Ca2+-selective Transient Receptor Potential V Channel Architecture and Function Require a Specific Ankyrin Repeat
2004; Elsevier BV; Volume: 279; Issue: 33 Linguagem: Inglês
10.1074/jbc.m404778200
ISSN1083-351X
AutoresIsabell Erler, Daniela Hirnet, Ulrich Wissenbach, Veit Flockerzi, Barbara A. Niemeyer,
Tópico(s)Herbal Medicine Research Studies
ResumoTransient receptor potential (TRP) proteins form cation-conducting ion channels with currently 28 known genes encoding TRP channel monomers in mammals. These monomers are thought to coassemble to form homo- or heterotetrameric channels, but the signals governing their assembly are unknown. Within the TRPV subgroup, TRPV5 and TRPV6 show exclusive calcium selectivity and play an important role in calcium uptake. To identify signals that mediate assembly of functional TRPV6, we screened domains for self-association using co-immunoprecipitation, sucrose gradient centrifugation, bacterial two-hybrid assays, and patch clamp analysis. Of the two identified interaction domains within the N-terminal region, we showed that the first domain encompassing the third ankyrin repeat is the stringent requirement for physical assembly of TRPV6 subunits and when transferred to an unrelated protein enables its interaction with TRPV6. Deletion of this repeat or mutation of critical residues within this repeat rendered nonfunctional channels that do not co-immunoprecipitate or form tetramers. Suppression of dominant-negative inhibitors of TRPV6-specific currents was achieved by deletion of ankyrin (ANK) 3. We propose that the third ANK repeat initiates a molecular zippering process that proceeds past the fifth ANK repeat and creates an intracellular anchor that is necessary for functional subunit assembly. Transient receptor potential (TRP) proteins form cation-conducting ion channels with currently 28 known genes encoding TRP channel monomers in mammals. These monomers are thought to coassemble to form homo- or heterotetrameric channels, but the signals governing their assembly are unknown. Within the TRPV subgroup, TRPV5 and TRPV6 show exclusive calcium selectivity and play an important role in calcium uptake. To identify signals that mediate assembly of functional TRPV6, we screened domains for self-association using co-immunoprecipitation, sucrose gradient centrifugation, bacterial two-hybrid assays, and patch clamp analysis. Of the two identified interaction domains within the N-terminal region, we showed that the first domain encompassing the third ankyrin repeat is the stringent requirement for physical assembly of TRPV6 subunits and when transferred to an unrelated protein enables its interaction with TRPV6. Deletion of this repeat or mutation of critical residues within this repeat rendered nonfunctional channels that do not co-immunoprecipitate or form tetramers. Suppression of dominant-negative inhibitors of TRPV6-specific currents was achieved by deletion of ankyrin (ANK) 3. We propose that the third ANK repeat initiates a molecular zippering process that proceeds past the fifth ANK repeat and creates an intracellular anchor that is necessary for functional subunit assembly. Transient receptor potential (TRP) 1The abbreviations used are: TRP, transient receptor potential; ANK, ankyrin; aa, amino acid(s); GFP, green fluorescent protein; EGFP, enhanced GFP; HEK, human embryonic kidney; HA, hemagglutinin; F, farad(s); wt, wild-type; PM, pore mutant. 