The Spectrin Cytoskeleton Influences the Surface Expression and

The Spectrin Cytoskeleton Influences the Surface Expressionand Activation of Human Transient Receptor PotentialChannel 4 Channels*□S

Received for publication, November 28, 2007 Published, JBC Papers in Press, November 29, 2007, DOI 10.1074/jbc.M709729200

Adam F. Odell1, Dirk F. Van Helden2, and Judith L. ScottFrom the School of Biomedical Sciences, Faculty of Health, University of Newcastle, Level 5, MSB, University Drive,New South Wales 2308, Australia

Despite over a decade of research, only recently have themechanisms governing transient receptor potential channel(TRPC) channel function begun to emerge, with an essentialrole for accessory proteins in this process. We previously iden-tified a tyrosine phosphorylation event as critical in the plasmamembrane translocation and activation of hTRPC4 channelsfollowing epidermal growth factor (EGF) receptor activation.Tofurther characterize the signaling events underlying this proc-ess, a yeast-two hybrid screen was performed on the C terminusof hTRPC4. The intracellular C-terminal region from proline686 to leucine 977 was used to screen a human brain cDNAlibrary. Twomembers of the spectrin family, �II- and �V-spec-trin, were identified as binding partners. The interaction ofhTRPC4 with �II-spectrin and �V-spectrin was confirmed byglutathione S-transferase pulldown and co-immunoprecipita-tion experiments. Deletion analysis identified amino acids 730–758 of hTRPC4 as critical for the interaction with this regionlocated within a coiled-coil domain, juxtaposing the Ca2�/cal-modulin- and IP3R-binding region (CIRB domain). This regionis deleted in the proposed �hTRPC4 splice variant form, whichfailed to undergo both EGF-induced membrane insertion andactivation, providing a genetic mechanism for regulating chan-nel activity. We also demonstrate that the exocytotic insertionand activation of hTRPC4 following EGF application is accom-panied by dissociation from �II-spectrin. Furthermore, deple-tion of�II-spectrin by small interference RNA reduces the basalsurface expression of �hTRPC4 and prevents the enhancedmembrane insertion in response to EGF application. Impor-tantly, depletion of �II-spectrin did not affect the expression ofthe � variant. Taken together, these results demonstrate that adirect interaction between hTRPC4 and the spectrin cytoskele-ton is involved in the regulation of hTRPC4 surface expressionand activation.

Recently, the seven mammalian members of the canonic orclassic TRPC3 subfamily (TRPC1–7) have emerged as strongcandidates for regulating non-excitable Ca2� entry. They canbe broadly classified into two subgroups based on homologyand activation mechanisms. The TRPC3/6/7 subgroupresponds to the generation of diacylglycerol following receptor-mediated activation of phospholipase C, whereas members oftheTRPC1/4/5 subgroupdemonstrate both store-operated andreceptor-mediated properties. The store-operated phenotypeis typified by the reduction in thapsigargin-induced Ca2� entryin endothelial cells derived from the TRPC4 null mouse (1, 2).An involvement in receptor-mediated Ca2� entry was alsodemonstrated in the TRPC4 null mouse as well as in HEK293cells treated with TRPC4 antisense oligonucleotides, whichexhibited impaired carbachol-mediated oscillations (1–3).TRPC4 has also recently been identified as an essential compo-nent of EGF receptor-stimulated Ca2� influx (4); however, theprecise nature of the signaling pathway regulating activation ofCa2� entry following receptor stimulation and store-depletionremains unclear. The organization of TRPC channels into sig-naling complexes is proposed as critical in regulating the acti-vation of TRPC channels and controlling their physiologicalfunctions (5). Through interactions with various protein andlipid molecules, some of which remain unknown, TRPC chan-nels are assembled into functional signaling microdomains (6).Definitive identification and characterization of the microdo-mains involved in TRPC-regulated Ca2� entry are necessary tofully elucidate themechanisms of store- and receptor-operatedCa2� entry. Recent efforts have identified several potentialcomponents of the signaling machinery involved in TRPC4-mediated Ca2� entry. These include the Na�/H�-exchangerregulatory factor 1 (NHERF), various IP3R subtypes, Ca2�-cal-modulin, immunophilins, protein 4.1, Homer and stromalinteracting protein 1 (7–11). Most proteins have been found tobind to discrete domains within TRPC4, such as the C-terminalCIRB domain for which Ca2�-calmodulin and IP3Rs compete(12, 13). Removal of the CIRB domain from the �TRPC4 splicevariant was reported to enhance carbachol-mediated Ca2�

* This work was supported by the National Health and Medical ResearchCouncil (NHMRC) of Australia, the Australian Research Council, and theHunter Medical Research Institute. The costs of publication of this articlewere defrayed in part by the payment of page charges. This article musttherefore be hereby marked “advertisement” in accordance with 18 U.S.C.Section 1734 solely to indicate this fact.

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Fig. S1 and Table S1.

1 To whom correspondence should be addressed (current address): Univer-sity of Leeds, Leeds, United Kingdom. Tel.: 44-113-343-7728; Fax: 44-113-343-6603; E-mail: [email protected].

2 An NHMRC Principal Research Fellow and Brawn Senior Fellow.

3 The abbreviations used are: hTRPC4, human transient receptor potentialchannel 4; EGF, epidermal growth factor; EGFR, epidermal growth factorreceptor; GST, glutathione S-transferase; CIRB, Ca2�/calmodulin- and IP3R-binding region; NHERF, Na�/H�-exchanger regulatory factor 1; STK, Src-family tyrosine kinase; aa, amino acid(s); CMV, cytomegalovirus; HA,hemagglutinin; siRNA, small interference RNA; RIPA, radioimmune precip-itation assay; GFP, green fluorescent protein; ERK, extracellular signal-reg-ulated kinase.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 283, NO. 7, pp. 4395–4407, February 15, 2008© 2008 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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entry in HEK293 cells, and led to the suggestion of the lessplentiful� variant being the active formof the channel (14–16).Additionally, the role of NHERF in controlling the surfaceexpression and localization of TRPC4 is also well documented(17–19), and recently a role for the spectrin cytoskeleton in thecontrol of endothelial ISOCwas identifiedwhere a spectrin-pro-tein 4.1 interaction influenced Ca2� entry following thapsigar-gin treatment (20).Spectrins are �200–400 kDa proteins that form extended,

flexible protein dimers of �200–260 nm in length and 3–6 nmacross and contain actin-binding domains at each end. Spectrindimers are composed of �- and �-subunits, which associatelaterally to form anti-parallel heterodimers. These het-erodimers then assemble head-to-head to form heterotetram-ers, which are organized in a polygonal network of 5–7extended spectrin molecules linked to short actin filaments(21). This spectrin-actin network is coupled to the plasmamembrane primarily by association with ankyrin, which in turnis bound to dimers of the Na�/H� exchanger (22). Additionalconnections are formed between band 4.2, protein 4.1, theMAGUK p55, and glycophorin C, which associate at the spec-trin-actin junction (21). Spectrin is now recognized as a ubiq-uitous scaffolding protein that acts in conjunctionwith a varietyof adaptor proteins to organize membrane microdomains onboth the plasmamembrane and on intracellular organelles (23–25). Spectrin appears to organize and control the localizationand function of integral membrane proteins by creating mesh-works with other proteins (21). A role for TRPC4 in mediatingthe interaction between the spectrin cytoskeleton and Ca2�

entry was recently proposed, with disruption of the protein 4.1-binding site on TRPC4 preventing activation of the endothelialISOC channel (10, 26).

