Identification and characterization of a Ca2+-sensitive interaction of the vanilloid receptor TRPV1...

Identification and characterization of a Ca2+-sensitive interactionof the vanilloid receptor TRPV1 with tubulin

C. Goswami,* M. Dreger,*,1 R. Jahnel,* O. Bogen,* C. Gillen� and F. Hucho*

*Freie Universitat Berlin, Institute fur Chemie-Biochemie, Berlin, Germany

�Research & Development Grunenthal GmbH, Aachen, Germany

Abstract

The vanilloid receptor TRPV1 plays a well-established func-

tional role in the detection of a range of chemical and thermal

noxious stimuli, such as those associated with tissue in-

flammation and the resulting pain. TRPV1 activation results in

membrane depolarization, but may also trigger intracellular

Ca2+-signalling events. In a proteomic screen for proteins

associated with the C-terminal sequence of TRPV1, we

identified b-tubulin as a specific TRPV1-interacting protein.

We demonstrate that the TRPV1 C-terminal tail is capable of

binding tubulin dimers, as well as of binding polymerized

microtubules. The interaction is Ca2+-sensitive, and affects

microtubule properties, such as microtubule sensitivity

towards low temperatures and nocodazole. Our data thus

provide compelling evidence for the interaction of TRPV1 with

the cytoskeleton. The Ca2+-sensitivity of this interaction sug-

gests that the microtubule cytoskeleton at the cell membrane

may be a downstream effector of TRPV1 activation.

Keywords: calcium dependence, capsaicin receptor,

cytoskeleton interaction, transient receptor potential V1.

J. Neurochem. (2004) 91, 1092–1103.

There is growing evidence that ion channel proteins oftenoccur in the cell membrane as parts of multiproteincomplexes. These complexes, such as the NMDA receptorcomplex (Husi et al. 2000), are termed signalling complexes,because components of effector and modulator systems ofion channel function are concentrated by protein–proteininteractions in close proximity to the ion channel proteins.Based on this concept of signalling complexes, considerableinsight into signalling events triggered by ion channelfunction is expected to be gained from the identification ofion channel-interacting proteins.

The vanilloid receptor TRPV1 is of specific importance inthermosensation and detection of noxious stimuli associatedwith tissue inflammation (Tominaga et al. 1998; Caterinaet al. 2000; Davis et al. 2000; Caterina and Julius 2001). Thision channel is a member of the transient receptor potential(TRP) family of non-selective ion channels and can beactivated by noxious heat, low pH, capsaicin, resiniferatoxinand endogenous lipid-derived mediators, such as anandamideand N-arachidonoyl-dopamine (NADA) (Huang et al. 2002),partly in a synergistic manner. The phosphoinositide PIP2(Prescott and Julius 2003) and direct phosphorylation ofintracellular sites of TRPV1 (Bhave et al. 2002, 2003;Numazaki et al. 2002; Jung et al. 2003; Mohapatra andNau 2003) contribute to the modulation of ion channel

function. It is believed that TRPV1 channel activation leads totwo lines of downstream events: membrane depolarizationand Ca2+ influx. The Ca2+ influx itself modulates channelfunction by triggering channel desensitization (Koplas et al.1997; Tominaga et al. 1998). The molecular basis of this,however, is not yet understood, though there is evidence thatthe phosphorylation status of TRPV1 and interaction withcalmodulin (CAM) might be involved (Numazaki et al. 2003;

Resubmitted manuscript received July 21, 2004; accepted July 23, 2004.Address correspondence and reprint requests to F. Hucho, Institut fur

Chemie-Biochemie, Freie Universitat Berlin, Thielallee 63, 14195 Ber-lin, Germany. E-mail: [email protected] or M. Dreger, Uni-versity Laboratory of Physiology, Parks Road, Oxford OX1 3PT, UK.E-mail: [email protected] Present address of M. Dreger is University Laboratory ofPhysiology, Parks Road, Oxford OX1 3PT, UK.Abbreviations used: BSA, bovine serum albumin; CAM, calmodulin;

DHB, 2,5-dihydroxybenzoic acid; FHIT, fragile histidine triad protein;IPTG, isopropyl thiogalactoside; MALDI-MS, matrix-assisted laserdesorption ionization-mass spectrometry; MBP, maltose binding protein;MBP-TRPV1-Ct, TRPV1 C-terminal cytoplasmic portion MBP fusionprotein; MBP-TRPV1-Nt, TRPV1 N-terminal cytoplasmic portion MBPfusion protein; MT, microtubules; NADA, N-arachidonoyl-dopamine;NEB, amylose resin; NGF, nerve growth factor; PBS, phosphate-buf-fered saline; PSD, post-source decay; RT, room temperature; SDS–PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis;TRP, transient receptor potential.

