Regulation of the Epithelial Mg2+ Channel TRPM6 by Estrogen and the Associated Repressor Protein of Estrogen Receptor Activity (REA)
2009; Elsevier BV; Volume: 284; Issue: 22 Linguagem: Inglês
10.1074/jbc.m808752200
ISSN1083-351X
AutoresGang Cao, Jenny van der Wijst, Annemiete van der Kemp, Femke van Zeeland, René J.M. Bindels, Joost G.J. Hoenderop,
Tópico(s)Plant Micronutrient Interactions and Effects
ResumoThe maintenance of the Mg2+ balance of the body is essential for neuromuscular excitability, protein synthesis, nucleic acid stability, and numerous enzymatic systems. The Transient Receptor Potential Melastatin 6 (TRPM6) functions as the gatekeeper of transepithelial Mg2+ transport. However, the molecular regulation of TRPM6 channel activity remains elusive. Here, we identified the repressor of estrogen receptor activity (REA) as an interacting protein of TRPM6 that binds to the 6th, 7th, and 8th β-sheets in its α-kinase domain. Importantly, REA and TRPM6 are coexpressed in renal Mg2+-transporting distal convoluted tubules (DCT). We demonstrated that REA significantly inhibits TRPM6, but not its closest homologue TRPM7, channel activity. This inhibition occurs in a phosphorylation-dependent manner, since REA has no effect on the TRPM6 phosphotransferase-deficient mutant (K1804R), while it still binds to this mutant. Moreover, activation of protein kinase C by phorbol 12-myristate 13-acetate-PMA potentiated the inhibitory effect of REA on TRPM6 channel activity. Finally, we showed that the interaction between REA and TRPM6 is a dynamic process, as short-term 17β-estradiol treatment disassociates the binding between these proteins. In agreement with this, 17β-estradiol treatment significantly stimulates the TRPM6-mediated current in HEK293 cells. These results suggest a rapid pathway for the effect of estrogen on Mg2+ homeostasis in addition to its transcriptional effect. Together, these data indicate that REA operates as a negative feedback modulator of TRPM6 in the regulation of active Mg2+ (re)absorption and provides new insight into the molecular mechanism of renal transepithelial Mg2+ transport. The maintenance of the Mg2+ balance of the body is essential for neuromuscular excitability, protein synthesis, nucleic acid stability, and numerous enzymatic systems. The Transient Receptor Potential Melastatin 6 (TRPM6) functions as the gatekeeper of transepithelial Mg2+ transport. However, the molecular regulation of TRPM6 channel activity remains elusive. Here, we identified the repressor of estrogen receptor activity (REA) as an interacting protein of TRPM6 that binds to the 6th, 7th, and 8th β-sheets in its α-kinase domain. Importantly, REA and TRPM6 are coexpressed in renal Mg2+-transporting distal convoluted tubules (DCT). We demonstrated that REA significantly inhibits TRPM6, but not its closest homologue TRPM7, channel activity. This inhibition occurs in a phosphorylation-dependent manner, since REA has no effect on the TRPM6 phosphotransferase-deficient mutant (K1804R), while it still binds to this mutant. Moreover, activation of protein kinase C by phorbol 12-myristate 13-acetate-PMA potentiated the inhibitory effect of REA on TRPM6 channel activity. Finally, we showed that the interaction between REA and TRPM6 is a dynamic process, as short-term 17β-estradiol treatment disassociates the binding between these proteins. In agreement with this, 17β-estradiol treatment significantly stimulates the TRPM6-mediated current in HEK293 cells. These results suggest a rapid pathway for the effect of estrogen on Mg2+ homeostasis in addition to its transcriptional effect. Together, these data indicate that REA operates as a negative feedback modulator of TRPM6 in the regulation of active Mg2+ (re)absorption and provides new insight into the molecular mechanism of renal transepithelial Mg2+ transport. Mg2+ is a central electrolyte important for many biological functions by its intervention in gene transcription, protein synthesis, nucleic acid stability, channel regulation, cell cycle, and numerous enzymatic systems (1Vetter T. Lohse M.J. Curr. Opin. Nephrol. Hypertens. 