J Biol Chem. 2009 Dec 4;284(49):34400-12. doi: 10.1074/jbc.M109.015149. Epub 2009 Oct 7.
A pathogenic C terminus-truncated polycystin-2 mutant enhances receptor-activated Ca2+ entry via association with TRPC3 and TRPC7.
Miyagi, K., Kiyonaka, S., Yamada, K., Miki, T., Mori, E., Kato, K., Numata, T., Sawaguchi, Y., Numaga, T., Kimura, T., Kanai, Y., Kawano, M., Wakamori, M., Nomura, H., Koni, I., Yamagishi, M., Mori, Y.,
["Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Japan."]
["Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Japan."]
Mutations in PKD2 gene result in autosomal dominant polycystic kidney disease (ADPKD). PKD2 encodes polycystin-2 (TRPP2), which is a homologue of transient receptor potential (TRP) cation channel proteins. Here we identify a novel PKD2 mutation that generates a C-terminal tail-truncated TRPP2 mutant 697fsX with a frameshift resulting in an aberrant 17-amino acid addition after glutamic acid residue 697 from a family showing mild ADPKD symptoms. When recombinantly expressed in HEK293 cells, wild-type (WT) TRPP2 localized at the endoplasmic reticulum (ER) membrane significantly enhanced Ca(2+) release from the ER upon muscarinic acetylcholine receptor (mAChR) stimulation. In contrast, 697fsX, which showed a predominant plasma membrane localization characteristic of TRPP2 mutants with C terminus deletion, prominently increased mAChR-activated Ca(2+) influx in cells expressing TRPC3 or TRPC7. Coimmunoprecipitation, pulldown assay, and cross-linking experiments revealed a physical association between 697fsX and TRPC3 or TRPC7. 697fsX but not WT TRPP2 elicited a depolarizing shift of reversal potentials and an enhancement of single-channel conductance indicative of altered ion-permeating pore properties of mAChR-activated currents. Importantly, in kidney epithelial LLC-PK1 cells the recombinant 679fsX construct was codistributed with native TRPC3 proteins at the apical membrane area, but the WT construct was distributed in the basolateral membrane and adjacent intracellular areas. Our results suggest that heteromeric cation channels comprised of the TRPP2 mutant and the TRPC3 or TRPC7 protein induce enhanced receptor-activated Ca(2+) influx that may lead to dysregulated cell growth in ADPKD.
PMID: 19812035

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Screening
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Experimental screening | Non-experimental screening | Reference | ||||||||
TRP channel construct | Interactor source | |||||||||
TRP channel | Interactor | Method | Species | Region | Species | Organ/tissue | Sample type | |||
TRPC3 |
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TRPP1 | Inference | Prediction | 19812035 | |||||
TRPC7 |
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TRPP1 | Inference | Prediction | 19812035 | |||||
TRPP1 |
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TRPC3 | Inference | Prediction | 19812035 | |||||
TRPP1 |
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TRPC7 | Inference | Prediction | 19812035 |
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click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)

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Validation: In vivo validation
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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 | ||||
TRPC1 |
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TRPP1 | Co-immunoprecipitation | HEK293 | Mouse | Full-length | Human | Full-length | 19812035 | |
TRPC3 |
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TRPP1 | Co-immunoprecipitation | HEK293 | Mouse | Full-length | Human | 1-697 | 19812035 | |
TRPC3 |
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TRPP1 | Co-immunofluorescence staining | LLC-PK1 | Not used | Human | 1-697 | 19812035 | ||
TRPC7 |
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TRPP1 | Co-immunoprecipitation | HEK293 | Mouse | Full-length | Human | 1-697 | 19812035 | |
TRPP1 |
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TRPC1 | Co-immunoprecipitation | HEK293 | Human | Full-length | Mouse | Full-length | 19812035 | |
TRPP1 |
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TRPC3 | Co-immunoprecipitation | HEK293 | Human | 1-697 | Mouse | Full-length | 19812035 | |
TRPP1 |
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TRPC3 | Co-immunofluorescence staining | LLC-PK1 | Human | 1-697 | Not used | 19812035 | ||
TRPP1 |
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TRPC7 | Co-immunoprecipitation | HEK293 | Human | 1-697 | Mouse | Full-length | 19812035 |
(
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click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)

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Characterization
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Binding region mapping | Stoichiometry | Affinity (Kd) | Reference | |||||||
TRP channel | Interactor | |||||||||
TRP channel | Interactor | Method | Species | Region | Species | Region | ||||
TRPC3 |
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TRPP1 | Fusion protein-pull down assay | Mouse | 659-753 | Human | 1-697 | 19812035 | ||
TRPP1 |
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TRPC3 | Fusion protein-pull down assay | Human | 1-697 | Mouse | 659-753 | 19812035 |
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click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)

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Functional consequence
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TRP channel | Interactor | Method | Post-translational modification | Subcellular trafficking | Activity | Reference | ||||||
TRPC3 |
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TRPP1 | Calcium measurement | Activation | 19812035 | |||||||
TRPC7 |
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TRPP1 | Calcium measurement | Activation | 19812035 |
(
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click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