Journal of Neurochemistry, 2004, 91, 1092–1103 doi:10.1111/j.1471-4159.2004.02795.x

1092 � 2004 International Society for Neurochemistry, J. Neurochem. (2004) 91, 1092–1103

Jung et al. 2004; Rosenbaum et al. 2004). In a hypothesis-driven approach based on the notion that nerve growth factor(NGF) signalling affects TRPV1 function, the high-affinityNGF receptor TrkA and phospholipase Cc have beenidentified as candidate components of a TRPV1-signallingcomplex in vitro (Chuang et al. 2001).

Though no familiar protein domains have been detectedwithin the C-terminal region of TRPV1, many functionalfeatures have been assigned to it, such as involvement inreceptor desensitization (Vlachova et al. 2003), PIP2 binding(Prescott and Julius 2003), CAM binding (Numazaki et al.2003), requirements for responses to heat (Vlachova et al.2003) and capsaicin (Jung et al. 2002).

Here we report results based on a proteomic screen forproteins that interact with the C-terminal cytoplasmic domainof TRPV1. We demonstrate that tubulin dimers specificallybind to the TRPV1 C-terminus in vitro. This interaction alsooccurs with polymerized microtubules and is affected by theconcentration of free Ca2+. This interaction exerts a stabil-izing effect on microtubules (MT) when exposed to condi-tions that favour depolymerization. We conclude that Ca2+

signalling through TRPV1 may affect the structure of the MTnetwork at the plasma membrane.

Materials and methods

Antibodies and reagents

The mouse monoclonal anti MBP antibody was purchased from

New England Biolabs (Beverly, MD, USA), the rabbit polyclonal

anti-N-terminal TRPV1 antibody and the respective blocking

peptide (sequence M1EQRASLDSEESESPPQENSC21 which cor-

respond to the first 21 amino acid residues of TRPV1) were from

Affinity Bioreagents (Golden, CO, USA) and from Alexis Bio-

chemicals (San Diego, CA, USA). The goat polyclonal anti-

C-terminal TRPV1 antibody was from Santa Cruz Biotechnology

(Santa Cruz, CA, USA). The sequence of the supplied blocking

peptide for the goat polyconal anti-C-terminal TRPV1 antibody was

found to contain the sequence stretch PEDAEVFKDSMV by

matrix-assisted laser desorption ionization-mass spectrometry

(MALDI-MS), which corresponds to the 823–834 amino acid

residues of TRPV1. The mouse monoclonal anti a-tubulin antibody

was from Oncogene (San Diego, CA, USA). The mouse monoclonal

anti b-tubulin antibodies were from Boehringer (Mannheim,

Germany), and also from Santa Cruz Biotechnolgy. Rat monoclonal

anti tubulin antibody YL1/2 was purchased from Abcam Ltd

(Cambridge, UK). The mouse monoclonal anti actin antibody was

from Oncogene. Alexa fluor 594 phalloidin was from Molecular

Probes (Eugene, OR, USA). Nocodazole and taxol were purchased

from Sigma-Aldrich (Taufkirchen, Germany). EZ-Link Sulfo-NHS-

LC-Biotin was purchased from Molecular Probes.

Expression and purification of TRPV1 fusion proteins

cDNA fragments corresponding to amino acid residues 1–431

(N-terminal cytoplasmic portion) of rat TRPV1 were amplified by

PCR using primers 5¢-GCGCGAATTCATGGAACAACGGGC-

TAG-3¢ and 5¢-GCGCTCAGATTACTTGACAAATCTG-3¢. A

cDNA fragment corresponding to amino acid residues 681–838 of

rat TRPV1 (C-terminal cytoplasmic portion) was amplified by PCR

using primers 5¢-GCGCGAATTCATGGGTGAGACCGTCAA-3¢and 5¢-GCGCTCTAGATTATTTCCCCTGGGAC-3¢. Amplified

fragments were subcloned into the EcoR1 and XbaI restriction sites

of the pMAL-c2x vector (New England Biolabs). A stop codon was

introduced into the pMAL-c2x vector in between EcoRI and HindIIIsites using a linker made by 5¢-AATTCGGTACCTGAA-3¢ and

5¢-AGCTTTCAGGTACCG-3¢ to express only maltose-binding

protein (MBP). All expression constructs were verified by automa-

ted nucleotide sequencing. Escherichia coli strain BL21DE3 was

transformed by heat shock with plasmid coding for the TRPV1

C-terminal cytoplasmic portion MBP fusion protein (MBP-TRPV1-

Ct), the TRPV1 N-terminal cytoplasmic portion MBP fusion protein

(MBP-TRPV1-Nt) and MBP only. E. coli cells were induced to

express the proteins by isopropyl thiogalactoside (IPTG) for 2 h.