2002; 11: 403-410Crossref PubMed Scopus (89) Google Scholar, 2Grubbs R.D. Biometals. 2002; 15: 251-259Crossref PubMed Scopus (140) Google Scholar, 3Romani A. Scarpa A. Arch. Biochem. Biophys. 1992; 298: 1-12Crossref PubMed Scopus (320) Google Scholar, 4Konrad M. Schlingmann K.P. Gudermann T. Am. J. Physiol. Renal Physiol. 2004; 286: 599-605Crossref PubMed Scopus (188) Google Scholar). In most species, serum Mg2+ levels are kept within a narrow range between 0.8 and 1.1 mm, while the free intracellular Mg2+ concentration [Mg2+]i has been estimated around 0.5–1.0 mm (5Chubanov V. Gudermann T. Schlingmann K.P. Pflugers Arch. 2005; 451: 228-234Crossref PubMed Scopus (85) Google Scholar). Regulation of the Mg2+ balance principally resides within the kidney where Mg2+ excretion tightly matches the intestinal absorption of Mg2+ (5Chubanov V. Gudermann T. Schlingmann K.P. Pflugers Arch. 2005; 451: 228-234Crossref PubMed Scopus (85) Google Scholar). The majority of Mg2+ in the renal ultrafiltrate is reabsorbed passively in the proximal tubule and the thick ascending limb of the loop of Henle, while the final Mg2+ excretion is determined in the distal convoluted tubule (DCT) 3The abbreviations used are: DCT, distal convoluted tubule; TRPM6, Transient Receptor Potential Melastatin 6; REA, repressor of estrogen receptor activity; GST, glutathione S-transferase; HA, hemagglutinin; ER, estrogen receptor.3The abbreviations used are: DCT, distal convoluted tubule; TRPM6, Transient Receptor Potential Melastatin 6; REA, repressor of estrogen receptor activity; GST, glutathione S-transferase; HA, hemagglutinin; ER, estrogen receptor. via an active reabsorption process (5Chubanov V. Gudermann T. Schlingmann K.P. Pflugers Arch. 2005; 451: 228-234Crossref PubMed Scopus (85) Google Scholar). The Transient Receptor Potential Melastatin 6 (TRPM6) localizes along the apical membranes of DCT and intestinal cells where it plays a crucial role in active Mg2+ (re)absorption. Mutations in TRPM6 lead to hypomagnesemia with secondary hypocalcemia (HSH) indicating that this channel is important for the maintenance of the Mg2+ balance (6Walder R.Y. Landau D. Meyer P. Shalev H. Tsolia M. Borochowitz Z. Boettger M.B. Beck G.E. Englehardt R.K. Carmi R. Sheffield V.C. Nat. Genet. 2002; 31: 171-174Crossref PubMed Scopus (459) Google Scholar, 7Schlingmann K.P. Weber S. Peters M. Niemann Nejsum L. Vitzthum H. Klingel K. Kratz M. Haddad E. Ristoff E. Dinour D. Syrrou M. Nielsen S. Sassen M. Waldegger S. Seyberth H.W. Konrad M. Nat. Genet. 2002; 31: 166-170Crossref PubMed Scopus (630) Google Scholar, 8Voets T. Nilius B. Hoefs S. van der Kemp A.W. Droogmans G. Bindels R.J. Hoenderop J.G. J. Biol. Chem. 2004; 279: 19-25Abstract Full Text Full Text PDF PubMed Scopus (499) Google Scholar). Previous studies demonstrated that expression of TRPM6 is regulated by dietary Mg2+ and its channel activity is strongly inhibited by the intracellular Mg2+ concentration ([Mg2+]i) (8Voets T. Nilius B. Hoefs S. van der Kemp A.W. Droogmans G. Bindels R.J. Hoenderop J.G. J. Biol. Chem. 2004; 279: 19-25Abstract Full Text Full Text PDF PubMed Scopus (499) Google Scholar, 9Groenestege W.M. Hoenderop J.G. van den Heuvel L. Knoers N. Bindels R.J. J. Am. Soc. Nephrol. 