1The abbreviations used are: TRP, transient receptor potential; ANK, ankyrin; aa, amino acid(s); GFP, green fluorescent protein; EGFP, enhanced GFP; HEK, human embryonic kidney; HA, hemagglutinin; F, farad(s); wt, wild-type; PM, pore mutant. channels have evolved as a family of ion channels defined by their relatedness to their seminal member, the Drosophila TRP channel. Members of the TRP family are involved in sensory processes such as invertebrate vision and temperature, pain, and gustatory sensation often restricted to specialized cells and in more ubiquitous processes such as sensing osmotic stress or regulating intracellular magnesium concentration (for reviews, see Refs. 1Montell C. Science's STKE. 2001; (http://stke.sciencemag.org/cgi/content/full/OC_sigtrans;2001/90/re1)PubMed Google Scholar and 2Clapham D.E. Nature. 2003; 426: 517-524Crossref PubMed Scopus (2109) Google Scholar). The general topology of a TRP subunit includes intracellular N- and C-terminal regions of variable length and six transmembrane-spanning domains with a pore loop between transmembrane domains 5 and 6. In analogy to voltage-gated potassium channels it is thought that four subunits need to assemble to form a functional channel. Tetramer formation has been shown for TRPV1 and TRPV5/6 (3Kedei N. Szabo T. Lile J.D. Treanor J.J. Olah Z. Iadarola M.J. Blumberg P.M. J. Biol. Chem. 2001; 276: 28613-28619Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar, 4Jahnel R. Dreger M. Gillen C. Bender O. Kurreck J. Hucho F. Eur. J. Biochem. 2001; 268: 5489-5496Crossref PubMed Scopus (90) Google Scholar, 5Hoenderop J.G. Voets T. Hoefs S. Weidema F. Prenen J. Nilius B. Bindels R.J. EMBO J. 2003; 22: 776-785Crossref PubMed Scopus (293) Google Scholar). Of the three major subfamilies of TRP channels, TRPV, TRPC, and TRPM, it has been shown that some members within the TRPC and within the TRPV family can form heteromeric channels (5Hoenderop J.G. Voets T. Hoefs S. Weidema F. Prenen J. Nilius B. Bindels R.J. EMBO J. 2003; 22: 776-785Crossref PubMed Scopus (293) Google Scholar, 6Montell C. Mol. Pharmacol. 1997; 52: 755-763Crossref PubMed Scopus (120) Google Scholar, 7Goel M. Sinkins W.G. Schilling W.P. J. Biol. Chem. 2002; 277: 48303-48310Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar, 8Amiri H. Schultz G. Schaefer M. Cell Calcium. 2003; 33: 463-470Crossref PubMed Scopus (64) Google Scholar, 9Strubing C. Krapivinsky G. Krapivinsky L. Clapham D.E. J. Biol. Chem. 2003; 278: 39014-39019Abstract Full Text Full Text PDF PubMed Scopus (346) Google Scholar).So far almost all of the TRP channel proteins form cation-conducting ion pores with a range of different selectivities (1Montell C. Science's STKE. 2001; (http://stke.sciencemag.org/cgi/content/full/OC_sigtrans;2001/90/re1)PubMed Google Scholar, 2Clapham D.E. Nature. 2003; 426: 517-524Crossref PubMed Scopus (2109) Google Scholar); TRPC, TRPN, and TRPV channels contain multiple ankyrin (ANK) repeats within their intracellular N termini. Although ANK repeats are common modular protein interaction motifs, not much is known regarding whether or not their specific sequence and/or number of repeats are required to form specialized interactions or about their role in the assembly of ion channels. Two members of the subgroup of TRPV channels, TRPV5 and TRPV6, show exquisite selectivity for calcium ions and are likely to be involved in epithelial calcium uptake (for a recent review, see Ref. 10den Dekker E. Hoenderop J.G. Nilius B. Bindels R.J. Cell Calcium. 2003; 33: 497-507Crossref PubMed Scopus (179) Google Scholar). Moreover it has been shown that expression of TRPV6 can act as an indicator for the malignancy and invasiveness of prostate cancer (11Wissenbach U. Niemeyer B.A. Fixemer T. Schneidewind A. Trost C. Cavalie A. Reus K. Meese E. Bonkhoff H. Flockerzi V. J. Biol. Chem. 2001; 276: 19461-19468Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar, 12Peng J.B. Zhuang L. Berger U.V. Adam R.M. Williams B.J. Brown E.M. Hediger M.A. Freeman M.R. Biochem. Biophys. Res. Commun. 