Thereafter, the cells were lysed by freezing and thawing cycles in

lysis buffer (20 mM Tris–HCl, pH 7.4, 150 mM NaCl, 0.2 g/mL

sucrose, lysozyme, benzonase and protease inhibitor cocktail). The

lysed extracts were cleared by spinning at 100 000 g in a TFT 45

rotor for 2 h. The cleared lysate was applied to the amylose resin

and washed throroughly. Bound protein was eluted with 10 mM

maltose in elution buffer (50 mM PIPES, pH 6.8, 100 mM NaCl,

1 mM EGTA and 0.2 mM MgCl2). All protein determinations were

performed according to Bradford (1976).

Purification of tubulin

ab-tubulin dimers were purified from porcine brain according to

Shelanski et al. (1973). In brief, two cycles of assembly from

soluble brain extract in the presence of glycerol and GTP and

disassembly by cold temperature, were followed by chromatography

on phosphocellulose.

Preparation of high speed spinal cord extract

Rat spinal cord was homogenized in 20 mM HEPES (pH 7.4), 1 mM

EGTA and 320 mM sucrose and protease inhibitor cocktail (Roche

Molecular Biochemicals, Indianapolis, IN, USA). After homogeni-

zation, the extract was cleared by centrifugation at 48 000 g for

30 min at 4�C, followed by solubilizationwith 1% (w/v) Tween-20 for

2 h at room temperature (RT; 20�C). The solubilized extract was

centrifuged at 100 000 g for 2 h at 25�C in fixed angle rotor

TFT65.13. The top layers that contained myelin and the bottom layers

that contained insoluble material were discarded. Myelin-free cleared

supernatant from the middle of the gradient was used for the assay.

Pulldown assay

MBP alone, MBP-TRPV1-Ct and MBP-TRPV1-Nt were expressed

in E. coli and the cleared cell lysates were applied to the amylose

resin (NEB), and incubated for 1 h at RT followed by washing.

Approximately 1 mL of amylose resin with the bound fusion protein

was incubated with 10 mL of cleared spinal cord extract (1 mg/mL

protein) overnight either in the presence or absence of Ca2+ (5 mM).

This was followed by washing three times with 10 mL each and

constant buffer conditions. The proteins were eluted by 10 mM

maltose. Eluted samples were concentrated by TCA precipitation

and analysed by 10% sodium dodecyl sulphate polyacrylamide gel

electrophoresis (SDS–PAGE) according to Laemmli (1970).

Interaction of tubulin with TRPV1 1093

� 2004 International Society for Neurochemistry, J. Neurochem. (2004) 91, 1092–1103

In experiments aimed at determining if there is a direct interaction

between tubulin and TRPV1, 40 lL of amylose beads bound to

MBP-TRPV1-Ct, MBP-TRPV1-Nt or MBP alone were incubated

with 50 lg of ab-tubulin under buffer conditions as given in the

figure legends.

Biotinylation of tubulin

Purified tubulin (1 mg/mL) in 1 mL PEM buffer (50 mM pH 6.8,

1 mM EGTA, 0.2 mM MgCl2) was labelled with 1 mg/mL of

EZ-Link Sulfo-NHS-LC Biotin at 4�C for 2 h. The labelling

reaction was terminated by adding Tris–HCl pH. 7.4 to the final

concentration of 50 mM. Unreacted biotinylation reagent was

separated from biotinylated tubulin by gel filtration.

MALDI-MS

The proteins of interest were digested in gel according to standard

procedures (Shevchenko et al. 1996). Measurements were per-

formed using 2,5-dihydroxybenzoic acid (DHB) as matrix sub-

stance. A Bruker Reflex (Bruker Daltonics, Bremen, Germany) mass

spectrometer was used to acquire peptide mass fingerprint spectra as

well as fragment ion spectra obtained from post-source decay (PSD)

of selected precursor ions. PSD spectra were assembled using the

FAST method (Bruker Daltonics). The search engines ProFound and

PepFrag as available at http://prowl.rockefeller.edu/ were used to

match peptide mass fingerprints and fragment ion patterns to NCB

Inr database entries.