2006; 17: 1035-1043Crossref PubMed Scopus (184) Google Scholar). Generally, Mg2+-free solutions are used to measure the characteristic outwardly rectifying TRPM6 currents (8Voets T. Nilius B. Hoefs S. van der Kemp A.W. Droogmans G. Bindels R.J. Hoenderop J.G. J. Biol. Chem. 2004; 279: 19-25Abstract Full Text Full Text PDF PubMed Scopus (499) Google Scholar, 10Li M. Du J. Jiang J. Ratzan W.J. Su L.T. Runnels L.W. Yue L. J. Biol. Chem. 2007; 282: 25817-25830Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 11Chubanov V. Schlingmann K.P. Waring J. Heinzinger J. Kaske S. Waldegger S. Mederos y Schnitzler M. Gudermann T. J. Biol. Chem. 2007; 282: 7656-7667Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). Remarkably, TRPM6 contains a C-terminal α-kinase domain, of which the regulatory role on channel activity remains elusive (12Runnels L.W. Yue L. Clapham D.E. Science. 2001; 291: 1043-1047Crossref PubMed Scopus (611) Google Scholar, 13Drennan D. Ryazanov A.G. Prog. Biophys. Mol. Biol. 2004; 85: 1-32Crossref PubMed Scopus (102) Google Scholar, 14Montell C. Curr. Biol. 2003; 13: R799-801Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). It has been shown that RACK1 can interact with this domain and inhibit channel activity in a phosphorylation-dependent manner (15Cao G. Thebault S. van der Wijst J. van der Kemp A. Lasonder E. Bindels R.J. Hoenderop J.G. Curr. Biol. 2008; 18: 168-176Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). A recent study demonstrated that the ATP binding motif in the α-kinase domain is required for modulation of TRPM6 channel activity by intracellular ATP (16Thebault S. Cao G. Venselaar H. Xi Q. Bindels R.J. Hoenderop J.G. J. Biol. Chem. 2008; 283: 19999-20007Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Previous studies suggested that Mg2+ uptake in DCT is stimulated by hormones, such as parathyroid hormone, calcitonin, arginine vasopressin, insulin and prostaglandins (17Dai L.J. Ritchie G. Kerstan D. Kang H.S. Cole D.E. Quamme G.A. Physiol. Rev. 2001; 81: 51-84Crossref PubMed Scopus (258) Google Scholar). A recent study reported that epidermal growth factor (EGF) acts as an autocrine/paracrine magnesiotropic hormone via stimulating its receptor on the basolateral membrane of DCT cells, thereby specifically increasing TRPM6 current (18Groenestege W.M. Thebault S. van der Wijst J. van den Berg D. Janssen R. Tejpar S. van den Heuvel L.P. van Cutsem E. Hoenderop J.G. Knoers N.V. Bindels R.J. J. Clin. Investig. 2007; 117: 2260-2267Crossref PubMed Scopus (287) Google Scholar). Further, Groenestege et al. (9Groenestege W.M. Hoenderop J.G. van den Heuvel L. Knoers N. Bindels R.J. J. Am. Soc. Nephrol. 2006; 17: 1035-1043Crossref PubMed Scopus (184) Google Scholar) demonstrated that the renal TRPM6 mRNA level in ovariectomized rats was significantly reduced, whereas 17β-estradiol treatment normalized TRPM6 mRNA levels. Next to this classical transcriptional pathway, accumulating evidence suggests also rapid estrogen effects that occur within minutes via a non-transcriptional route (19Moriarty K. Kim K.H. Bender J.R. Endocrinology. 2006; 147: 5557-5563Crossref PubMed Scopus (187) Google Scholar, 20Fu X.D. Simoncini T. Semin. Reprod. Med. 2007; 25: 178-186Crossref PubMed Scopus (46) Google Scholar, 21Simoncini T. Mannella P. Genazzani A.R. Ann. N. Y. Acad. Sci. 2006; 1089: 424-430Crossref PubMed Scopus (42) Google Scholar, 22Zheng F.F. Wu R.C. Smith C.L. O'Malley B.W. Mol. Cell Biol. 2005; 25: 8273-8284Crossref PubMed Scopus (64) Google Scholar). Estrogen has the ability to facilitate rapid membrane-initiated signaling cascades through activation of plasma membrane-associated receptors, such as the recently discovered G protein-coupled receptor 30 (GPCR30) (23Revankar C.M. Cimino D.F. Sklar L.A. Arterburn J.B. Prossnitz E.R. Science. 