Here, we have identified a direct coupling between �II-spec-trin and hTRPC4. This is dependent on a coiled-coil domainlocated near the proline-rich, protein 4.1-binding motif ofhTRPC4 and is mediated by the C-terminal spectrin repeats of�II-spectrin. Previous experiments demonstrated enhancedinsertion of hTRPC4 into the plasmamembrane following acti-vation of the EGF receptor (27). Here we report that this inser-tion is accompanied by the dissociation of spectrin-hTRPC4interaction. Channels lacking the spectrin-binding site failed toundergo membrane insertion and were unable to be activatedby EGF. This suggests that a direct associationwith the spectrincytoskeleton may influence the signaling events involved incontrolling the level of active hTRPC4 channels expressed atthe cell surface.

EXPERIMENTAL PROCEDURES

Molecular Biology—The C terminus of hTRPC4 (CT, aa686–977) and various truncations (CT1, aa 686–784; CT2, aa686–859; CT3, aa 759–859; CT4, aa 759–977; and CT5,aa 846–977) were amplified by PCR from pCMVSport-hTRPC4(kindly provided by Dr. J. W. Putney (28)) and cloned into theBamH1 and Sal1 restriction sites of pBTM116 in frame withLexA DNA-binding domain. A full list of primers can be foundin supplemental Table S1. Truncations of hTRPC4 were co-transformed with pACT2-�II-spectrin into L40 yeast andassessed for reporter gene activation. For GST fusion protein

construction, the C-terminal fragments of hTRPC4 (CT andCT1-CT5) were restriction-digested from pBTM116 withEcoR1 and Sal1 and subcloned into the corresponding sites inpGEX-4T-1. The C terminus of �II-spectrin (aa 1998–2474)was amplified by PCRusing the primers outlined in supplemen-tal Table S1 and the insert ligated into the Sal1 restriction site ofpBTM116. The C terminus of �II-spectrin was amplified byPCR from clone 18 and ligated into the EcoR1 and Sal1 restric-tion sites of pAS2.1. The C-terminal fragments of �V-spectrinand �II-spectrin were amplified by PCR from clones 52 and 18,respectively, using the primers outlined in supplemental TableS1 and the PCR fragments cloned into pGEX-4T-1 vector forexpression of GST fusion proteins.For the generation of the �II-spectrin GST fusion protein

truncations, various fragments were amplified by PCR frompBluescript-�II-spectrin (kindly supplied by Dr. J. S. Morrow).See supplemental Table S1 for primers where, shown in brack-ets, are the corresponding beginning and final �II-spectrinamino acids of the truncations. PCR inserts were digested withthe relevant restriction enzymes, cloned into pGEX-4T-1 andconstructs assessed by DNA sequencing and the induction ofprotein synthesis. The further �II-spectrin truncation, �II-(951–2472), was generated by PCR from pBluescript-�II-spec-trin. PCR products were ligated into Sal1 and Not1 restrictionsites of pGEX-4T-1.To generate the bacterial constructs of full-length �II-spec-

trin for GST-tagged protein generation, a 1000-bp PCR frag-ment corresponding to the 5�-end of the �II-spectrin wasamplified and ligated into pBluescript digested with Kpn1 andXba1 restriction sites using the following primer pair; For, ACG-GGTACCAGTCGACATGGACCCAAGTGGGGTCAAA-GTGCTG and Rev, TGCTCTAGAGCAGCAAGATCTCTC-TCGAGACCCTC. For the generation of full-length untagged�II-spectrin GST fusion protein, an Sal1 site was used after the5� Kpn1 restriction site. This produced pBluescript constructscontaining the 5� end of �II-spectrin with either Kpn1 or Sal1restriction sites. The pBluescript-�II-spectrin construct alsocontained a Not1 restriction site at the 3� end. Full-length �II-spectrin was subsequently subcloned from pBluescriptII-�II-spectrin with Sal1 and Not1 and ligated into pGEX-4T-1. Allconstructs were confirmed by DNA sequencing and assessedfor full-length protein expression by Western blotting.The � and � splice variants of hTRPC4 as well as the M1

(MUT1), M2 (MUT2), and M3 (MUT3)-hTRPC4 deletionmutants were generated using a modified linker-scanningmutation approach. Briefly, primers were designed flanking theregions to be excised and twoPCR fragments generated encom-passing the 5� and 3� segments on either side of the proposeddeletion. These two PCR products were combined and utilizedas the template for a second round of PCR amplification withthe forward (containing the FLAG epitope), and reverse prim-ers were utilized in the first PCR round covering the start andstop codons of hTRPC4. The primers are outlined in supple-mental Table S1. The resultant products were assessed for theabsence of intendedC-terminal regions by restriction digestionand DNA sequencing. Correct expression of the protein prod-uct was assessed by transient transfection andWestern blottingwith anti-FLAG antibody.

Interactions between hTRPC4 and Spectrin

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Yeast Media and Manipulation—The L40, Y187, and Y190Saccharomyces cerevisiae yeast strains were grown at 30 °C onYPDmedia (10 g/liter yeast extract, 20 g/liter peptone, 0.1 g/li-ter adenine, 2% dextrose) or synthetic minimal media with theappropriate supplements. Synthetic minimal media consistedof Dropout base powder (1.7 g/liter yeast nitrogen base, 20 g/li-ter dextrose, 5 g/liter ammonium sulfate, BIO 101, Vista, CA)supplemented with the appropriate synthetic media formula-tion (CSM, BIO 101). The synthetic (YC) complete media wasthen autoclaved at 121 °C for 15–20 min with or without agar(25 g/liter) and stored at 4 °C in the dark. Tryptophan wasremoved to select the bait vectors, pBTM116, pFBL23, andpAS2.1, and leucine was omitted to select for the cDNA librarypACT2 vector. Lysine and uracil were omitted from all YC syn-thetic media to maintain selection of the integrated HIS3 andlacZ reporter genes, respectively, in L40 and Y190 yeast. Histi-dine was removed when selecting for interacting bait and preyproteins. In all yeast YCmedia, lysine and uracil are absent. Allthe initial characterizations of bait constructs and libraryscreens were performed in the L40 strain of Saccharomyces cer-evisiae (29). Two bait vectors were utilized to investigatehTRPC4 binding partners: the LexA-based bait vector,pBTM116, and theGAL4-based pAS2.1 vector (Clontech). Theprey vector used was the pACT2 GAL4-based DNA-activationdomain vector (Clontech). The human brain cDNA library waspurchased already cloned into the GAL4 DNA-activationdomain of pACT2 prey plasmid and titered according to themanufacturer’s instructions.Antibodies—Anti-M2 FLAG antibody and anti-�-tubulin

antibodies were obtained from Sigma-Aldrich. Anti-PY20 anti-body was supplied by BD Transduction Laboratories, and the4G10 and anti-�1-integrin antibodies were from Upstate Bio-technology. Anti-TRPC4 rabbit polyclonal antibody was fromAlomone Laboratories (Israel). The anti-TRPC4 (C20) goat andanti-FYN (FYN3) rabbit polyclonal antibodies were purchasedfrom Santa Cruz Biotechnology. Anti-EGFR mouse mono-clonal antibody was kindly supplied by Dr. M. Hibbs. Anti-HAmonoclonal, rabbit polyclonal anti-�II-spectrin, and rabbitanti-phospho-ERK1/2 monoclonal antibodies were from CellSignal Technologies. The rabbit polyclonal anti-NHERF anti-body was from Abcam Laboratories. All secondary antibodies,anti-GST antibody and streptavidin-horseradish peroxidasewere from Amersham Biosciences, GE, except anti-goatAlexaFluor488, which was from Molecular Probes.Cell Culture, Transfection, and Immunofluorescence