2001; 282: 729-734Crossref PubMed Scopus (152) Google Scholar, 13Fixemer T. Wissenbach U. Flockerzi V. Bonkhoff H. Oncogene. 2003; 22: 7858-7861Crossref PubMed Scopus (201) Google Scholar). TRPV6 contains six putative ankyrin repeats within its N-terminal region (11Wissenbach U. Niemeyer B.A. Fixemer T. Schneidewind A. Trost C. Cavalie A. Reus K. Meese E. Bonkhoff H. Flockerzi V. J. Biol. Chem. 2001; 276: 19461-19468Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar), and it has been shown that TRPV6 and TRPV5 can form homotetramers and heterotetramers when overexpressed alone or together, respectively (5Hoenderop J.G. Voets T. Hoefs S. Weidema F. Prenen J. Nilius B. Bindels R.J. EMBO J. 2003; 22: 776-785Crossref PubMed Scopus (293) Google Scholar). Analyzing the structure responsible for physical and functional assembly of these channel proteins is essential for understanding their function and may facilitate designing potential drug targets.In this study we analyzed the domains within TRPV6 that are involved in assembling functional ion channels. We found that an N-terminal region (aa 116–191) is sufficient to enable self-interaction; a construct containing this region had a dominant-negative effect on TRPV6 currents. Moreover transfer of this region to a protein unrelated to TRPV proteins, the γ1 subunit of voltage-activated calcium channels, enabled this protein to physically interact with TRPV6. Conversely deletion of this region was able to suppress the dominant-negative phenotype of a mutant subunit with an altered ionic pore. The interacting region contains the third ANK repeat, and we could show that specific amino acids in the second helical segment of this ANK repeat are necessary for assembly of functional TRPV6 channels. This is particularly important as no data exist on the physiological function of the ANK repeats of TRP channels, and we may now be able to establish rules for subunit assembly.EXPERIMENTAL PROCEDURESSite-directed Mutagenesis—All experiments were conducted using hTRPV6 (GenBank™ accession number NM_018646). To obtain the three TRPV6 deletion mutants, a MluI restriction site (ACGCGT) was introduced by PCR at nucleotide positions 333–335 resulting in replacement of serine at position 112 by an arginine. The mutation was introduced using a reverse PCR primer containing the downstream SacI site and the indicated base pairs and a PCR forward primer that included the SacI site of the pCAGGS-IRES-EGFP vector. The SacI-cut amplified fragment was ligated into SacI-cut pCAGGS-TRPV6 vector. Deletion constructs were made by PCR with different forward primers containing an MluI recognition site and a reverse primer covering a unique TRPV6 EcoRV restriction site. The MluI/EcoRV fragments were ligated into the MluI and EcoRV sites of TRPV6-pCAGGS-IRES-EGFP and for precipitation experiments were recloned into TRPV6-EGFP-pCDNA3. The D542A and 138AR139 to 138EA139 mutations were obtained by using the QuikChange XL kit (Stratagene). The template for this reaction was the EGFP-tagged full-length TRPV6. TRPV6 antisense construct was obtained by cloning the entire coding sequence of TRPV6 in antisense orientation into pCAGGS-IRES-EGFP vector. All amplified DNA used for expression constructs was sequenced.Recombinant Proteins—All C-terminal EGFP-tagged constructs were obtained by inserting PCR-amplified TRPV6 fragments including the consensus sequence for initiation of translation in vertebrates followed by a start ATG into pCDNA3 vector that was modified to include an in-frame EGFP coding sequence.Electrophysiology—Patch clamp recordings on single transfected HEK 293 or HEK-TRPV6 cells were performed 48–72 h after transfection as described previously (11Wissenbach U. Niemeyer B.A. Fixemer T. Schneidewind