2005; 307: 1625-1630Crossref PubMed Scopus (1809) Google Scholar). In addition, it has been demonstrated that the classical estrogen receptor α (ERα)) can be localized to the plasma membrane in response to estrogen or by interaction with adaptor proteins like Shc and p130Cas (24Cabodi S. Moro L. Baj G. Smeriglio M. Di Stefano P. Gippone S. Surico N. Silengo L. Turco E. Tarone G. Defilippi P. J. Cell Sci. 2004; 117: 1603-1611Crossref PubMed Scopus (106) Google Scholar, 25Levin E.R. Steroids. 2002; 67: 471-475Crossref PubMed Scopus (289) Google Scholar). The aim of the present study was to investigate the regulation of the α-kinase domain on TRPM6 channel activity by identification of proteins specifically interacting with the α-kinase domain. Using a combined approach including biochemical, immunohistochemical, mass spectrometry, and electrophysiological analyses, we demonstrated a novel operation mode for rapid estrogen regulation on TRPM6 channel activity via attenuating the inhibitory effect of the TRPM6-associated protein, repressor of estrogen receptor activity (REA). Cell Culture and Transfection—HEK293 cells were grown and transfected as previously described (26Topala C.N. Groenestege W.T. Thebault S. van den Berg D. Nilius B. Hoenderop J.G. Bindels R.J. Cell Calcium. 2007; 41: 513-523Crossref PubMed Scopus (55) Google Scholar), and electrophysiological recordings were performed 48-h post-transfection. DNA Constructs and cRNA Synthesis—The kinase domain of mouse (1759–2028) and human (1750–2022) TRPM6 was cloned into pGEX6p-2 (Amersham Biosciences, Uppsala, Sweden) by PCR using mouse kidney cDNA or human TRPM6 in pCINeo/IRES-GFP (8Voets T. Nilius B. Hoefs S. van der Kemp A.W. Droogmans G. Bindels R.J. Hoenderop J.G. J. Biol. Chem. 2004; 279: 19-25Abstract Full Text Full Text PDF PubMed Scopus (499) Google Scholar) as template. The α-kinase domain (1750–2022) of human TRPM6 was subcloned into the pEBG vector (27Okada T. Inoue R. Yamazaki K. Maeda A. Kurosaki T. Yamakuni T. Tanaka I. Shimizu S. Ikenaka K. Imoto K. Mori Y. J. Biol. Chem. 1999; 274: 27359-27370Abstract Full Text Full Text PDF PubMed Scopus (396) Google Scholar). Full-length mouse REA cDNA was cloned into pT7Ts, pCB7 and pEBG by PCR using mouse kidney cDNA. Wild-type TRPM6 in the pCINeo/IRES-GFP vector was HA-tagged at the N-terminal tail as described previously (8Voets T. Nilius B. Hoefs S. van der Kemp A.W. Droogmans G. Bindels R.J. Hoenderop J.G. J. Biol. Chem. 2004; 279: 19-25Abstract Full Text Full Text PDF PubMed Scopus (499) Google Scholar). GST-α-kinase truncants (TRPM6 1759–1993, 1759–1885, 1759–1857, 1857–1885, and 1759–1813) mutants, TRPM6 phosphotransferase-deficient mutant (K1804R) and GST-α-kinase K1804R mutant were created using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer's protocol. All constructs were verified by sequence analysis. REA cRNA was synthesized in vitro using T7 RNA polymerase as described previously (28Hoenderop J.G. van der Kemp A.W. Hartog A. van Os C.H. Willems P.H. Bindels R.J. Biochem. Biophys. Res. Commun. 1999; 261: 488-492Crossref PubMed Scopus (93) Google Scholar). Identification of Proteins by Nano Liquid Chromatography Tandem Mass Spectrometry—After SDS-PAGE, GST, and GST-TRPM6 α-kinase interacting proteins were treated with dithiothreitol and iodoacetamide, and digested in-gel by trypsin as previously described (29Olsen J.V. Ong S.E. Mann M. Mol. Cell Proteomics. 2004; 3: 608-614Abstract Full Text Full Text PDF PubMed Scopus (846) Google Scholar). Peptide identification experiments by liquid chromatography tandem mass spectrometry were performed using a nano-HPLC Agillent 1100 nanoflow system connected online to a 7-Tesla linear quadrupole ion trap-Fourier Transform Ion Cyclotron Resonance (LTQ-FT) mass spectrometer (Thermo Electron, Bremen, Germany) as described previously (29Olsen J.V. Ong S.E. Mann M. Mol. Cell Proteomics. 