Microscopy—Cell lines were maintained in Dulbecco’s modi-fied Eagle’s medium containing 2 mM L-glutamine, 10% fetalbovine serum at 37 °C in 5% CO2. Cell culture reagents wereobtained from Invitrogen. HEK293 cells stably expressingFLAG-hTRPC4 constructsweremaintained in the abovemediacontaining 400�g/ml Zeocin (Invitrogen). COS-7 andHEK293cells were transfected with expression plasmids with Lipo-fectamine 2000 (Invitrogen) according to manufacturer’sinstructions, and 35–60% transfection efficiency was routinelyachieved. siRNA depletion of �II-spectrin was performed byreverse transfection using Lipofectamine RNAiMAX reagent(Invitrogen). Control and �II-spectrin (sc-36549) siRNA wereobtained from Santa Cruz Biotechnology. For immunofluores-

cence detection, transfected cells were fixed in 4% paraformal-dehyde and stained with anti-TRPC4 C20 antibody followed byanti-goat AlexaFluor488 secondary antibody. Nuclei were visu-alized by 4�,6-diamidino-2-phenylindole staining. Cells weremounted with Anti-Fade (DakoCytomation) and observedunder 63� oil-immersion objective on a Zeiss Axiovert 100Mconfocal microscope and analyzed with Zeiss LSM510.In Vitro Binding Assays, Immunoprecipitation, and

Immunoblotting—GST fusion proteins were generated as out-lined in a previous study (27). For GST-pulldown assays equalvolumes of immobilized GST fusion proteins were incubatedwith mammalian cell lysates containing 1–3 mg of protein at4 °C for 2 h or overnight. Beads were washed four times withlysis buffer, and bound proteins were resolved by 8% SDS-PAGE and visualized by Western blotting on nitrocellulose asindicated. For preparation of lysates of COS-7, HEK293, andA431 cells, cells were washed with ice-cold phosphate-bufferedsaline, lysed with RIPA buffer, and equal amounts of proteinsubjected to immunoprecipitation with protein G-Sepharose(GE, Amersham Biosciences) as outlined previously (27). Forimmunoprecipitation from rat brain membranes, lysates werepre-cleared with protein G-Sepharose followed by centrifuga-tion. Immune complexes were washed four times with lysisbuffer and resolved, along with lysates by 8% SDS-PAGE fol-lowed by Western blotting onto Hybond ECL nitrocellulosemembrane (GE, Amersham Biosciences). Membranes wereblocked and probed with indicated antibodies for 2 h or over-night and visualized with ECL Plus reagent on Typhoon Imag-ing System (GE, Amersham Biosciences). For quantification,the protein bands were analyzed with ImageQuaNT software.Biotinylation of Cell Surface Proteins—Following treatment,

indicated cells were biotinylated with EZ-Link Sulfyl-NHS-Bi-otin (Pierce Biotechnology) as outlined previously (27). Lysateswere then prepared by addition of modified RIPA buffer, andequal quantities of protein were either incubated with 50%NeutrAvidin-agarose slurry (Pierce Biotechnology) or immu-noprecipitated with the appropriate antibody for 2 h at 4 °C.Beads were washed three times with lysis buffer, subjected toSDS-PAGE, and analyzed by Western blotting.Purification of Rat Brain Microsomes—Adult Sprague-Daw-

ley rats were killed by decapitation according to InstitutionalAnimal Ethics, and cortex tissues were dissected and homoge-nized in 10 volumes of ice-cold homogenization buffer (320mMsucrose, 1 mM EDTA (pH 7.5)). The homogenate was centri-fuged at 1,000 � g for 10 min, and the supernatant at 15,000 �g for 30 min at 4 °C. The pellet (P2) was resuspended in RIPAbuffer, and insolublematerial was removed by centrifugation. 1mg of soluble rat brain protein was used for immunoprecipita-tion with 4 �g of the indicated antibodies, and 25 �l of proteinG-Sepharose overnight at 4 °C. Washed immunocomplexeswere analyzed as above.Ca2� Imaging—Ca2� imaging was performed as outlined

previously (27). Briefly, cells were cultured on glass coverslipsand loaded with 2–4 �M fura-2 acetoxymethyl ester in Dulbec-co’smodified Eagle’smedium at 37 °C for 30min and incubatedin HEPES-buffered saline (140 mM NaCl, 5 mM KCl, 0.5 mMMgCl2, 5.5 mMHEPES, 10mM glucose, 2 mMCaCl2, pH 7.4) for30minprior to stimulation. In experiments performed inCa2�-




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free medium, cells were placed in HEPES-buffered saline with-out CaCl2 containing 200 �M EGTA. Changes in [Ca2�]i weremonitored as 340/380 nm fluorescence ratio, and single cellimaging was performed on transfected HEK293 cells illumi-natedwith LambdaDG-4 lamp, capturedwith anOrca ER cam-era, and analyzed with MetaFluor program (Universal ImagingDevices) under 40� objective lens. All statistics were per-formed using Prism, one-way analysis of variance, and theTukey post test.

RESULTS

The C Terminus of hTRPC4 Binds to �II-Spectrin and�V-Spectrin—A yeast two-hybrid screen in L40 yeast of ahuman brain cDNA library generated in pACT2was performedusing a LexA fusion protein of the C terminus of hTRPC4(hTRPC4-CT, aa 686–977) to identify intracellular proteinsthat interact with hTRPC4 and regulate its activation. A num-ber of potential binding partners were identified, including thepreviously isolated hTRPC4-interacting protein, Na�/H�-ex-changer regulatory factor or NHERF (1 out of 128 positiveclones). A novel putative interaction was also identifiedbetween the cytoskeletal proteins, �II-spectrin (2 out of 128positive clones) and �V-spectrin (10 out of 128 positive clones)and the C terminus of hTRPC4. The interactions betweenhTRPC4 and both �II-spectrin and �V-spectrin were con-firmed by re-introduction of the constructs into L40 yeast incombination with control vectors (Table 1). Isolation andsequence analysis of the interacting spectrin clones revealedthat hTRPC4-CT was binding to the C termini of both mole-cules; aa 1998–2472 of �II-spectrin and aa 3232–3674 of�V-spectrin. The interacting amino acids corresponded tospectrin repeats 19–21 and theC-terminal EF-hand domains of�II-spectrin and spectrin repeats 28–30 and PH domain of�V-spectrin.

