelia [12, 13, 16, 17]. The cpk gene, whose mutations cause renal cystic disease, encodes cystin [13], a novel protein that is expressed and co-localizes with polaris in primary cilia of renal epithelial cells. Data are the mean Ϯ S.E. of four experiments (p Ͻ 0.05)
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