2004; 3: 608-614Abstract Full Text Full Text PDF PubMed Scopus (846) Google Scholar). Peptides and proteins were identified using the Mascot 2.1 (Matrix Science, Boston, MA) algorithm to search a local version of the Uniprot data base. First ranked peptides were parsed from Mascot data base search html files with MSQuant to generate unique first ranked peptide lists and internal calibration of measured ion masses. GST Fusion Proteins and Pull-down Assay—TRPM6 α-kinase GST fusion protein was purified as previously described (30van de Graaf S.F. Hoenderop J.G. Gkika D. Lamers D. Prenen J. Rescher U. Gerke V. Staub O. Nilius B. Bindels R.J. EMBO J. 2003; 22: 1478-1487Crossref PubMed Scopus (241) Google Scholar). REA protein was labeled with [35S]methionine using a reticulocyte lysate system (Promega, Madison, WI) and added to purified GST fusion proteins, immobilized on glutathione-Sepharose 4B beads. After 2 h of incubation at room temperature, beads were washed extensively with pull-down buffer (10 mm Tris-HCl, pH 7.4), 150 mm NaCl, 0.33% (v/v) Triton X-100). The bound proteins were eluted with SDS-PAGE loading buffer, separated on SDS-PAGE gel and visualized by autoradiography. Sequence Analysis and Structure Modeling—The structural model of TRPM6 α-kinase domain was built based on the crystal structure of TRPM7 α-kinase domain (31Yamaguchi H. Matsushita M. Nairn A.C. Kuriyan J. Mol. Cell. 2001; 7: 1047-1057Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar) (Protein Data Bank under ID codes: 1IAJ (apo), 1IAH (ADP complex), and 1IA9 (AMP·PNP complex)) using SWISS-MODEL and analyzed by DeepView Swiss-PdbViewer (Version 3.7) and Yasara. RT-PCR—Total RNA isolation from mouse tissue and reverse transcription were performed as described previously (30van de Graaf S.F. Hoenderop J.G. Gkika D. Lamers D. Prenen J. Rescher U. Gerke V. Staub O. Nilius B. Bindels R.J. EMBO J. 2003; 22: 1478-1487Crossref PubMed Scopus (241) Google Scholar). REA and β-actin were amplified by PCR and subsequently analyzed by agarose gel electrophoresis. Electrophysiology—Patch clamp experiments were performed in the tight seal whole-cell configuration at room temperature using an EPC-10 patch clamp amplifier computer controlled by the Patchmaster software (HEKA Elektronik, Lambrecht, Germany). Electrode resistances were 2–5 MΩ, and capacitance and access resistance were monitored continuously. A ramp protocol, consisting of linear voltage ramps from –100 to +100 mV (within 450 ms), was applied every 2 s from a holding potential of 0 mV. Current densities were obtained by normalizing the current amplitude to the cell membrane capacitance. The time course of current development was determined by measuring the current at +80 and –80 mV. I/V relations were established from the ramp protocols. The analysis and display of patch clamp data were performed using Igor Pro software (WaveMetrics, Lake Oswego). The standard pipette solution contained 150 mm NaCl, 10 mm EDTA, and 10 mm HEPES-NaOH, pH 7.2. The extracellular solutions contained 150 mm NaCl, 10 mm HEPES-NaOH, and 1 mm Ca2+, pH 7.4. To investigate the effect of purified GST-REA or GST on TRPM6-mediated currents, proteins were added to the standard pipette solution and thereby infused via the patch pipette for 7 min before starting the recordings. Co-precipitation—HEK293 cells were transiently co-transfected with REA and pEBG-TRPM6 α-kinase or pEBG empty vector. Cells were treated with 50 nm 17β-estradiol or vehicle for 10 min at 37 °C and were lysed for 1 h on ice in lysis buffer (150 mm NaCl, 5 mm EDTA, 50 mm Tris-NaOH, pH 7.5, 0.33% (v/v) Triton including the protease inhibitors leupeptin (0.01 mg/ml), pepstatin (0.05 mg/ml), and phenylmethylsulfonyl fluoride (1 mm)). After centrifugation, supernatants of the lysates were incubated overnight with glutathione-Sepharose 4B beads at 4 °C. After extensive washing in lysis buffer, the bound proteins were eluted and separated by SDS-PAGE. The co-precipitation was analyzed using the anti-REA antibody (Abcam, Cambridge, UK). Cell Surface Labeling with Biotin—HEK293 cells were transiently transfected with 15 μg HA-TRPM6 in poly-l-lysine (Sigma)-coated 10-cm dishes. 