The interactions between hTRPC4-CT and �II-spectrin and�V-spectrin were confirmed by co-transformation of the inter-acting cDNAs into L40 yeast expressing the C terminus ofhTRPC4 (pBTM116-CT). Neither clone isolated from the yeasttwo-hybrid screen interacted with the control LexA-LaminCprotein when co-expressed with pBTM116-LaminC; however,both �II-spectrin and �V-spectrin activated expression of thelacZ and HIS3 reporter genes in L40 yeast, thereby confirmingthe specificity of their interaction with hTRPC4 (Table 1). Alsoshown in Table 1 are the negative (any assay with an empty

vector) and positive control (pBTM116-Fos and pACT2-Jun)reactions. To further confirm the interaction between hTRPC4and the spectrin molecules, the C terminus of hTRPC4 wascloned into an alternative bait vector, the GAL4 DNA-bindingdomain-based vector, pAS2.1 (pAS2.1-CT), and co-expressedwith the pACT2-spectrin plasmids in Y187 yeast. Under theseconditions, both �II-spectrin (Fig. 1A) and �V-spectrin (datanot shown) induced activation of the lacZ reporter gene. Therewas no lacZ expression when either prey construct was co-expressed with empty pAS2.1 vector (Fig. 1A). Similarly,pAS2.1-CT failed to activate the reporter gene in the absence ofspectrin fusion proteins (data not shown). Negative controltransformations did not activate lacZ reporter gene expression(Fig. 1A). In addition, a domain exchange assay was performedbetween hTRPC4-CT transferred to pACT2 and �II-spectrinbait generated in pAS2.1. Upon co-transformation intoY187 yeast strain, only pAS2.1-�II-spectrin and pACT2-hTRPC4-CT bait-prey combinations induced reporter geneactivation as assessed by colony-lift filter assay (Fig. 1A). Noother control transformations induced lacZ gene activation

FIGURE 1. The C terminus of hTRPC4 interacts with �II-spectrin and�V-spectrin. A, colony-lift filter assay was performed between �II-spectrinand hTRPC4. Y187 yeast were transformed with the indicated bait/prey plas-mid combinations and selected on YC-Trp-Leu drop-out media. Single colo-nies were selected and re-streaked onto the same selective media prior toanalysis of lacZ gene activation by colony-lift filter assay. B, GST-pulldownanalysis of the interaction between hTRPC4 and �II-spectrin/�V-spectrin.Purified and immobilized GST, GST-�II-spectrin-CT, or GST-�V-spectrin wereincubated with 1% Nonidet P-40-solubilized cell lysates of HEK293 cells tran-siently expressing FLAG-hTRPC4. Bound proteins were analyzed by Westernblotting using anti-FLAG M2 antibody. Also shown is a Coomassie stain of GSTfusion proteins. C, purified GST or GST-hTRPC4-CT were incubated with RIPA-solubilized HEK293 cell lysates, and bound proteins were analyzed by West-ern blotting with anti-�II-spectrin antibody.

TABLE 1Summary of confirmatory transformations between hTRPC4 andeither �II-spectrin or �V-spectrinL40 yeast were co-transformedwith the indicated bait and prey plasmids and platedonto both YC-Trp-Leu- and YC-Trp-Leu-His-selective drop-outmedia. Yeast wereassayed for lacZ reporter gene activation by colony-lift filter assays. Indicated inparentheses are the corresponding amino acids of each protein analyzed in theassay.

Bait Prey HIS3 lacZpBTM116 pACT2 � �pBTM116-Fos pACT2-Jun � ��pBTM116-LaminC pACT2 � �pBTM116-CT pACT2 � �pBTM116-CT-(686–977) pACT2-�II-(1998–2472) � ��pBTM116-LaminC pACT2-�II-(1998–2472) � �pBTM116-CT-(686–977) pACT2-�V-(3232–3674) � �pBTM116-LaminC pACT2-�V-(3232–3674) � �




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(Fig. 1A). Similar resultswere obtainedupon co-transformationof pBTM116-�II-spectrin and pACT2-CT in L40 yeast (datanot shown). Taken together, these results confirm that the Cterminus of hTRPC4 interacts specifically with �II-spectrinand �V-spectrin regardless of bait-prey combination and yeaststrain utilized.GST fusion protein pulldown assays were also performed to

confirm and biochemically characterize the interactionbetween�II-spectrin,�V-spectrin, and hTRPC4. RecombinantGST fusion proteins of �II-spectrin (GST-�II-spectrin-CT)and �V-spectrin (GST-�V-spectrin-CT) regions were isolatedfrom the two-hybrid screen, generated in BL21 Escherichia coli,and incubated overnight with Nonidet P-40 lysates of HEK293cells transiently transfected with FLAG-tagged hTRPC4.Bound proteins were examined for the presence of hTRPC4 bySDS-PAGE and immunoblottingwith anti-M2 FLAGantibody.As shown in Fig. 1B, both C-terminal �II-spectrin and�V-spectrin constructs isolated from the yeast two-hybridscreen were able to bind FLAG-hTRPC4 under the conditionsexamined. No significant interaction was detected betweenGSTalone andhTRPC4 (Fig. 1B). The affinity ofGST-�II-spec-trin for FLAG-hTRPC4 was greater than that of �V-spectrindespite similar fusion protein input, suggesting a stronger asso-ciation between the former proteins (Fig. 1B). This, combinedwith the known role of�II-spectrin in vesicle trafficking (30, 31)and the interaction of �II-spectrin with numerous Ca2� signal-ing proteins, provided the impetus for further investigation ofthis putative interaction. Thus, the interaction betweenhTRPC4-CT and endogenous full-length �II-spectrin was alsoinvestigated in reverse pulldown assays using the immobilizedC terminus of hTRPC4 (GST-hTRPC4-CT) incubated withRIPA-solubilized HEK293 extracts. Bound proteins were ana-lyzed by SDS-PAGE and immunoblotting for �II-spectrin. Asshown in Fig. 1C, full-length�II-spectrin (230 kDa)was presentin precipitates from GST-hTRPC4-CT but not in those per-formedwithGST alone. Equal quantities of fusion protein wereincluded in each reaction.The Interaction between hTRPC4 and �II-Spectrin Is

Mediated by Spectrin Repeats in the C Terminus of

�II-Spectrin—The interaction offull-length�II-spectrin with hTRPC4was also studied in GST pulldownassays. Full-length �II-spectrin(1–2474, 230 kDa) and truncatedforms of the molecule (aa 951–2472and 1998–2474) expressed as GSTfusion proteins were able to bindFLAG-hTRPC4 (Fig. 2A). However,a decrease in the affinity of�II-spec-trin for hTRPC4was observed as thelength of fusion protein increased(Fig. 2A), possibly indicating com-petition between other proteins inthe HEK293 lysates and FLAG-hTRPC4 for �II-spectrin-bindingsites. An interaction between full-length �II-spectrin and TRPC4 wasalso sought through co-immuno-

precipitation experiments of FLAG-tagged murine TRPC4from transiently transfected HEK293 cells. Proteins bound toanti-FLAG antibodies incubated with either control or FLAG-mTRPC4-transfected HEK293 cells were immunoblotted forthe presence of �II-spectrin. Under these conditions �II-spec-trin was shown to co-immunoprecipitate from cells expressingFLAG-mTRPC4 and not from control HEK293 cells trans-fected with empty vector (Fig. 2B) indicating the interaction ispreserved between human and mouse. Additionally, NHERFwas also present in the FLAG-mTRPC4 precipitates, indicatingthe formation of a complex between mTRPC4, �II-spectrin,and NHERF in HEK293 cells (Fig. 2B).Given the strong interaction between �II-spectrin and