Immunoprecipitation assay and western blot analysis

F11 cells were transfected with the construct coding for full-length

TRPV1 by lipofectamine (Invitrogen, Carlsbad, CA, USA). Two

days after transfection, cells were extracted with 1% (w/v) dodecyl

maltoside at RT for 2 h. The extract was cleared by centrifugation at

28 000 g for 30 min. The extraction buffer contained 20 mM PIPES

pH 6.8, 1 mM EGTA, 1 mM CaCl2, 0.2 mM MgCl2, 1% (w/v)

dodecyl maltoside and 150 mM NaCl along with the protease

inhibitor cocktail and benzonase (Merck, Rahway, NJ, USA). Then,

600 lg of cleared cell extract at a protein concentration of 3 mg/mL

were incubated with 2 lg of antibodies. Immunocomplexes were

isolated with protein G sepharose (20 lL) and separated by SDS–

PAGE. For immunoprecipitation of TRPV1 from spinal cord, 1 g

(wet weight) of spinal cord tissue from rat were homogenized in the

same buffer as used in the homogenization of F11 cells, followed by

extraction with 1% (w/v) dodecyl maltoside and clearance of the

extract by centrifugation. Then, 6 mL of cleared extract at a protein

concentration of roughly 1 mg/mL were incubated with 20 lg of

the N-terminus-specific anti-TRPV1 antibody, and immunocom-

plexes were collected with 25 lL protein G sepharose. For

competition experiments using the blocking peptide, a roughly

300-fold molar excess of peptide over antibody was used. The

proteins were separated by SDS–PAGE, and transferred to a

nitrocellulose membrane by electroblotting. For western blot

analysis, the membranes were blocked with 5% non-fat milk in

TBS-T (20 mM Tris, 150 mM NaCl. 0.1% Tween-20) buffer

followed by probing with the respective primary antibody,

and horseradish peroxidase-conjugated antibodies were used as

secondary antibodies. The ECL detection system (Amersham

Biosciences, Freiburg, Germany) was used for the visualization of

the immunoreactivity.

Co-sedimentation assay with taxol-stabilized MTs

A total of 140 lg of purified ab-tubulin dimer in a total volume of

90 lL were incubated in modified PEM Buffer (20 mM PIPES,

pH 6.8, 0.2 mM MgCl2 and 1 mM EGTA supplemented by 1 lMtaxol, 5 mM GTP and 1 mM ATP) for 30 min at 37�C, to form MT.

After MT formation, 5 lg of purified MBP-TRPV1-Ct, MBP-

TRPV1-Nt and MBP were incubated with taxol-stabilized MT for

40 min at RT either in the presence or absence of 1 mM CaCl2,

followed by centrifugal separation of pellet (MT) and supernatant

(free dimer) at 70 000 g/30 min/35�C.For MT formation under taxol-free conditions, 40 lg of tubulin

dimer were used in PEM buffer with 1 mM CaCl2 (optional), 5 mM

GTP, 1 mM ATP in the absence of taxol and incubated for 30 min at

RT.

For cold-induced depolymerization of MT, first the MTs were

allowed to form in PEM-S buffer (PEM buffer with 0.1 M NaCl)

without taxol as described above. Ca2+ and MBP-TRPV1-Ct, or

MBP-TRPV1-Nt, were added along with the tubulin dimer

during the first cycle of polymerization. Polymerized MT

(pellets) were isolated by centrifugation, resuspended in ice-cold

PEM-S buffer by repeated pipet aspiration, and kept on ice for

another 30 min. This was followed by the centrifugal separation

of dimers (supernatant) and polymer MT (pellet) at 70 000 g(30 min, 4�C).

Immunocytochemistry

F11 cells were grown and transfected on glass coverslips. Two

days after transfection, the cells were fixed with 2% paraformal-

dehyde for 10 min, permeabilized with 0.4% Triton X-100 in

phosphate-buffered saline (PBS) for 10 min, followed by incuba-

tion with 100 mM glycin. The cells were blocked with 5% normal

goat serum or bovine serum albumin (BSA). They were probed

with the anti-N-terminal TRPV1 antibody (Affinity Bioreagents)

at a 1 : 1000 dilution and rat monoclonal tubulin antibody YL1/2

(Abcam Ltd) at a 1 : 750 dilution. Anti-rat Alexa fluor 594-

conjugated secondary and Cy2-labelled anti-rabbit secondary

antibody were used to visualize the tubulin and TRPV1,

respectively. Alexa Fluor 594 phalloidin (Molecular Probes) was

used for visualizing actin fibres. The coverslips were mounted on

glass slides with fluromount G. Images were taken by a confocal

microscope with a 63· objective.

