Interaction of the epithelial Ca2+ channels TRPV5 and TRPV6 with the intestine- and kidney-enriched PDZ protein NHERF4
- 217 Downloads
The epithelial Ca2+ channels TRPV5 and TRPV6 constitute the apical Ca2+ influx pathway in epithelial Ca2+ 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-Pi 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 Ca2+ channels.
KeywordsECaC1 CaT1 Calcium homeostasis PDZ Protein–protein interactions
The authors thank Dr. Arjen Mensenkamp for the TRPV4 construct. This work was sponsored by the Dutch Organization of Scientific Research (Zon-Mw 016.006.001, Zon-Mw 902.18.298, NWO-ALW 810.38.004, NWO-ALW 805.09.042, NWO-ALW 814.02.001, and NWO-Talent S91-282).
- 4.Bianco S, Peng JB, Takanaga H, Kos CH, Crescenzi A, Brown EM, Hediger MA (2004) Mice lacking the epithelial calcium channel CaT1 (TRPV6) show a deficiency in intestinal calcium absorption depite high plasma levels of 1,25-dihydroxyvitamin D. FASEB J 18:A706Google Scholar
- 7.Nilius B, Prenen J, Hoenderop JG, Vennekens R, Hoefs S, Weidema AF, Droogmans G, Bindels RJ (2002) Fast and slow inactivation kinetics of the Ca2+ channels ECaC1 and ECaC2 (TRPV5 and 6): role of the intracellular loop located between transmembrane segment 2 and 3. J Biol Chem 277:30852–30858CrossRefPubMedGoogle Scholar
- 27.Palmada M, Poppendieck S, Embark HM, Van de Graaf SFJ, Boehmer C, Bindels RJM, Lang F (2005) Requirement of PDZ domains for the stimulation of the epithelial Ca2+ channel TRPV5 by the NHE regulating factor NHERF2 and the serum and glucocorticoid inducible kinase SGK1. Cell Physiol Biochem 15:175–182CrossRefPubMedGoogle Scholar
- 33.Embark HM, Setiawan I, Poppendieck S, Van de Graaf SFJ, Boehmer C, Palmada M, Wieder T, Gerstberger R, Cohen P, Yun CC, Bindels RJM, Lang F (2004) Regulation of the epithelial Ca2+ channel TRPV5 by the NHE regulating factor NHERF2 and the serum and glucocorticoid inducible kinase isoforms SGK1 and SGK3 expressed in Xenopus oocytes. Cell Physiol Biochem 14:203–212CrossRefPubMedGoogle Scholar
- 41.Hoenderop JG, Vaandrager AB, Dijkink L, Smolenski A, Gambaryan S, Lohmann SM, de Jonge HR, Willems PH, Bindels RJ (1999) Atrial natriuretic peptide-stimulated Ca2+ reabsorption in rabbit kidney requires membrane-targeted, cGMP-dependent protein kinase type II. Proc Natl Acad Sci U S A 96:6084–6089CrossRefPubMedGoogle Scholar
- 43.Markert T, Vaandrager AB, Gambaryan S, Pohler D, Hausler C, Walter U, De Jonge HR, Jarchau T, Lohmann SM (1995) Endogenous expression of type II cGMP-dependent protein kinase mRNA and protein in rat intestine. Implications for cystic fibrosis transmembrane conductance regulator. J Clin Invest 96:822–830PubMedCrossRefGoogle Scholar