J Biol Chem. 2006 Dec 8;281(49):37566-75. Epub 2006 Sep 1.

Polycystin-2 cation channel function is under the control of microtubular structures in primary cilia of renal epithelial cells.External 2231691f894ba696de1310221b0a0dbbb31a7251e75115c265587c3d9d5f507c

Li, Q., Montalbetti, N., Wu, Y., Ramos, A., Raychowdhury, M. K., Chen, X. Z., Cantiello, H. F.,
["Department of Physiology, University of Alberta, Edmonton, Alberta T6G 2H7, Canada."]
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.
PMID: 16950792External 2231691f894ba696de1310221b0a0dbbb31a7251e75115c265587c3d9d5f507c
Screening Toggle 893349bafcc528f8346c51dc3420151d67b0126b2c122dd1017121c03fa0f69b
  Experimental screening Non-experimental screening Reference
TRP channel construct Interactor source
TRP channel Interactor Method Species Region Species Organ/tissue Sample type
TRPP1 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 KIF3A Inference Prediction 16950792
TRPP1 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 KIF3B Yeast two-hybrid Human 682-968 Human Heart cDNA library 16950792
(Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4: click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
Validation: In vitro validation Toggle 893349bafcc528f8346c51dc3420151d67b0126b2c122dd1017121c03fa0f69b
  Assay with recombinant proteins Reference
TRP channel construct Interactor construct
TRP channel Interactor Method Species Region Expression system Species Region Expression system
TRPP1 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 KIF3A Yeast two-hybrid Human 682-968 Yeast Human Full-length Yeast 16950792
TRPP1 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 KIF3A Fusion protein-pull down assay Human 682-968 E. coli Human Full-length In vitro translation 16950792
(Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4: click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
Validation: In vivo validation Toggle 893349bafcc528f8346c51dc3420151d67b0126b2c122dd1017121c03fa0f69b
  Assay with endogenous proteins Assay with overexpressed proteins Reference
Cell or tissue Cell or tissue TRP channel construct Interactor construct
TRP channel Interactor Method Species Region Species Region
TRPP1 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 KIF3A Co-immunofluorescence staining Mouse Inner medullary collecting duct cell 16950792
TRPP1 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 KIF3A Co-immunofluorescence staining MDCK 16950792
TRPP1 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 KIF3A Co-immunofluorescence staining LLC-PK1 16950792
(Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4: click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
Characterization Toggle 893349bafcc528f8346c51dc3420151d67b0126b2c122dd1017121c03fa0f69b
  Binding region mapping Stoichiometry Affinity (Kd) Reference
TRP channel Interactor
TRP channel Interactor Method Species Region Species Region
TRPP1 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 KIF3A Yeast two-hybrid Human 872-927 Human 403-702 16950792
(Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4: click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
Functional consequence Toggle 893349bafcc528f8346c51dc3420151d67b0126b2c122dd1017121c03fa0f69b
TRP channel Interactor Method Post-translational modification Subcellular trafficking Activity Reference
TRPP1 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 KIF3A Patch clamp Activation 16950792
(Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4: click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
TRP / Interactor

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