Results

Construction and expression of MBP-TRPV1-Ct

The cytoplasmically located N- and C termini of TRPV1are believed to play a role in protein–protein interactions.Recent studies on the proteomic screening for interactionpartners of ligand-gated ion channels led us to searchsystematically for TRPV1-C terminus-interacting proteinsby pulldown assays. As a bait, we used the cytoplasmicC-terminal portion of TRPV1 (amino acid residues 681–838) fused to the MBP, with the MBP at the fusionprotein’s N-terminus. The MBP portion served to immo-bilize the fusion protein to an amylose resin. The fusion

1094 C. Goswami et al.


protein was expressed in E. coli. The MBP-TRPV1-Ctappeared on SDS–PAGE as a single protein band at�65 kDa (Fig. S1a) as assessed using an anti C-terminalTRPV1 or anti-MBP western blot. Additional major

degradation products or incompletely translated proteinspecies were not observed (Fig. S1a). The bulk of fusionprotein was readily soluble under native extractionconditions.

Detection of Ca2+-sensitive interactions of the TRPV1

C-terminal fragment

Ca2+ plays an important role in the desensitization of theTRPV1, and Ca2+ influx through TRPV1 may triggerintracellular signalling events. Assuming that at least someof the protein–protein interactions of the TRPV1C-terminus might be Ca2+-sensitive, we conducted pull-down assays both in the presence and absence of Ca2+.The MBP-TRPV1-Ct fusion protein was for that purposebound to an amylose resin, incubated with protein extracts,and the fusion protein along with interacting proteins waseluted from the resin with maltose. The soluble eluate wasthen precipitated with TCA and subjected to SDS–PAGE.We used extracts that predominantly contained solubleproteins from rat or pig spinal cord, or from the DRG-derived cell line F11, as sources for potential MBP-TRPV1-Ct-interacting proteins. In the presence of Ca2+, anumber of spinal cord proteins were observed to be pulleddown with MBP-TRPV1-Ct, while some of these proteinswere not pulled down to the same extent in the absence ofCa2+ (Fig. 1a). None of these potential interacting proteinswas observed in the absence of fusion protein in thefractions eluted from only amylose resin either in presenceor in the absence of Ca2+, or when MBP alone was usedas a bait (Fig. 1a). These observations were reproducedfour times in independent experiments.

By mass spectrometry, a protein pulled down by MBP-TRPV1-Ct in a Ca2+-sensitive manner, corresponding to a55 kDa protein band observable on the gel, was identified astubulin b chain 15 from rat (Fig. S2; a total of 16 of peptidescovering 39% of the full protein sequence matched NCBI nrA25113). This result was confirmed by western blot analysis(Fig. 1b). Monoclonal antibodies specific for either a- orb-tubulin used for the western blot analysis revealed also thepresence of a-tubulin. Both tubulins bound to the MBP-TRPV1-Ct predominantly in the presence of Ca2+, anda-tubulin was virtually only detected when the pulldown wasconducted in the presence of Ca2+ (Fig. 1b). The pulldown ofthe tubulins by the MBP-TRPV1-Ct was observed from pigspinal cord extracts as well as from F11 cell extracts (data notshown).

The identification of other potential TRPV1-interactingproteins is ongoing. For the present study, we focussed on thecharacterization of the TRPV1–tubulin interaction.

Tubulin, but not actin, co-precipitates with full-length

TRPV1 in anti-TRPV1 immunoprecipitation

To confirm the interaction between TRPV1 and tubulin by anindependent method, we expressed full-length TRPV1 in F11

Fig. 1 Identification of a- and b- tubulin as a C-terminal of TRPV1-

interacting partner. (a) MBP-TRPV1-Ct (indicated by arrow) interacts

with a 55 kDa protein (indicated by asterisk). MBP-TRPV1-Ct immo-

bilised on amylose beads (lane 2 and 3) or MBP alone immobilised on

amylose beads (lane 4 and 5) were incubated with spinal cord extract

over night in the presence of 5 mM Ca2+ or absence of Ca2+. Bound

proteins were eluted with 10 mM maltose, precipitated with trichloro-

acetic acid, resuspended in SDS–PAGE sample buffer, and subjected

to 10% SDS–PAGE. Proteins were visualised by silver stain, and

subsequently subjected to in-gel digestion and MALDI-MS analysis.

Lane 1, spinal cord extract, lane 2, pulldown eluate of proteins bound

in the presence of Ca2+, lane 3, pulldown eluate in the absence of

Ca2+, lanes 4 and 5 represent negative controls in the presence or

absence of Ca2+, showing non-specific binding to MBP alone immo-

bilized on amylose beads. (b) Western blot analysis reveals a Ca2+-

sensitive interaction between both a- and b-tubulin with MBP-TRPV1-

Ct. The proteins separated by SDS–AGE and transferred to nitrocel-

lulose membranes were probed with monoclonal antibodies against a-

tubulin (left panel) and b-tubulin (right panel) of spinal cord extract

(lane 1), proteins bound to MBP-TRPV1-Ct in the presence of Ca2+

(lane 2) or in the absence of Ca2+ (lane 3), proteins bound to MBP

alone with Ca2+ (lane 4) and without Ca2+ (lane 5).