A. Trost C. Cavalie A. Reus K. Meese E. Bonkhoff H. Flockerzi V. J. Biol. Chem. 2001; 276: 19461-19468Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar). Pipette solution contained 140 mm aspartic acid, 10 mm EGTA, 10 mm NaCl, 1 mm MgCl2, 10 mm Hepes (pH 7.2 with CsOH). Bath solution contained 115 mm triethylammonium chloride, 10 mm CsCl, 2.8 mm KCl, 2 mm MgCl2, 10 mm glucose, 20 mm Hepes (pH 7.4 with NaOH), and 30 mm CaCl2. After obtaining whole cell configuration, whole cell currents were recorded every 10 s by applying 100-ms voltage clamp ramps from –100 mV to +100 mV from a holding potential of +70 mV until currents at –80 mV reached maximum values. Currents (pA) were divided by the whole cell capacitance (pF). Data are given as mean ± S.E. Values were not corrected for liquid junction potentials.Co-immunoprecipitation Experiments—TRPV6 stable HEK 293 (TRPV6s) cells were transfected with pCDNA3-TRPV6-EGFP expression constructs, cultured for 24–72 h depending on the condition and fluorescence of the cells, washed once with phosphate-buffered saline, treated with trypsin for 1 min, pooled after adding medium, centrifuged for 7 min at 1000 rpm (4 °C), washed once with ice-cold phosphate-buffered saline, centrifuged for 7 min at 1000 rpm, and resuspended depending on the size of the cell pellets in 1–2 ml of ice-cold RIPA buffer (150 mm NaCl, 50 mm Tris-HCl, pH 8.0, 0.5% sodium deoxycholate, 1% Nonidet P40, 0.1% SDS, 5 mm EDTA) with added protease inhibitors (1 μg/ml leupeptin, 0.1 mm phenylmethylsulfonyl fluoride, 1 mm pepstatin Aor1 μg/ml antipain, 0.3 μm aprotinin, 1 mm benzamidine). Cells were lysed by sequential pipetting through three different gauge syringe needles (0.7, 0.55, and 0.4 inner diameters). After lysis, 50–100 μlofthe lysate were precipitated with trichloroacetic acid, washed with acetone, and resuspended in loading buffer; the remaining lysate was centrifuged at 14,000 rpm for 15 min at 4 °C. Supernatants were incubated with 2.2 μg of affinity-purified polyclonal anti-TRPV6 antibody (antibody 429) or 3 μg of anti-HA antibody at 4 °C overnight followed by an incubation with protein-A-Sepharose beads for 1–2 h. After four to five washes with RIPA buffer, beads were resuspended in an equal volume of loading buffer, treated for 3 min at 95 °C, subjected to SDS-PAGE, and analyzed by Western blot analysis.Cell Surface Biotinylation—Dishes with confluent transfected HEK cells were washed and treated exactly as described in Ref. 14Mery L. Strauss B. Dufour J.F. Krause K.H. Hoth M. J. Cell Sci. 2002; 115: 3497-3508Crossref PubMed Google Scholar. Controls included monitoring the input of TRPV6 protein as well as stripping and reprobing the blot with an anti-GFP antibody to control that only the TRPV6-EGFP fusion protein (+5p2) but not the EGFP that is co-expressed from the pCAGGS-IRES-EGFP for TRPV6-wt or TRPV6-ΔANK 3 + 4 was precipitated by avidin-agarose.Sedimentation by Sucrose Gradient Centrifugation—HEK 293 cells transfected with TRPV6-EGFP or TRPV6-M5-EGFP were treated with trypsin 48 h after transfection, solubilized in buffer (150 mm NaCl, 50 mm Tris-HCl, pH 8, 0.5% sodium deoxycholate, 5 mm EDTA, 1 μg/ml leupeptin, 0.1 mm phenylmethylsulfonyl fluoride, 1 mm pepstatin A), incubated for 1 h at 4 °C, and centrifuged for 30 min at 52,000 rpm (TLA100) to pellet undissolved material. Samples were loaded onto a continuous 10–20% sucrose gradient (in 20 mm Tris, 5 mm EDTA, 0.1% Triton-X-100, and protease inhibitors as above) and subjected to 50,000 rpm (NVT65) for 1 h and 30 min. 660-μl fractions were then collected, and 35 μl of each fraction were resolved on SDS-polyacrylamide gels and analyzed by immunoblotting. All markers were obtained from Sigma.Antibodies and Reagents—Polyclonal (antibody 429) and monoclonal anti-TRPV6 (26B3) antibodies were generated in our laboratory (15Hirnet D. Olausson J. Fecher-Trost C. Bodding M. Nastainczyk W. Wissenbach U. Flockerzi V. Freichel M. Cell Calcium. 