Elsevier

Cellular Signalling

Volume 17, Issue 8, August 2005, Pages 951-960
Cellular Signalling

Tyrosine phosphatase PTP1B interacts with TRPV6 in vivo and plays a role in TRPV6-mediated calcium influx in HEK293 cells

https://doi.org/10.1016/j.cellsig.2004.11.012Get rights and content

Abstract

This study investigates the role of tyrosine phosphorylation and dephosphorylation in the regulation of the Ca2+ permeant TRPV6 channel.

HEK293 cells co-transfected with TRPV6 and the tyrosine phosphatase PTP1B show a constitutive Ca2+ entry which was independent of tyrosine phosphorylation under resting conditions. Following depletion of intracellular Ca2+ stores, TRPV6-mediated Ca2+ entry could be increased in the presence of a tyrosine phosphatase inhibitor (bis-(N,N-dimethyl-hydroxamido) hydroxo-vanadate; DMHV). Inhibition of Src-kinases completely abolished DMHV-induced increase in TRPV6-mediated Ca2+ influx. Co-transfection with Src led to tyrosine phosphorylation of TRPV6 which could be dephosphorylated by PTP1B.

In vivo interaction of TRPV6 with PTP1B was visualized using the bimolecular fluorescence complementation (BiFC) method and proved by co-immunoprecipitation of both proteins.

These data indicate that tyrosine phosphorylation is involved in the regulation of the TRPV6 channel protein.

Introduction

Transient receptor potential (TRP) channels are a family of ion channels whose members are involved in ubiquitous and also more specialized processes such as the vision process in Drosophila, regulation of osmolarity or intracellular magnesium homeostasis in mammalian cells (for review see Refs. [1], [2]).

Recently two new closely related members of the “TRPV subfamily” were cloned [3], [4] and are named TRPV5 and TRPV6 [5]. Both are highly selective Ca2+ channels and are expressed in several tissues including intestine, kidney, pancreas, prostate and testis [6], [7], [8], [9]. TRPV6 is permeant for monovalent and divalent cations [10] with high selectivity for Ca2+ [11] and it has been shown to be involved in Ca2+ (re)absorption in Ca2+ transporting tissues including the small intestine and kidney [6]. It was therefore speculated that the main function of these channels is Ca2+ (re)absorption. Indeed mice lacking TRPV5 showed renal Ca2+ wasting and a reduced bone thickness [11]. This led to the conclusion that the knockouts had lost the ability to retain Ca2+.

Beside its role in Ca2+ (re)absorption TRPV5 and TRPV6 may have additional functions in the body. Wissenbach et al. [12] described that TRPV6 expression correlates with prostate cancer in humans and therefore could be a candidate to serve as a therapeutic target. Beside its well-established role in intestinal Ca2+ transport, TRPV6 has been also suggested to mediate “capacitative” or “store-operated”-Ca2+-entry (SOCE). Release of Ca2+ from inositol-1,4,5-trisphosphate (IP3)-sensitive Ca2+ stores leads to activation of Ca2+ entry in different cell types [13], [14]. Neither the mechanism that links store depletion to activation of SOCE nor the molecular identity of this channel has yet been clarified. Several members of the TRP family have been discussed to be responsible for SOCE [15], [16], [17], [18], [19]. Recently, Yue et al. [20] reported that the human TRPV6 shows properties of a specific SOCE channel (so-called Ca2+ release-activated Ca2+-channel, CRAC; [21]) when transfected in Chinese hamster ovary cells. TRPV6 was activated by depletion of Ca2+ stores and showed whole cell current kinetics and ion selectivity for cations similar as CRAC. However, recent data describe clear differences in endogenous and TRPV6-induced currents [22], [23]. In connection with the here described study the different behavior of CRAC and TRPV6-induced Ca2+ entry to a blocker of CRAC is of particular interest: 2-aminoethyldiphenyl borate (2-APB), which had been reported to inhibit CRAC at concentrations >10 μM [24] did not effect Ca2+ currents through TRPV6 [23]. The authors concluded from their study that the pores of TRPV6 and CRAC are not identical and that TRPV6 and CRAC are two distinct channels with overlapping properties but most likely encoded by different genes. We have used 2-APB in this study to discriminate between intrinsic and TRPV6-induced Ca2+ entry. Taken together, it seems to be unlikely that TRPV6 is identical with CRAC. Nevertheless, a store dependent modulation of TRPV6 activity may be an intrinsic feature of this channel [25]. This allows to speculate that TRPV6 has features of CRAC although it is not identical to it.

In order to obtain insight into the regulatory mechanism of TRPV6-mediated Ca2+ influx, we tested interaction of TRPV6 with PTP1B and the effects of tyrosine phosphorylation and dephosphorylation on TRPV6-induced Ca2+ entry. Our data give strong evidence for tyrosine phosphorylation of TRPV6 as a regulatory mechanism in TRPV6-mediated Ca2+ influx.

Section snippets

Cell culture, transfection and construction of TRPV6-, PTP1B-, and Src-plasmids

HEK293 cells grown on glass coverslips in DMEM/Ham's F-12 (1:1) medium (PAA Laboratories, Cölbe, Germany) supplemented with 2 mM l-glutamine and 10% FCS at 37 °C, 95% O2, 5% CO2, 100% humidity were transfected with different DNA constructs 24 h after seeding. Transfection was performed in Petri dishes (35 mm diameter) with 1 μg DNA/dish using “Fugene 6” for transfection (Roche, Indianapolis).

Rat trpv6 gene cloned into a pTracer-CMV2-vector (Invitrogen, Groningen, The Netherlands) was kindly

TRPV6-mediated Ca2+ influx in transiently transfected HEK293 cells under control conditions and after Ca2+-store depletion

The TRPV6-mediated Ca2+ entry into HEK293 cells was determined by the fura-2 fluorescence method as changes of the cytoplasmic Ca2+ concentration ([Ca2+]cyt). TRPV6 gene cloned into a pTracer-CMV2-vector (Invitrogen) was used in a first set of experiments to estimate TRPV6-mediated Ca2+ influx into HEK293 cells. As shown in Fig. 1, basal [Ca2+]cyt was higher in HEK293 cells transfected with TRPV6 (120±5 nM) as compared to control cells (untransfected or transfected with empty vector, 63±6 nM).

Discussion

This study demonstrates that both proteins, TRPV6 and PTP1B, interact in vivo and that TRPV6 can be tyrosine phosphorylated and dephosphorylated by Src and PTP1B, respectively. For other TRP family members, TRPV4 and TRPC3 tyrosine phosphorylation has also been described [35], [36]. We give evidence, that tyrosine phosphorylation by Src and dephosphorylation by PTP1B can modulate TRPV6 induced Ca2+ entry in transiently transfected HEK293 cells.

Blocking these enzymes with PP1 or DMHV resulted in

Acknowledgements

This work was supported by the Deutsche Forschungsgemeinschaft: Sonderforschungsbereich 530 and Graduiertenkolleg “Zelluläre Regulation und Wachstum”. Part of the work was supported by HOMFOR (No. A/2003/17).

The authors wish to thank Dr. Matthias Hediger/Boston for the rTRPV6 gene and Dr. Benjamin Neel/Boston for the cDNA of PTP1B. We also thank Prof. Olaf Pongs for valuable discussions.

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