cells, and performed immunoprecipitation experiments. Thiscell line, due to its origin as a fusion cell line from mouseneuroblastoma cells and rat DRG neurones, is a favourablecellular system to mimick the TRPV1 molecular environ-ment in the spinal cord (Jahnel et al. 2001). b-Tubulinco-precipitated with TRPV1-specific antibodies, both whenN-terminus-specific anti-TRPV1 antibodies or C-terminus-specific anti-TRPV1 antibodies were used for immunopre-cipitation (Fig. 2a, see also Fig. S3 for antibody specificity).Co-precipitation of tubulin from the same extract was notobserved with non-specific antibodies, suggesting that theTRPV1 and tubulin interaction is specific. The anti-N-terminal TRPV1 antibody did not precipitate the tubulinsfrom non-transfected cells (Fig. 2b), confirming further thetubulin–TRPV1 interaction observed.

Tubulin was also co-precipitated with TRPV1 from spinalcord extracts, suggesting that the tubulin–TRPV1 interactionnot only occurs in transfected cells, but also in native tissue(Fig. 2c).

We tested whether monoclonal antibodies against a-tubulin and b-tubulin are capable of co-precipitating TRPV1with the tubulins. However, TRPV1 was not co-precipitatedin those experiments (data not shown). We conclude fromthis observation that only a small subset of tubulin mightinteract with TRPV1, and TRPV1 was much less abundant inthe preparation than tubulin.

Actin is another major constituent of cytoskeletal struc-tures. In order to assess whether actin co-precipitates in anti-TRPV1 immunoprecipitation, we probed the anti-TRPV1immunoprecipitate for the presence of actin. However, wedid not detect any actin (Fig. 2d). Taken together, our resultssuggest that tubulin, but not actin, is a component of theTRPV1 complex, and links the receptor to the cytoskeleton.

TRPV1 co-localizes in the cell with microtubular

structures

To assess the TRPV1–tubulin interaction at the cellular level,we performed co-localization experiments with indirect

Fig. 2 Tubulin can be co-immunoprecipitated with TRPV1.

(a) Co-immunoprecipitation of b-tubulin and TRPV1 from TRPV1-

transfected F11 cells. From extracts of TRPV1-transfected F11 cells

(lane 1), proteins were immunoprecipitated by the anti-N-terminal

TRPV1 antibody (lane 2), anti C-terminal TRPV1 antibody (lane 3) or a

non-specific antibody (lane 4). Immunocomplexes were separated by

10% SDS–PAGE, followed by the detection of TRPV1 (left) and

b-tubulin (right) by western blot. (b) Western blot analysis using anti-

tubulin antibody (lane 1) failed to detect tubulin in the anti-N-terminal

TRPV1 immunoprecipitate from non-transfected F11 cells, which do

not express TRPV1. (c) Tubulin co-immunoprecipitates with TRPV1

from spinal cord extract. TRPV1 could be detected by western blots

directly in the tissue extract in some preparations, not in others (see

also Fig. S3e). However, detection of TRPV1 was possible after anti-

TRPV1 immunoprecipitation. From detergent-solubilized extract of rat

spinal cord (lane 1), proteins were immunoprecipitated by the anti-N-

terminal TRPV1 antibody (lane 2), or a non-specific antibody (lane 3).

Immunocomplexes were separated by 10% SDS–PAGE, followed by

the detection of TRPV1 (left) and b-tubulin (right) by western blot.

(d) Actin is not co-immunoprecipitated with TRPV1. Western blot

analysis using an anti-actin antibody (left panel) and anti TRPV1

antibody (right panel) of an extract of F11 cells transiently expressing

TRPV1 (lane 1), proteins that co-precipitate with TRPV1 from trans-

fected F11 cells (lane 2), and proteins co-precipitated with a non-

specific antibody (lane 3).



immunofluorescence. We transiently expressed full-lengthTRPV1 in F11 cells, and visualized TRPV1 and endogenoustubulin. We observed that TRPV1 was localized both inintracellular membranes and the plasma membrane. Weobserved co-localization of anti-TRPV1 immunoreactivitywith anti tubulin immunoreactivity (Fig. 3a, see alsoFig. S4a).In neurite-like extensions of F11 cells, we observed a dot-likeanti-TRPV1 immunoreactivity.Many of these distinct dot-likeTRPV1-immunoreactive structures also stained positive withanti tubulin antibodies. Interestingly, we did not observe suchdot-like anti tubulin immunoreactivity pattern in non-trans-fected F11 cells (Fig. S4c). In contrast to what we observedwith tubulin and TRPV1, we observed only minimal co-localization of TRPV1 with actin filaments and, in particular,no overlap in dot-like areas, when we stained F-actin withfluorescent phalloidin (Fig. 3b, see also Fig. S4b).