72 h after transfection, cells were treated with 50 nm 17β-estradiol or vehicle for 10 min at 37 °C. Cell surface labeling with NHS-LC-LC-biotin (Pierce, Etten-Leur, The Netherlands) was performed as previously described (32Gkika D. Topala C.N. Hoenderop J.G. Bindels R.J. Am. J. Physiol. Renal Physiol. 2006; 290: 1253-1259Crossref PubMed Scopus (34) Google Scholar). 1 h after homogenizing, biotinylated proteins were precipitated using neutravidin-agarose beads (Pierce, Etten-Leur, The Netherlands). TRPM6 expression was analyzed by immunoblot for the precipitates (plasma membrane fraction) and for the total cell lysates using the anti-HA antibody (Sigma). Immunoblotting—Protein samples were denatured by incubation for 30 min at 37 °C in Laemmli buffer and then subjected to SDS-PAGE. Immunoblots were incubated with either mouse anti-HA (Sigma) or rabbit anti-REA (Abcam, Cambridge, UK) antibody. Subsequently, blots were incubated with sheep horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG (Sigma) and then visualized using the enhanced chemiluminescence system. Immunohistochemistry—Immunohistochemistry was performed as previously described (33Hoenderop J.G. Dardenne O. Van Abel M. Van Der Kemp A.W. Van Os C.H. St -Arnaud R. Bindels R.J. Faseb J. 2002; 16: 1398-1406Crossref PubMed Scopus (200) Google Scholar). Briefly, mouse kidney serial sections were incubated for 16 h at 4 °C with rabbit anti-REA (Abcam, Cambridge, UK) and guinea pig anti-TRPM6 (8Voets T. Nilius B. Hoefs S. van der Kemp A.W. Droogmans G. Bindels R.J. Hoenderop J.G. J. Biol. Chem. 2004; 279: 19-25Abstract Full Text Full Text PDF PubMed Scopus (499) Google Scholar). To visualize TRPM6, tyramide signal amplification kit (NEN Life Science Products, Zaventem, Belgium) was used after incubation with biotin-coated goat anti-mouse secondary antibody. Images were taken with a Bio-Rad MRC 100 confocal laser scanning microscope. In Vitro Phosphorylation Assays—HA-TRPM6 was precipitated by using the anti-HA antibody. The precipitates were incubated in a total volume of 30 μl of kinase reaction buffer (50 mm HEPES-KOH, pH 7.4, 4 mm MnCl2, 0.5 mm CaCl2, 100 μm ATP) and 2 μCi of [γ32P]ATP for 30 min at 30 °C. The reaction was terminated by three washing steps with phosphorylation washing buffer (50 mm HEPES-KOH, pH 7.4, 4 mm MnCl2, 0.5 mm CaCl2). Phosphorylation was analyzed after gel electrophoresis by autoradiography. Statistical Analysis—Values are expressed as mean ± S.E. Statistical significance between groups was determined by analysis of variance (ANOVA). In the case of significance, differences between the means of two groups were analyzed by unpaired Student's t test. p < 0.05 was considered statistically significant. REA Associates with TRPM6—To identify proteins potentially interacting with the TRPM6 α-kinase domain, the combination of a GST pull-down with the α-kinase domain of TRPM6 in mouse kidney lysate followed by Fourier Transform Mass Spectrometry (FTMS), was performed. REA (34Montano M.M. Ekena K. Delage-Mourroux R. Chang W. Martini P. Katzenellenbogen B.S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6947-6952Crossref PubMed Scopus (242) Google Scholar), was identified as an interacting protein of the TRPM6 α-kinase domain. Next, GST pull-down binding assays were used to confirm the interaction between REA and