hTRPC4, mutation deletion analysis of �II-spectrin cDNA wasused to define the region of the spectrin molecule responsiblefor binding to hTRPC4. The �II-spectrin fragment isolatedfrom the yeast two-hybrid screen contained the final threespectrin repeats and the C-terminal EF1 and EF2 Ca2�-bindingdomains (see Fig. 3A, schematic). Previous reports have impli-cated spectrin repeats in mediating a variety of protein-proteininteractions. For example, the interaction between the spectrinrepeat-containing protein�-actinin-2 and the Kv1.5 potassiumchannel was dependent upon the presence of spectrin repeatsbut independent of the EF1 and EF2 domains of �-actinin (32).Therefore, it was of interest to investigate the role of the spec-trin repeats in binding to hTRPC4. A series of C-terminal trun-cated�II-spectrin constructs was generated as GST fusion pro-teins: GST-S1, spectrin repeat 19; GST-S2, spectrin repeats 19and 20; GST-S3, spectrin repeats 19, 20, and 21; GST-S4, spec-trin repeat 21; and GST-S5, containing the EF1 and EF2domains (Fig. 3A), and incubated with lysates of HEK293 cellstransfected with FLAG-hTRPC4. As shown in Fig. 3, A (indi-cated by the � or under hTRPC4) and B, all fusion proteinscontaining spectrin repeats (S1, S2, S3, and S4) bound FLAG-hTRPC4. Furthermore, regardless of the number or type ofspectrin repeats included, there was no significant difference inthe level of hTRPC4 binding observed among the truncations(Fig. 3B). GST fusion proteins containing the EF1 and EF2domains alone, did not bindhTRPC4, however removal of these

FIGURE 2. hTRPC4 forms a complex with �II-specrin. A, GST, GST-�II-spectrin (aa 951–2472), GST-�II-spectrin(aa 1998 –2472), and full-length GST-�II-spectrin (aa 1–2472) were incubated with lysates from HEK293 tran-siently expressing FLAG-hTRPC4, and bound proteins were analyzed by immunoblotting with anti-FLAG anti-body. Total lysate, corresponding to 6% total input, is shown as a positive control. Also shown is a Coomassiestain of the GST fusion proteins. B, mTRPC4 was immunoprecipitated with anti-FLAG antibody from untrans-fected HEK293 cell lysates and cells expressing mTRPC4 and immunoblotted with anti-�II-spectrin, anti-FLAG,and anti-NHERF antibodies.




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domains from the spectrin repeats reduced hTRPC4 bindingcompared with the �II-spectrin fragment isolated in the origi-nal two-hybrid screen (Fig. 3B). These results indicate that theEFdomains strengthenormodulate the interaction of�II-spec-trin with hTRPC4 rather than directly participate in the bind-ing. Taken together, these results suggest that the spectrinrepeats of �II-spectrin are responsible for mediating the inter-action with hTRPC4. There appears to be a redundancy inbinding, with a single spectrin repeat able to mediate the asso-ciation with hTRPC4. Moreover, the repeats do not act in anadditive manner to strengthen the interaction betweenhTRPC4 and �II-spectrin.The Spectrin Binding Site Is Absent in the � Splice Variant of

hTRPC4—To define the region of hTRPC4 responsible for �II-spectrin binding, DNA constructs encoding fragments of the Cterminus of hTRPC4, CT1-hTRPC4 (aa 759–977), CT2-hTRPC4 (aa 846–977), CT3-hTRPC4 (aa 759–859), CT4-hTRPC4 (686–859), and CT5-hTRPC4 (aa 686–784), weregenerated by PCR and cloned in-frame with the LexA DNAbinding domain of pBTM116 (Fig. 4A). Yeast two-hybrid assayswere then performed between the hTRPC4 C-terminal trunca-tions and theC-terminal�II-spectrin (Fig. 4A) and�V-spectrin

(data not shown) fragments isolatedfrom the library screen. Under theseconditions only the CT4-hTRPC4(aa 686–859) and CT5-hTRPC4 (aa686–784) fragments interactedwith �II-spectrin and �V-spectrin(Fig. 4A), as determined by reportergene activation, indicating that theinteracting domain in hTRPC4potentially lies between amino acids686 and 784 (Fig. 4A). Amino acids686–784 consist of a coiled-coildomain �95 amino acids upstreamof the EWKFAR TRP box and inde-pendent of the recently describedprotein 4.1 binding site and theestablished Ca2�-calmodulin/IP3R-binding domain (CIRB domain), thelatter of which is absent in the�hTRPC4 splice variant (14, 33).

The novel spectrin-binding sitecoincides with a recently describedadditional calmodulin-binding do-main, located 95 amino acidsdownstream of the CIRB domainresponsible for facilitating theactions of Ca2�/calmodulin (34).This region was absent in the postu-lated �hTRPC4 splice variant,which lacks 141 amino acids (aa730–870) in the C-terminal tail.Because the potential �II-spectrinbinding site of hTRPC4 partiallyoverlaps with splice sites for the�hTRPC4 variant, it was of interestto determine whether �hTRPC4

could bind�II-spectrin. FLAG-tagged �hTRPC4was generatedby linker-scanning PCR, and the sequence was verified by DNAsequencing. The �hTRPC4 variant (lacking aa 785–868) wassimilarly generated for use as a control. A schematic of theregions deleted from each variant is shown in Fig. 4B. Full-length hTRPC4 (� variant) and the � and � variants were tran-siently transfected into HEK293 cells, and lysates were incu-bated with either immobilized GST, GST-�II-spectrin(C-terminal domain), or GST-�V-spectrin (C-terminaldomain) fusion proteins. Bound proteins were analyzed byimmunoblotting for the presence of FLAG-hTRPC4 variants.As shown in Fig. 4B, �hTRPC4 and �hTRPC4 bound to bothGST-�II-spectrin and�V-spectrin, however the �hTRPC4 var-iant failed to bind to either GST-�II-spectrin or GST-�V-spec-trin. These data, taken together with the results obtained fromthe yeast two-hybrid andGST-pulldown studies with truncatedhTRPC4, further defined the spectrin binding site in hTRPC4to amino acids Gly-730 through Glu-784. Despite lacking thespectrin-binding domain, the cellular distribution of the�hTRPC4 and �hTRPC4 variants were not noticeably differentwhen expressed in COS-7 cells and examined by confocalimmunofluorescencemicroscopy (Fig. 4C), suggesting that this

FIGURE 3. The interaction of �II-spectrin with hTRPC4 is dependent on the final three spectrinrepeats of �II-spectrin and independent of the Ca2�-binding EF domains. A, schematic of GST-�II-spectrin fusion proteins utilized in binding assays in vitro. Indicated is the relative strength of the hTRPC4association. B, various GST-�II-spectrin mutants were immobilized and incubated with FLAG-hTRPC4-transfected HEK293 lysates. Bound proteins were analyzed by SDS-PAGE and Western blotting with anti-FLAG M2 antibody along with 6% of the pulldown input. Coomassie staining is shown for demonstrationof equal fusion protein loading. Right, FLAG immunoreactivity was quantified from four independentassays, collated, and expressed as a percentage of hTRPC4 binding to the complete C terminus of �II-spectrin isolated from the yeast two-hybrid screen.