Purified tubulin alone binds to the C-terminal sequence of

TRPV1, but not with the N-terminal portion of TRPV1

In the MBP-TRPV1-Ct pulldown assay with proteins fromspinal cord extracts we observed that tubulin interacts withthe C-terminal domain of the TRPV1 along with otherproteins. In order to test if the tubulin–TRPV1 interaction is

direct and independent of other proteins, we prepared asoluble tubulin dimer fraction from porcine brain andperformed the pulldown assay with this purified tubulinpreparation. We observed that a considerable amount ofpurified tubulin was pulled down with MBP-TRPV1-Ct bothin the presence and absence of Ca2+, but not with MBPalone. Purified tubulin was also not pulled down by MBP-TRPV1-Nt (Fig. 4a). We confirmed this by western blotanalysis using antibodies directed against tubulin (Fig. 4b).In reverse pulldown experiments using biotinylated tubulinimmobilized on streptavidin agarose as a bait, we found thatMBP-TRPV1-Ct, but neither MBP-TRPV1-Nt nor MBPalone, bound to the tubulin (Figs 4c and d). This suggeststhat the TRPV1–tubulin interaction is direct and specific andis mediated by the TRPV1 C-terminal cytoplasmic portion,not by the N-terminal cytoplasmic sequence.

MBP-TRPV1-Ct, but not MBP-TRPV1-Nt, interacts also

with polymerized forms of tubulin

In order to assess if the C-terminal domain of TRPV1 alsointeracts with polymerized forms of tubulin, we performed aco-assembly/co-sedimentation experiment. MTs can beformed from preparations of soluble dimers by adding

Fig. 3 TRPV1 co-localizes with tubulin in transfected F11 cells.

(a) Confocal images (i–iv) and enlarged sections of the same images

(v–viii) of F11 cells transiently expressing TRPV1. There is some dis-

tinct co-localization (yellow in the overlay) of TRPV1 (green) and tub-

ulin (red) immunoreactivity. TRPV1 (i and v) and tubulin (ii and vi) were

visualized by indirect immunofluorescence with an anti-TRPV1 anti-

body and an anti tubulin antibody. Regions of co-localization appear as

yellow (iii and vii). Distinct spots of co-localization near the plasma

membrane are marked with arrows. Phase-contrast pictures (iv and

viii) of the same are shown. Scale bar: 20 lM. (b) Confocal images of

F11 cells (i–iv) transiently expressing TRPV1 probed for TRPV1

(green; i) and actin fibres (red; ii) using the anti-TRPV1 antibody and

Alexa Fluor 594 phalloidin. Scale bar: 20 lM. No significant overlap (iii)

of the immunofluorescence signals was observed. Phase-contrast

pictures (iv) of the same is shown. For serial Z-stacks views, see also

Fig. S4.



GTP. The addition of taxol favours MT formation and alsostabilizes the MTs formed. MBP-TRPV1-Ct, MBP-TRPV1-Nt, or MBP were incubated with pre-formed MTs in thepresence or absence of free Ca2+ at room temperature. TheMTs that formed along with bound proteins were separatedfrom soluble dimers and unbound proteins by centrifuga-tion, and the corresponding supernatants and pellets wereanalysed by staining of proteins and western blots usinganti-MBP antibodies. We observed that approximately 50%of the total MBP-TRPV1-Ct co-sedimented with thepolymerized MTs (Fig. 5a). In contrast, MBP-TRPV1-Nt,and MBP alone, failed to co-sediment with polymerizedMTs, and all of the MBP-TRPV1-Nt, or MBP, respectively,

remained in the supernatants. This result was furtherconfirmed by western blot analysis of the same samplesusing an anti-MBP antibody (Fig. 5b). These data suggestthat MBP-TRPV1-Ct, but not MBP-TRPV1-Nt, is capableof interaction not only with tubulin dimers, but also withpolymerized MTs. In the absence of polymerized MTs,MBP-TRPV1-Ct was not found in the pellet fraction.Instead, it remained in the supernatant after centrifugationunder the same conditions (Fig. 5c). This supports ourconclusion that the presence of the MBP-TRPV1-Ct in thepellet fraction is due to its direct interaction with polymer-ized MT. In this experiment, we observed no significanteffect of Ca2+ on the interaction.

