Pflugers Arch. 2006 Jul;452(4):407-17. Epub 2006 Mar 25.

Interaction of the epithelial Ca2+ channels TRPV5 and TRPV6 with the intestine- and kidney-enriched PDZ protein NHERF4.External 2231691f894ba696de1310221b0a0dbbb31a7251e75115c265587c3d9d5f507c

van de Graaf, S. F., Hoenderop, J. G., van der Kemp, A. W., Gisler, S. M., Bindels, R. J.,
["Cell Physiology, Department of Physiology, 286, Nijmegen Center for Molecular Life Sciences, Radboud University Nijmegen Medical Center, P.O. Box 9101, NL-6500 HB, Nijmegen, The Netherlands."]
The epithelial Ca(2+) channels TRPV5 and TRPV6 constitute the apical Ca(2+) influx pathway in epithelial Ca(2+) transport. PDZ proteins have been demonstrated to play a crucial role in the targeting or anchoring of ion channels and transporters in the apical domain of the cell. In this study, we describe the identification of NHERF4 (Na-P(i) Cap2/IKEPP/PDZK2) as a novel TRPV5- and TRPV6-associated PDZ protein. NHERF4 was identified using two separate yeast two-hybrid screens with the carboxyl termini of TRPV5 and TRPV6 as bait. Binding of the carboxyl termini of TRPV5 and TRPV6 with NHERF4 was confirmed by GST pull-down assays using in-vitro-translated NHERF4 or lysates of Xenopus laevis oocytes expressing NHERF4. Furthermore, the interaction was confirmed by GST pull-down and co-immunoprecipitation assays using in-vitro-translated full-length TRPV5 and Xenopus oocytes or HEK293 cells co-expressing NHERF4 and TRPV5/TRPV6, respectively. The fourth PDZ domain of NHERF4 was sufficient for the interaction, although PDZ domain 1 also contributed to the binding. The binding site for NHERF4 localized in a conserved region in the carboxyl terminus of TRPV5 and was distinct from the binding site of the PDZ protein NHERF2. NHERF4 predominantly localized at the plasma membrane of X. laevis oocytes and HeLa cells. This localization was independent of the presence of TRPV5. Therefore, we hypothesize a role for this novel PDZ protein as a putative plasma membrane scaffold for the epithelial Ca(2+) channels.
PMID: 16565876External 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
TRPV4 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 NHERF4 Inference Prediction 16565876
TRPV5 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 NHERF4 Yeast two-hybrid Mouse C-terminus Mouse Kidney cDNA library 16565876
TRPV6 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 NHERF4 Yeast two-hybrid Mouse C-terminus Mouse Kidney cDNA library 16565876
(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
TRPV4 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 NHERF4 Fusion protein-pull down assay Mouse C-terminus E. coli Mouse Full-length In vitro translation 16565876
TRPV5 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 NHERF2 Fusion protein-pull down assay Mouse Full-length E. coli Not specified Full-length In vitro translation 16565876
TRPV5 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 NHERF4 Fusion protein-pull down assay Mouse C-terminus E. coli Mouse Full-length In vitro translation 16565876
TRPV6 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 NHERF4 Fusion protein-pull down assay Mouse C-terminus E. coli Mouse Full-length In vitro translation 16565876
(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
TRPV5 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 NHERF4 Co-immunoprecipitation HEK293 Mouse Full-length Mouse Full-length 16565876
TRPV5 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 NHERF4 Co-immunofluorescence staining Xenopus oocyte Mouse Full-length Mouse Full-length 16565876
TRPV5 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 NHERF4 Co-immunofluorescence staining Mouse kidney section 16565876
TRPV6 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 NHERF4 Co-immunoprecipitation HEK293 Mouse Full-length Mouse Full-length 16565876
(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
TRPV5 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 NHERF4 Fusion protein-pull down assay Mouse 579-617 Mouse Not determined 16565876
(Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4: click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
TRP / Interactor

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