2003; 33: 509-518Crossref PubMed Scopus (65) Google Scholar). Monoclonal anti-GFP and anti-HA antibodies were from Roche Applied Science, and horseradish peroxidase-coupled anti-mouse antibody was from Jackson Laboratories.Bacterial Two-hybrid Assay—Experiments were performed by using the Bacteriomatch two-hybrid system (Stratagene) with the following modifications. DNA encoding a bait or a target protein was cloned in-frame into pTRG or the modified pBT vector (pBTL) containing a (Gly4-Ser)3 linker. All constructs were sequenced. Electrocompetent bacteria (Bacteriomatch two-hybrid system reporter strain, Stratagene) were thawed on ice and electroporated in the presence of 50 ng of pTRG and 50 ng of pBTL (Bio-Rad; 2.5 kV, 200 ohms, 25 μF). After adding 1 ml of SOC (LB + 10 mm MgCl2 + 10 mm MgSO4 + 20 mm glucose) medium, cells were incubated at 30 °C with shaking (225 rpm) for 1.5 h. 100 μl of each transformation were plated on CTCK-agar plates (600 μg/ml carbenicillin, 15 μg/ml tetracycline, 34 μg/ml chloramphenicol, 50 μg/ml kanamycin, 20 mm isopropyl-1-thio-β-d-galactopyranoside, and 4 mg/ml 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-gal) were added to each plate shortly before plating the bacteria) and incubated at 30 °C for 24 h. Interactions were detected by growth and number of blue colonies.Stable Cell Lines and Cell Culture Conditions—HEK 293 (CRL-1573, ATCC, Manassas, VA) cells were maintained in minimal essential medium (Invitrogen) containing 10% fetal calf serum in a 37 °C, 5% CO2 incubator and passaged by treatment with trypsin. An hTRPV6-containing pCDNA3 vector was transfected into the cells by using Super-Fect transfection reagent (Qiagen) and selected in the presence of 500 μg/ml Geneticin (Invitrogen). Surviving cells were isolated, subcloned, and tested by Northern and Western blot for the presence of TRPV6 transcript and protein.Immunofluorescence of EGFP-tagged Constructs—HEK 293 cells or HEK 293 cells stably expressing TRPV6 were grown on poly-d-lysine-coated coverslips and transfected with different EGFP-tagged cDNA constructs using SuperFect. 24–48 h after transfection, cells were washed once with phosphate-buffered saline, mounted in phosphate-buffered saline on glass slides, and imaged in vivo using fluorescence microscopy (Nikon E600, PlanApo 60× A/1.40 oil with attached DN100 or Olympus BX-50, UPlanApo 100×/1.35 with attached imago camera (T.I.L.L. Photonics)) and analySIS aquisition software (Soft Imaging System). Mitochondria were made visible by prior incubation for 30 min with 0.1 μm Texas Red Mito-Tracker (Molecular Probes).RESULTSIdentification of N-terminal Interaction Domains—To identify regions within the TRPV6 protein that are required for homomultimeric subunit assembly, we investigated protein-protein interactions using bacterial two-hybrid interaction screens and co-immunoprecipitation experiments. These approaches have the advantage of being able to investigate interactions both in a mammalian cell environment and in a system that allows a more rapid and independent dissection of binding sites.For co-immunoprecipitation experiments, we made use of a HEK 293 cell line stably expressing TRPV6 (TRPV6s cells) into which we transiently transfected different EGFP-tagged constructs. We used a polyclonal TRPV6 antibody that is directed against an epitope localized at the very C terminus (antibody 429, see Ref. 15Hirnet D. Olausson J. Fecher-Trost C. Bodding M. Nastainczyk W. Wissenbach U. Flockerzi V. Freichel M. Cell Calcium. 