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domain does not dramatically affect the cellular targeting of thechannel.Deletion mutations of the spectrin-binding site of hTRPC4

were generated by linker-scanning PCR. FLAG-tagged,M1-hTRPC4 (�G730-E784), M2-hTRPC4 (�G730-K758), andM3-hTRPC4 (�K758-E784) (see Fig. 4B, schematic) were tran-siently expressed in HEK293 cells, and solubilized lysates wereincubated with immobilized GST-�II-spectrin (Fig. 5A).Immunoblot analysis of bound proteins indicated that bothM1-hTRPC4 and M2-hTRPC4 were unable to interact withGST-�II-spectrin. However, the M3-hTRPC4 construct, lack-ing Lys-758 through Glu-784, bound �II-spectrin with similaraffinity towild-type�hTRPC4 (Fig. 5A), thereby demonstratingthat the �II-spectrin binding motif of hTRPC4 is locatedbetween amino acids Gly-730 and Lys-758 (Fig. 5A). Gly-730

through Leu-753 is a highly conserved region between hTRPC4and hTRPC5 with 22 out of 23 residues identical between thetwo members (Fig. 5B). Other members of the TRPC familydisplay limited homology across the putative spectrin-bindingdomain, and together thismay indicate that a direct interactionwith spectrin is unique to the TRPC4 and TRPC5 subfamily ofTRPC channels.

�II-Spectrin Associates with hTRPC4 Signaling Complexesand Modulates EGF-induced hTRPC4 Exocytotic Insertionand Activation—The co-localization of signaling proteinswithin apparent microdomains in cells may be important forthe assembly of hTRPC4 channels into signaling complexesand their subsequent activation by EGF receptor stimula-tion. The role of �II-spectrin in this event was investigatedinitially by studying �II-spectrin interactions with TRPC4,

FIGURE 4. The � splice variant of hTRPC4 does not associate with �II-spectrin. A, schematic of pBTM116 fusion proteins and a summary of lacZ geneactivation in the presence of pACT2-�II-spectrin. Shown darker is the region of hTRPC4 C terminus responsible for mediating �II-spectrin and �V-spec-trin binding. Also indicated to the right is a colony-lift filter assay of lacZ expression from L40 yeast co-transformed with the opposing pBTM116construct and pACT2-�II-spectrin. B, GST fusion proteins of the C termini of �II-spectrin and �V-spectrin were incubated with lysates from eitherFLAG-tagged �hTRPC4-, �hTRPC4-, or �hTRPC4-expressing HEK293 cells. Bound proteins were analyzed by Western blotting with anti-FLAG antibody.C, COS-7 cells grown on coverslips were transiently transfected with either FLAG-�hTRPC4 or FLAG-�hTPRC4, fixed, and stained with anti-TRPC4 C20antibody following by anti-goat AlexaFluor 488 and 4�,6-diamidino-2-phenylindole. Cells were visualized by Zeiss LSM510 confocal microscope under63� objective.




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NHERF, and Fyn in a variety of cell lines and tissues. Fyn immu-noprecipitates from rat brain microsomal fractions were foundto contain both TRPC4 and �II-spectrin (Fig. 6A), demonstrat-ing the possible presence of a multiprotein complex within thebrain. Further, anti-hemagglutinin (HA) precipitates fromFLAG-hTRPC4 expressing COS-7 cells transiently transfectedwith HA-tagged NHERF contained �II-spectrin and EGFR, inaddition to hTRPC4, when examined by immunoblotting (Fig.6B). These results suggest that �II-spectrin and �hTRPC4 arefound together in macromolecular complexes within variouscell types.We have also demonstrated previously that EGF-induced

activation of hTRPC4 is dependent on tyrosine phosphoryla-tion of hTRPC4 by the STK, Fyn, and the subsequent associa-tion of hTRPC4 with the scaffolding protein NHERF (27). Theparticipation of �II-spectrin in the EGF-dependent exocytoticmovement of hTRPC4 to the plasma membrane was thusexamined using COS-7 cells transfected with FLAG-hTRPC4.The cells were stimulated with EGF and biotinylated, and anti-FLAG-precipitated lysates were immunoblotted for the pres-

ence of �II-spectrin and biotin-labeled hTRPC4. EGF stimula-tion (100 ng/ml and 20 min) was found to induce thedissociation of�II-spectrin fromhTRPC4 concomitant with anincrease in plasma membrane expression of hTRPC4 (Fig. 6C).In addition, when biotinylated proteins from hTRPC4-trans-fected COS-7 cells stimulated with EGF were isolated withNeutrAvidin-agarose and analyzed for the presence of �II-spectrin, a decrease in the association of �II-spectrin with inte-gral membrane proteins was observed (Fig. 6D).The importance of the �II-spectrin-hTRPC4 interaction in

hTRPC4 exocytic insertion and channel activation followingEGF stimulation was confirmed using COS-7 cells transfectedwith FLAG-�hTRPC4, which lacks the putative �II-spectrinbinding site. Following EGF application, �hTRPC4 failed toundergo furthermembrane translocation despite an increase intyrosine phosphorylation (Fig. 7A). This was in contrast to the�hTRPC4 variant, which exhibited a robust membrane inser-tion and tyrosine phosphorylation response (Fig. 7, A and B).Furthermore, �hTRPC4 channels exhibited elevated and morevariable basal plasma membrane expression than �hTRPC4

FIGURE 5. The �II-spectrin binding domain of hTRPC4 is located between amino acids 730 and 758. A, deletion mutants of hTRPC4 were generated bylinker scanning PCR. M1-hTRPC4, M2-hTRPC4, and M3-hTRPC4 were expressed as FLAG-tagged proteins in HEK293 cells and incubated with immobilized GSTor GST-�II-spectrin (C terminus). Bound proteins were analyzed by Western blotting with anti-FLAG and anti-GST antibodies. Also shown are the percentagesof mutant hTRPC4 bound to �II-spectrin as quantified from three separate experiments and expressed as a percentage of WT-hTRPC4 binding. B, alignment ofthe final 300 amino acids at the C-terminal of the 6 expressed human TRPC proteins. Note the putative spectrin-binding region highlighted in yellow. Alignmentwas performed with the ClustalW program.




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channels (Fig. 7B) suggesting spectrin binding may influenceplasma membrane levels in non-stimulated cells. Importantly,co-expression of �hTRPC4 in HEK293 cells with the EGFreceptor failed to produce a further increase in intracellularCa2� upon EGF application, in contrast to the �hTRPC4 vari-ant, which showed a larger, sustained increase in intracellularCa2� (Fig. 7C). Expression ofGFP alone, with the EGF receptor,produced a profile similar to the �hTRPC4 variant (Fig. 7C).Additionally, the stimulation of HEK293 cells expressing boththe EGF receptor and various hTRPC4 constructs or GFP, withEGF in the absence of extracellularCa2� for 8min prior toCa2�

re-addition, produced a larger sustained Ca2� influx in�hTRPC4-expressing cells (Fig. 7D). The smaller increase inintracellular Ca2� was similar in cells expressing either

�hTRPC4 orGFP (Fig. 7D), suggest-ing that the � variant is unable tobind spectrin and thus fails toundergo significant additionalmembrane insertion or activation inresponse to EGF. Taken together,these results indicate that spectrintethers hTRPC4-signaling com-plexes in intracellular microdo-mains and influences both the leveland activation of hTRPC4 channelsfollowing EGF stimulation.To further examine the role of