TRPM6 α-kinase domain. [35S]methionine-labeled REA protein was incubated with GST and GST-α-kinase immobilized on glutathione-Sepharose 4B beads. REA interacted specifically with the GST-α-kinase, but not with GST alone (Fig. 1A). Next, the association between TRPM6 and REA was further substantiated in mammalian cells by co-precipitation studies of GST and GST-α-kinase in REA-expressing HEK293 cells. REA co-precipitated with the GST-α-kinase, but not with GST alone (Fig. 1B). REA Binds to the 6th, 7th, and 8th β-Sheets in the TRPM6 α-Kinase Domain—To determine the REA binding site within TRPM6, a series of deletion mutants in the α-kinase domain was constructed. Truncated TRPM6 α-kinase mutants were expressed as GST-fused proteins and evaluated for their interaction with [35S]methionine-labeled REA protein (Fig. 1C). Truncation at position 1857 abolished the interaction between the two proteins, whereas truncation at position 1885 had no effect on the interaction with REA. Moreover, a GST fusion protein containing only the short peptide between the amino acids 1857 and 1885 of the TRPM6 α-kinase domain bound to REA (Fig. 1C). Therefore, the REA binding site within TRPM6 is restricted to the region between the positions 1857 and 1885. The integrity and quantity of the GST fusion proteins was analyzed and confirmed by Coomassie Blue staining of SDS-PAGE gel (Fig. 1D). The lower abundant product bands of different sizes might be due to binding of so far unknown proteins to the bigger truncation mutants or degradation products. To explore the REA binding site in the TRPM6 α-kinase domain, the tertiary structure of this domain was modeled by SWISS-MODEL based on the homology between TRPM6 and TRPM7 α-kinase domains (15Cao G. Thebault S. van der Wijst J. van der Kemp A. Lasonder E. Bindels R.J. Hoenderop J.G. Curr. Biol. 2008; 18: 168-176Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 31Yamaguchi H. Matsushita M. Nairn A.C. Kuriyan J. Mol. Cell. 2001; 7: 1047-1057Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar). Because of the significant homology between the TRPM6 and TRPM7 α-kinase domains (84%), these two domains share a well-conserved secondary structure. The surface and accessibility analysis of the REA binding site (6th, 7th, and 8th β-sheets) using Yasara software suggested that, among the 28 amino acids of the REA binding site, 18 are localized at the surface of the TRPM6 α-kinase domain. Because RACK1 also binds to this region, we investigated whether RACK1 can affect the interaction between TRPM6 and REA. As shown in Fig. 1E, REA co-precipitated in similar amounts with GST α-kinase in the presence and absence of RACK1. REA Co-expresses with TRPM6 in Kidney—To address the tissue distribution of REA, reverse transcriptase polymerase chain reaction (RT-PCR) analysis was performed on mouse tissues. The expected DNA fragment of 916 bp for REA was detected in most tested tissues as indicated in Fig. 2A. The β-actin fragment was present in all the tissues evaluated. Of note, REA is present in kidney where TRPM6 is predominantly expressed (8Voets T. Nilius B. Hoefs S. van der Kemp A.W. Droogmans G. Bindels R.J. Hoenderop J.G. J. Biol. Chem. 2004; 279: 19-25Abstract Full Text Full Text PDF PubMed Scopus (499) Google Scholar). To further study the co-expression between REA and TRPM6 in kidney, immunohistochemistry was performed on serial mouse kidney sections. This analysis indicated immunopositive staining for REA in the TRPM6-expressing DCT cells that have been implicated in active Mg2+ reabsorption (8Voets T. Nilius B. Hoefs S. van der Kemp A.W. Droogmans G. Bindels R.J. Hoenderop J.G. J. Biol. Chem. 2004; 279: 19-25Abstract Full Text Full Text PDF PubMed Scopus (499) Google Scholar) (Fig. 2B). REA Inhibits