2003; 33: 509-518Crossref PubMed Scopus (65) Google Scholar) to precipitate TRPV6 from TRPV6s cells but not from the parental HEK 293 cells (Fig. 1A). Due to post-translational glycosylation, TRPV6 protein can be detected at molecular masses of around 75 up to 85–100 kDa in a denaturing SDS-polyacrylamide gel (see Refs. 5Hoenderop J.G. Voets T. Hoefs S. Weidema F. Prenen J. Nilius B. Bindels R.J. EMBO J. 2003; 22: 776-785Crossref PubMed Scopus (293) Google Scholar and 15Hirnet D. Olausson J. Fecher-Trost C. Bodding M. Nastainczyk W. Wissenbach U. Flockerzi V. Freichel M. Cell Calcium. 2003; 33: 509-518Crossref PubMed Scopus (65) Google Scholar). To avoid detecting the rabbit immunoglobulins used for the immunoprecipitation in the Western blot, we used a mouse monoclonal antibody (26B3) that detects a distinct epitope of TRPV6 further upstream of the 429 site (see “Experimental Procedures”) as a detection antibody. To determine whether we can co-immunoprecipitate individual TRPV6 subunits that are only transiently expressed in the stable cell line we transfected a C-terminally EGFP-tagged TRPV6 that lacks the last 32 amino acids (TRPV6-Δ-EGFP) and thus the epitope for antibody 429. This almost full-length construct with an estimated molecular mass of ∼ 110 kDa could be co-immunoprecipitated from TRPV6-expressing cells using antibody 429 as the precipitating antibody and either a monoclonal anti-GFP antibody (Fig. 1B) or monoclonal anti-TRPV6 (26B3) as detection antibody. The very C-terminal 32 amino acid residues of TRPV6 (aa 694–725) that are critical for binding the regulatory calmodulin (16Niemeyer B.A. Bergs C. Wissenbach U. Flockerzi V. Trost C. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3600-3605Crossref PubMed Scopus (148) Google Scholar) thus do not contribute to TRPV6-TRPV6 interaction. No signal was detected if the EGFP-tagged construct was expressed in HEK 293 cells and antibody 429 was used as the precipitating antibody (Fig. 1B, right lane).As a separate control for the specificity of the co-immunoprecipitation experiments, we used a cell line that stably expresses HA-tagged TRPC3 protein and transiently expresses EGFP-tagged full-length TRPV6. As expected, the anti-HA antibody could precipitate TRPC3, but it did not co-immunoprecipitate the EGFP-tagged full-length TRPV6 construct (Fig. 1, C and D). Conversely the polyclonal antibody 429 did not co-immunoprecipitate hTRPC3 (Fig. 1D).Using the monoclonal anti-GFP antibody as precipitating antibody, we could also co-immunoprecipitate TRPV6 from the stable cell line transfected with interacting EGFP-tagged constructs (data not shown). However, because the monoclonal antibody was not as efficient in immunoprecipitating, we screened for interacting domains using antibody 429 as the precipitating antibody.To narrow down the protein domain within TRPV6 that is responsible for TRPV6-TRPV6 interaction, we repeated the experiment described above after transient transfection and expression of a series of EGFP-tagged fusion proteins encompassing different protein domains of TRPV6. First we focused on the intracellular C-terminal (aa 584–694) and the N-terminal regions (aa 1–328) that contains six rather than three ANK repeats (17Peng J.B. Chen X.Z. Berger U.V. Weremowicz S. Morton C.C. Vassilev P.M. Brown E.M. Hediger M.A. Biochem. Biophys. Res. Commun. 2000; 278: 326-332Crossref PubMed Scopus (183) Google Scholar). Aligning the N-terminal TRPV6 sequence with both an idealized ANK repeat (18Mosavi L.K. Minor Jr., D.L. Peng Z.Y. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16029-16034Crossref PubMed Scopus (282) Google Scholar) and an ANK repeat consensus (19Michaely P. Tomchick D.R. Machius M. Anderson R.G. EMBO J. 2002; 21: 6387-6396Crossref PubMed Scopus (169) Google Scholar) revealed these six ANK repeats (Fig. 2A) with the fifth ANK repeat being a somewhat atypical one that was not recognized as such using the protein prediction program SMART (smart.embl-heidelberg.de/embl), which detected five ANK repeats. Subsequently we subdivided the N-terminal constructs using the ANK repeats as guideposts (Fig. 2B). The check marks in Fig. 2B indicate which constructs were subcloned with a C-terminal EGFP tag and which were subcloned into vectors used for bacterial two-hybrid analysis.Fig. 2A, structure and consensus of TRPV6 ANK repeats. Ank c1 shows the consensus sequence of a designed 33-amino acid repeat (18Mosavi L.K. Minor Jr., D.L. Peng Z.Y. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16029-16034Crossref PubMed Scopus (282) Google Scholar), and Ank c2 shows the repeat consensus after Michaely et al. (19Michaely P. Tomchick D.R. Machius M. Anderson R.G. EMBO J. 2002; 21: 6387-6396Crossref PubMed Scopus (169) Google Scholar). Dark gray boxes indicate identical residues, lighter gray boxes indicate conserved residues, framed amino acids indicate non-conserved sites, the + in c2 indicates a non-polar amino acid. Underlined amino acids are positions that face the convex surface of the three-dimensional ANK repeat structure (19Michaely P. Tomchick D.R. Machius M. Anderson R.G. EMBO J. 2002; 21: 6387-6396Crossref PubMed Scopus (169) Google Scholar). Cylinders indicate the position of the two helical segments within the ANK repeat. Subscript numbers indicate position of the repeats within the TRPV6 sequence (V6) and number pairs to the right indicate the length in amino acid residues of the predicted helical segments. B, schematic representation of N-terminal TRPV6 expression constructs; check marks to the right indicate which construct contains a C-terminal EGFP tag and which one was investigated with the bacterial two-hybrid system (B2H). Numbered boxes indicate the position of putative ANK repeats.View Large Image Figure ViewerDownload (PPT)In the co-immunoprecipitation experiments, we were able to detect interactions of the full-length TRPV6 with the N-terminal region (aa 1–328) but neither with the C-terminal region (aa 584–694) nor with the intracellular loop between transmembrane domains 4 and 5 (aa 466–493) (Fig. 3A). We therefore subdivided the N-terminal domain into shorter fragments and narrowed down one interaction domain to the region around amino acids 116–163 that contains the third ANK repeat. The EGFP-tagged fragment covering ANK 1 and ANK 2 (aa 1–115) was not immunoprecipitated, but the fragment covering in addition ANK 3 and part of the adjacent sequence (aa 1–163) showed positive interaction (Fig. 3A). Controls for a comparable amount of expressed construct included monitoring the trichloroacetic acid-precipitated input (Fig. 3A, bottom) and the immunoprecipitant-supernatant of the expressed fusion protein and the amount of precipitated TRPV6 wild-type protein (by stripping and reprobing blots with antibody 26B3). Due to weak expression of construct 192–328 (ANK 5 + 6), see Fig. 3A, **, we could not exclude a further binding site downstream of amino acid 163. Construct 158–328 (ANK 4–6) also showed a positive interaction albeit weaker than construct 1–191, indicating that a second interaction site exists downstream of the ANK 3 domain (Fig. 3A). A short construct containing ANK domains 3 and 4 (aa 116–191) that ought to contain the first N-terminal interaction site co-immunoprecipitated only very weakly (Fig. 3B), ANK 3 alone showed an even weaker interaction, and no interaction was seen for ANK 4 alone (Fig. 3B). As all constructs contain a C-terminal EGFP tag, we used fluorescence microscopy to monitor their