�II-spectrin in regulating the sur-face expression of �hTRPC4,siRNA was used to deplete endog-enous �II-spectrin from COS-7cells expressing either �hTRPC4or �hTRPC4. RNA interferenceknockdown produced an 86%reduction in �II-spectrin expres-sion (n � 7), without any effect onthe expression of EGFR, �1-inte-grin, transferrin receptor, �-tubulinor either transfected �hTRPC4 or�hTRPC4 as assessed by Westernblotting (Fig. 8A). However, thedepletion of �II-spectrin resulted ina 51% (n � 12) decrease in the basalsurface expression of �hTRPC4 asdetermined by biotinylation ofmembrane proteins followed byNeutrAvidin pulldown assays (Fig.8B). In addition,�hTRPC4 channelswhen expressed in siRNA-treatedcells also failed to undergo EGF-in-duced membrane insertion (Fig.8B). In contrast, both the basal sur-face expression of �hTPRC4 and itsresponse to EGF application wereunaffected by �II-spectrin knock-down, in agreement with the previ-ous results. Furthermore, althoughthe treatment of COS-7 cells with

�II-spectrin siRNA did not significantly alter the surfaceexpression of transferrin receptor, there was variability inreceptor surface levels, showing both increased and unchangedlevels in various experiments, upon �II-spectrin depletion (n�18, S.E. � 10.6). This variability may reflect the importance ofthe spectrin cytoskeleton in controlling the general organiza-tion of the plasma membrane (35–37). Importantly, �II-spec-trin depletion did not alter the autophosphorylation of theEGFR (pEGFR) or the downstream activation of ERK1/2 asdetermined by immunoblotting for the dually phosphorylatedform (pERK1/2) (Fig. 8C). Taken together, these findings sug-gest that �II-spectrin not only regulates the EGF-dependentinsertion of �hTRPC4 channels but is also involved in regulat-ing the basal plasma membrane levels of the channel.

FIGURE 6. EGF stimulation disrupts the interaction between hTRPC4 and �II-spectrin. A, rat brainmicrosomes solubilized in RIPA buffer were immunoprecipitated with anti-FYN or anti-rabbit antibody asindicated and Western blotted for �II-spectrin, TRPC4, and Fyn. B, COS-7 cells expressing FLAG-hTRPC4were transiently transfected with HA-tagged NHERF as indicated, and lysates were immunoprecipitatedwith anti-HA antibody. Bound proteins were analyzed by Western blotting as indicated. C, COS-7 cellstransiently expressing hTRPC4 were serum-starved, stimulated with EGF (100 ng/ml for 15 min), andbiotinylated prior to immunoprecipitation of hTRPC4 from RIPA-solubilized lysates. Bound proteins wereimmunoblotted for the presence of �II-spectrin and biotinylated hTRPC4 as indicated. Shown is a repre-sentative of three individual experiments. D, lysates from biotinylated COS-7 cells stimulated with EGFwere precipitated with NeutrAvidin-agarose and analyzed by immunoblotting with anti-�II-spectrin andanti-TRPC4 antibodies.




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DISCUSSION

Stimulation of the EGFR was previously shown to induceCa2� entry through the activation of hTRPC4 channels (4, 27).A role for NHERF and STKs in hTRPC4 regulation was estab-lished in this regulation, however other proteins may also beinvolved in controlling the expression and activation ofhTRPC4 channels at the cell surface (27). In particular, variouscytoskeletal proteins, including actin (38, 39) and �-tubulin(40), have recently been implicated as important components

of TRPC signaling pathways. Utilizing the yeast two-hybrid sys-tem, two members of the spectrin family of cytoskeletal pro-teins, �II-spectrin and �V-spectrin, were identified as novelhTRPC4-binding proteins. The spectrin-binding site ofhTRPC4 was localized to between residues 730 and 758, com-prising a coiled-coil domain structure previously associatedwith protein dimerization. This region was responsible forassociating with the final three spectrin repeats of �II- and�V-spectrin. The direct interaction between hTRPC4 and �II-

FIGURE 7. Formation of a functional signaling complex between �II-spectrin and hTRPC4 involved in channel activation. A, the �hTRPC4 variant doesnot undergo EGF-dependent translocation. Serum-starved COS-7 cells transiently expressing either �hTRPC4 or �hTRPC4 were stimulated with EGF (100 ng/mlfor 15 min) prior to biotinylation. Lysates were immunoprecipitated with anti-FLAG antibody and analyzed by Western blotting as indicated. B, shown are thecollated data of Fig. 7A from five independent experiments. C, HEK293 cells expressing either GFP, �hTRPC4, or �hTRPC4 were loaded with Fura-2 andstimulated with EGF in the presence of 2 mM Ca2�. Ca2� transients were measured as the ratio of 340/380 nm wavelength using an ORCA-ER camera. Shownare averaged traces from 40 –50 cells. D, Fura-2-loaded HEK293 cells expressing either GFP, �hTRPC4, or �hTRPC4 were stimulated with EGF in the absence ofexternal Ca2� for 8 min prior to the re-addition of 2 mM Ca2�. Shown are the averaged Ca2� re-entry profiles from 50 – 60 individual cells.




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spectrin was important in controlling the translocation ofhTRPC4 channels to the plasma membrane following EGFstimulation, resulting in activation of the channel.Previous studies have implicated the sub-membranous spec-

trin cytoskeleton in the control of store-operated Ca2� entry,with disruption of the spectrin-protein 4.1G interaction inendothelial cells reducing the thapsigargin-activated Ca2�-se-lective ISOC current (20, 26). TRPC4was suggested as a compo-nent of the channels mediating ISOC, and a direct associationbetween TRPC4 and protein 4.1G was proposed to link this

channel with the spectrin and actincytoskeleton and thereby regulateCa2� entry (10). The putative pro-tein 4.1G-binding domain was pre-dicted to reside between aminoacids 675 and 685 of TRPC4, �40amino acids downstream of theinvariant EWKFAR region. Impor-tantly, the spectrin-interactingdomain of hTRPC4 was foundwithin amino acids 730 and 758,which lies �50 residues C-terminalof the proposed protein 4.1-bindingregion, which is similar to the pro-tein 4.1 binding sequences found inmembrane-associated guanylatekinases, such as Discs Large (Dlg)(10, 41). This fosters the proposalthat formation of macromolecularsignaling scaffolds is required tocommunicate changes in cellularsignaling to the plasma membraneand regulate hTRPC4 channels.Similar to the protein 4.1-TRPC4

interaction, the association betweenhTRPC4 and �II-spectrin appearsto be constitutively present inunstimulated cells. However, unlikethe protein 4.1-TRPC4 interactionreported previously as required foractivation of ISOC (10), the associa-tion of hTRPC4 with �II-spectrin isdisrupted by stimulation of cellswith EGF and coincides with Ca2�

entry. The disruption of �II-spec-trin-hTRPC4 binding may reflectthe Ca2�-dependent remodeling ofthe spectrin cytoskeleton, in a man-ner similar to the redistributionreported to occur following stimu-lation of exocytosis in chromaffincells (42). This discrepancy betweenthe association of �II-spectrin-hTRPC4 and protein 4.1-hTRPC4may indicate that the interactionwith protein 4.1, and consequentlyspectrin and the actin cytoskeleton,coordinates the formation of stable

signaling complexes surrounding TRPC4, with this complexlikely to be additionally stabilized by a direct associationbetween the channel and �II-spectrin. Cellular stimulation withCa2�-mobilizing ligands may then induce the dissociation of the�II-spectrin interaction, allowing TRPC4 channels to undergotranslocation to the plasma membrane. Here, they may be addi-tionally stabilized by the remaining association with protein 4.1.This coordinated responsewould allowTRPC4channel activity tobe tightly regulated within the cell and therebyminimize deleteri-ous fluctuations in intracellular Ca2�.

