Biochem Biophys Res Commun. 2007 Sep 21;361(2):433-8. Epub 2007 Jul 16.
PDZ domain-containing protein as a physiological modulator of TRPV6.
Kim, H. J., Yang, D. K., So, I.,
["Department of Physiology, Seoul National University College of Medicine, 28 Yongon-Dong, Chongno-Gu, Seoul 110-799, Republic of Korea."]
["Department of Physiology, Seoul National University College of Medicine, 28 Yongon-Dong, Chongno-Gu, Seoul 110-799, Republic of Korea."]
The epithelial Ca(2+) channel TRPV6 constitutes the apical Ca(2+) entry mechanism in active Ca(2+) transport in kidney and intestine, but little is known about regulation mechanism of TRPV6. We performed yeast two-hybrid screen with TRPV6 C-terminus since TRPV6 has PDZ (Post-synaptic density-95, Drosophila discs-large protein, Zonula occludens protein 1) binding motif at its C terminal end. As a result, we found that 4 PDZ domain-containing protein, PDZK2, interacts with TRPV6 through its fourth PDZ domain. Glutathione S-transferase pull-down assay shows that TRPV6 and PDZK2 directly interact and that TRPV6 C-terminal PDZ binding motif is primarily responsible for this interaction. Mutant Delta4 lacking last 4 amino acid EYQI did not interact with PDZK2. Heterologous overexpression of both TRPV6 and PDZK2 did not show any effect on the activation of TRPV6. On the other hand, the peak current amplitude of mutant Delta4 decreased compared with that of WT TRPV6. When introduced into HEK293 cells expressing TRPV6, PDZ binding motif peptide (EYQI) markedly reduced the peak current amplitude in divalent free (DVF) solution. Knocking down the endogenous PDZK2 of HEK293 cells by RNAi significantly decreased DVF current density. Taken together, we propose that PDZK2 is an essential TRPV6 interacting protein as a physiological modulator of TRPV6.
PMID: 17645868

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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 | |||
TRPV6 |
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NHERF4 | Yeast two-hybrid | Mouse | 575-727 | Mouse | Kidney | cDNA library | 17645868 |
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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 vitro validation
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Assay with recombinant proteins | Reference | |||||||||
TRP channel construct | Interactor construct | |||||||||
TRP channel | Interactor | Method | Species | Region | Expression system | Species | Region | Expression system | ||
TRPV6 |
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NHERF4 | Fusion protein-pull down assay | Mouse | 575-727 | E. coli | Mouse | Full-length | In vitro translation | 17645868 |
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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 | ||||
TRPV6 |
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NHERF4 | Fusion protein-pull down assay | Mouse | 724-727 | Mouse | Not determined | 17645868 |
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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 | ||||||
TRPV6 |
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NHERF4 | Patch clamp | Activation | 17645868 |
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click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