expression and localization. Interestingly all three constructs shown in Fig. 3B were strongly expressed; however, ANK 3 + 4 (aa 116–191) and ANK 3 alone (aa 116–163) were targeted to mitochondria. Fig. 3C shows the fusion protein EGFP fluorescence of living transfected cells in the left panel and Texas Red fluorescently labeled mitochondria of the same cells in the right panel. The finding that most of the recombinant fusion proteins covering ANK 3 and ANK 3 + 4 reside primarily within the mitochondria might explain why only minor amounts are available close to TRPV6-containing membranes for interaction with full-length TRPV6. In addition, this finding may indicate that the interactions observed after immunoprecipitation are not due to an artificial association of overexpressed proteins or protein fragments during solubilization of cells.Fig. 3Identification of the interaction domain.A and B, co-immunoprecipitation of different TRPV6 domains each tagged with a C-terminal (C-Term) EGFP with TRPV6 from TRPV6s cells using 429 as precipitating antibody and anti-GFP as detecting antibody, *, see Fig. 1; **, weak input. C, in vivo co-localization of EGFP-tagged constructs with mitochondria. The left panel shows EGFP fluorescence, and the right panel shows fluorescence of mitochondria marked with Texas Red Mito-Tracker.View Large Image Figure ViewerDownload (PPT)Analysis of 116–191—Is the TRPV6 region 116–191 including ANK 3 and 4 alone sufficient for subunit interaction, and can the interaction with full-length TRPV6 be restored by forcing the interaction domain construct out of the mitochondria? To answer these questions, a foreign protein, the four-transmembrane γ1 subunit of voltage-gated calcium channels was inserted between the ANK 3 + 4 (aa 116–191) fragment of TRPV6 and the EGFP tag (Fig. 4). γ1-EGFP alone, when expressed in TRPV6s cells, localized to the plasma membrane (data not shown) but could not be co-immunoprecipitated with TRPV6 (Fig. 4A). However, fusing the ANK 3 + 4 domain (aa 116–191) to the N terminus of γ1 indeed was sufficient to both restore plasma membrane localization of 116–191 and, more importantly, to enable an interaction between the γ subunit and TRPV6 channels as seen by co-immunoprecipitation of TRPV6 and ANK 3 + 4-γ1-EGFP (Fig. 4B). The ANK 3 + 4 region thus can act as a strong association signal when localized to TRPV6-expressing membranes.Fig. 4ANK 3 + 4 (aa 116–191) when fused to the calcium channel γ1 subunit is sufficient to enable interaction between the γ1 subunit and TRPV6.A, the γ1-EGFP fusion protein alone does not interact with TRPV6; no EGFP signal can be detected after immunoprecipitation of TRPV6 with antibody 429. B, adding ANK 3 + 4 to γ1-EGFP confers TRPV6 binding. Blots were stripped and reprobed with a monoclonal anti-TRPV6 antibody (26B3) to assure equal amounts of immunoprecipitated TRPV6. C, ANK 3 + 4 is a multimerization domain. Cell extracts of transfected HEK 293 cells were either solubilized in loading buffer in the presence (+) or absence (–) of 10 mm dithiothreitol, 1.43 m β-mercaptoethanol and treatment for 3 min at 95 °C. Fusing ANK 3 + 4 to EGFP results in the formation of higher molecular mass complexes with apparent dimers and trimers visible (arrows). Expected molecular masses of ANK 3 + 4-GFP are as follows: monomer, 35.2 kDa; dimer, 70.4 kDa; trimer, 105.6 kDa. IP, immunoprecipitation; WB, Western blot.View Large Image Figure ViewerDownload (PPT)We also investigated multimerization of EGFP alone and ANK 3 + 4-EGFP. Solubilizing transfected cells with an EGFP expression vector both under weakly denaturing conditions (–: 4% SDS, 37 °C) and under denaturing conditions (+: 4% SDS, 10
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