FIGURE 8. Depletion �II-spectrin by siRNA reduces the surface expression of �hTRPC4 and preventsEGF-stimulated membrane insertion. A, COS-7 cells reverse transfected with either scrambled or �II-spectrinsiRNA were transiently transfected with FLAG-�hTPRC4 or FLAG-�hTRPC4 and assessed for the effect on theexpression of the indicated proteins by immunoblotting. Right: protein expression for �II-spectrin, �hTRPC4,and �hTRPC4 were quantified and expressed as a percentage of scrambled siRNA levels (n 4 independentexperiments). B, siRNA-treated COS-7 cells transiently expressing either �hTRPC4 or �hTPRC4 were serum-starved, biotinylated, and lysed prior to incubation of cleared lysated with NeutrAvidin-agarose. Bound pro-teins were separated by SDS-PAGE and analyzed by immunoblotting for TRPC4 and transferrin receptor (TfR)levels. Below: membrane levels were quantified and expressed as a percentage of unstimulated, scrambledsiRNA-treated levels (n 6 independent experiments). C, cell lysates used in Fig. 8B were analyzed by Westernblotting for levels of the indicated proteins. Shown are representatives of at least six independent experiments.***, p 0.001.




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The localization of the spectrin-binding region within acoiled-coil domain of hTRPC4 appears important for mediat-ing the interaction with the spectrin repeats of �II- and�V-spectrin. Coiled-coil domains are highly versatile and fre-quently observed in protein interaction modules often associ-ated with oligomerization (43). They are foundwithin cytoskel-etal proteins, ion channels, and motor proteins, and theymediate the assembly of the soluble NSF attachment proteinreceptor complexes to regulate vesicle fusion (44). Importantly,spectrin repeats are also composed of coiled-coil domainsallowing spectrins to be classified as membrane-bound coiled-coil proteins (45). The formation of an interface between thecoiled-coil domains of hTRPC4 and the spectrin repeats of �II-spectrinmay therefore bemediating their interaction.Whetherthis interaction is modulated by protein modifications such asphosphorylation and protease cleavage remains to be deter-mined. Indeed, the cleavage of �II-spectrin by calpain is inhib-ited by Src-dependent phosphorylation of the SH3 domain in�II-spectrin at Tyr-1146 (46). This may stabilize �II-spectrinand promote its association with other proline-rich proteins tostimulate dissociation fromhTRPC4. Alternatively, amodifica-tion on the C terminus of hTRPC4 may disrupt �II-spectrinbinding and release the channel from the cytoskeleton.Intriguingly, a novel hTRPC4 splice variant identified in the

NCBI data base, the � variant, was found to lack the�II-spectrinbinding domain and as such an interaction between hTRPC4with �II-spectrin was absent. Importantly, the absence of thisinteraction correlated with a failure of these channels to trans-locate to the plasma membrane in response to EGF, despiteundergoing a robust tyrosine phosphorylation response. Thismay reflect a requirement for the �II-spectrin association tocouple the channel to the translocation machinery. Alterna-tively, �II-spectrin may sequester nascent inactive hTRPC4channels beneath the cell surface, to await an activating stimu-lus, which the � isoform is unable to respond to. The absence ofa consistent membrane translocation event in response to EGFresulting in a deficiency in the Ca2� entry profile of the � vari-ant, supports both possibilities. Thus, the cellular expression ofthe �hTRPC4 variant may represent a novel mechanism formodulating the Ca2� entry profile of cells containing hTRPC4.It is important to note that the gross cellular localization of

both splice variants is not inherently different, with both chan-nels forming punctuate structures consistent with their local-ization to lipid raft domains.4 Thus their different Ca2�-signal-ing profiles must be due to an alternate structural feature of thechannel, such as the loss of a protein interaction site, whichaffects their ability to respond to stimuli. Consistent with thishypothesis, depletion of endogenous �II-spectrin from COS-7cells resulted in a decrease in both membrane insertion andresting plasmamembrane levels of �hTRPC4while exerting noeffect on the � variant of the channel. However, the absence of�II-spectrin also altered the surface expression of other mem-brane proteins such as �1-integrin4 and transferrin receptor toa variable and non-significant extent, suggesting that spectrinsplay a role in modulating the plasma membrane structure in

addition to participating in direct protein-protein interactions.The effect on �hTRPC4 surface expression is similar to thatreported in nodes of Ranvier, where nodalNa� channel clusterswere reduced when �II-spectrin was disrupted (37). It was sug-gested that �II-spectrin is involved in stabilizing the nascentchannel clusters at nodal sites. We propose that �II-spectrinmay perform a similar function here, where �hTRPC4 channelclusters are both arranged and stabilized at, and adjacent to, theplasmamembrane sites of Ca2� entry. The presence or absenceof the spectrin-binding domainmay thus control the activity ofthe channel by allowing cytoskeletal control of channel com-plex surface expression and stabilization.The ability of �II-spectrin to act as a scaffold protein linking

hTRPC4 channels to intricate signaling complexes is enhancedby the large number of potential binding partners of spectrin.Through interactions with other cytoskeletal components,including F-actin and ankyrin, as well as signalingmolecules suchas synapsin I, membrane-associated guanylate kinases, and phos-pholipids, spectrins are ideally positioned to coordinate cellularresponses culminating in Ca2� entry (21). Spectrins are alsoknown to form complexes with various Ca2� regulators,including IP3Rs, ryanodine receptors, Na�/K�-ATPase, vari-ous ion channels, N-methyl-D-aspartic acid receptor subunits,and the epithelial Na� channel, ENaC (23, 47–51), positioningthem as potential critical regulators of Ca2� levels. Interactionsbetween hTRPC4 and the spectrin cytoskeleton may also pro-vide a functional bridge between the endoplasmic reticulumand plasma membrane, forming a mechanism for linking Ca2�

release from intracellular stores with Ca2� entry through theplasma membrane. Spectrins have also been identified inTRPC5 and TRPC6 immunoprecipitates from rat brain (52),and we have now shown that �II-spectrin is a component of amultiprotein signaling complex surrounding hTRPC4, alongwith NHERF, Fyn, and the EGFR. The latter three proteins areintegral in the phosphorylation and activation of hTRPC4 byEGF and the addition of�II-spectrin provides a stabilizing plat-form for the assembly of the complex at the plasmamembrane.Together with the recently identified interactions with Homerand stromal interacting protein 1, thismacromolecular channelcomplex may be critical in the activation of TRPC4 by variousstimuli (11, 53).

Acknowledgments—We acknowledge Drs. J. W. Putney and R. R.McKay for hTRPC4 cDNA and Dr. J. S. Morrow for kindly providing�II-spectrin cDNA.

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Adam F. Odell, Dirk F. Van Helden and Judith L. ScottHuman Transient Receptor Potential Channel 4 Channels

The Spectrin Cytoskeleton Influences the Surface Expression and Activation of

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The Spectrin Cytoskeleton Influences the Surface Expression and