TRPM6 Channel Activity in a Phosphorylation-dependent Manner—The functional role of REA on TRPM6 channel activity was investigated by whole-cell patch clamp recordings in HEK293 cells. Steeply outward rectifying currents carried by Na+ were measured in HEK293 cells co-expressing TRPM6 and empty vector (mock) (Fig. 3, A and B). These currents were significantly inhibited in cells co-expressing TRPM6 and REA (Fig. 3, A and B). Of note, the inhibitory effect of REA was specific for TRPM6, because REA did not affect the current in TRPM7-expressing HEK293 cells (Fig. 3, C and D). To further analyze the inhibitory effect of REA on TRPM6, GST and GST-REA were expressed in HEK293 cells and purified using glutathione-Sepharose 4B beads (Fig. 4A). These purified GST and GST-REA proteins were subsequently perfused into TRPM6-expressing HEK293 cells via the patch pipette before whole-cell recording. As shown in Fig. 4, B and C, REA significantly inhibited the TRPM6-mediated current, but the associated protein did not change the electrophysiological characteristics of the recorded currents. GST alone did not affect the TRPM6-mediated current. Next, the phosphotransferase-deficient mutant (K1804R) was used to investigate the role of TRPM6 phosphorylation activity in this process. REA was unable to inhibit the K1804R mutant-mediated current, similar as GST alone (Fig. 4, D and E). Importantly, the co-precipitation assay showed that the K1804R mutation did not influence the binding between TRPM6 and REA (Fig. 4F). Importantly, our in vitro phosphorylation assay demonstrated equal autophosphorylation of TRPM6 with or without REA co-expression (Fig. 4G). Next, we investigated the effect of PKC activation by phorbol 12-myristate 13-acetate-PMA (PMA) on the regulatory effect of REA, since it has been shown that RACK1 inhibition can be prevented by 5 min of preincubation of 100 nm PMA (15Cao G. Thebault S. van der Wijst J. van der Kemp A. Lasonder E. Bindels R.J. Hoenderop J.G. Curr. Biol. 2008; 18: 168-176Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Pretreatment of cells co-expressing TRPM6 and REA significantly decreased TRPM6 channel activity. Of note, the current amplitude in the presence of PMA was not altered in cells expressing TRPM6 alone (Fig. 4H). Finally, we investigated whether REA is a phosphorylation target of TRPM6. The in vitro phosphorylation assay showed that REA is not phosphorylated by TRPM6 (Fig. 4G).FIGURE 4REA inhibits TRPM6 channel activity in a rapid and phosphorylation-dependent manner. A, Coomassie Blue staining of purified GST and GST-REA. B, current-voltage (I/V) relation of TRPM6-transfected cells infused with GST (solid trace) or GST-REA (dashed trace). C, averaged values of the current density at +100 mV after 600 s of GST infused (n = 16) and GST-REA infused (n = 18) cells. * indicates p < 0.05. D, current-voltage (I/V) relations of TRPM6 K1804R transfected cells infused with GST (solid trace) or GST-REA (dashed trace). E, averaged values of the current density at +100 mV after 600 s of GST infused (n = 26) and GST-REA infused (n = 21) cells. F, co-precipitation studies of GST, GST-α-kinase, and GST-α-kinase K1804R in REA-expressing HEK293 cells (top panel). REA input (1%) expression was analyzed by immunoblotting (bottom panel). G, in vitro protein kinase assay of TRPM6 and TRPM6 and REA (left panel), analyzed for total expression by immunoblotting (right panels). H, effect of PKC activation by PMA (100 nm, 5 min pretreatment) on the current density at +80 after 200 s of TRPM6 and mock (n = 17–44), and TRPM6 and REA (n = 17–36). * indicates p < 0.05 compared with TRPM6 and mock without pretreatment, and # indicates p < 0.05 compared with TRPM
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