Fig. 4 Purified tubulin directly interacts with MBP-TRPV1-Ct in a

Ca2+-sensitive way, but not with MBP-TRPV1-Nt. (a) MBP-TRPV1-Nt,

MBP-TRPV1-Ct and MBP alone, all immobilized on amylose resin

material, were incubated with purified tubulin dimers. Lane 1 MBP-

TRPV1-Nt; lane 2, MBP-TRPV1-Ct; lane 3, MBP alone; lane 4, tubulin

dimers. Tubulin dimers did not bind to the MBP-TRPV1-Nt (lanes 5

and 6) nor to the MBP alone (lanes 9 and 10), but bound to the MBP-

TRPV1-Ct (lanes 7 and 8). Binding of tubulin dimers to MBP-TRPV1-

Ct appeared to be slightly more in presence of Ca2+ (+) as compared

to the situation in the absence of Ca2+ (–). The proteins were eluted

from the amylose resin with 10 mM maltose and resolved by 10%

SDS–PAGE. Proteins were stained with silver stain. (b) Western blot

analysis of corresponding samples with anti tubulin antibdy YL1/2,

confirming the presence of tubulin only in the MBP-TRPV1-Ct pull-

down elutes. (c) MBP-TRPV1-Ct is pulled down by biotinylated tubulin

immobilized with streptavidin agarose. Biotinylated tubulin immobilized

on streptavidin agarose was incubated with MBP-TRPV1-Nt (lane 1–

2), MBP-TRPV1-Ct (lane 3–4), or with MBP alone (lane 5–6) in the

presence of Ca2+ (lane 1, 3 and 5) or in the absence of Ca2+ (lane 2, 4

and 6). Lanes 7–9 show the input of MBP-TRPV1-Nt, MBP-TRPV1-Ct

and MBP, respectively. Only MBP-TRPV1-Ct was pulled down with

the biotinylated tubulin. (d) Western blot analysis of samples corres-

ponding to those in (c) probed with the anti-MBP antibody. Among all

the pulldown samples, anti-MBP immunoreactivity shows up only in

lanes 3 and 4, confirming that MBP-TRPV1-Ct, but not MBP-TRPV1-

Nt or MBP alone, were pulled down with biotinylated tubulin.



Tubulin interaction with MBP-TRPV1-Ct, but not MBP-

TRPV1-Nt, allows MT formation under conditions that

otherwise destabilize MTs

To explore if MBP-TRPV1-Ct interaction with polymerizedMT changes MT physico-chemical properties, especiallytheir ‘stability as polymers’, we assessed MT formation fromtubulin dimers in the absence of fusion proteins, or in thepresence of MBP-TRPV1-Ct, MBP-TRPV1-Nt, under con-ditions which interfere with MT formation, i.e. in thepresence of the MT-destabilizing agent nocodazole and Ca2+.In the presence of nocodazole or nocodazole/Ca2+, MTformation from tubulin dimers in the absence of fusionproteins was poor (Fig. 6, upper panel). Strikingly, whenMBP-TRPV1-Ct was present, MT formation was consider-ably enhanced even in the presence of nocodazole ornocodazole/Ca2+, and a significant portion of MBP-TRPV1-Ct was found in the MT pellet (Fig. 6, middlepanel). In contrast, in the presence of MBP-TRPV1-Nt, MTpolymer formation was not altered as compared to the‘tubulin-only’ sample, and no fusion protein was observed inthe MT pellet. The entire amount of MBP-TRPV1-Ntremained in the supernatant (Fig. 6, bottom panel).

These data suggest that MBP-TRPV1-Ct is able to providestability to the polymers even in the presence of nocodazole.

MBP-TRPV1-Ct, but not MBP-TRPV1-Nt, stabilizes

MTs against cold-induced depolymerization in a Ca2+-

sensitive manner

MTs are known to be susceptible to cold-induced depo-lymerization. In order to test if MBP-TRPV1-Ct affectsthis property, we performed tubulin polymerization from

tubulin dimers in the absence of fusion proteins, or in thepresence of MBP-TRPV1-Ct or MBP-TRPV1-Nt, with orwithout Ca2+, followed by depolymerization at lowtemperature (see Fig. 7a for a flow chart of the experi-ment). In the ‘tubulin-only’ sample (Fig. 7b, upper panel),a portion of tubulin dimers polymerized to form MTpolymers at 37�C, the latter of which could be pelletted bycentrifugation (Fig. 7b, lane 2). When subjected to ice-coldtemperatures, the MT polymers were partially depolymer-ized under the experimental conditions used, and tubulin

Fig. 5 MBP-TRPV1-Ct, but not MBP-TRPV1-Nt, or MBP alone,

co-sediments with polymerized microtubules (MTs). Purified ab-tubulin

dimers were incubated with taxol and GTP to form MTs. Taxol-sta-

bilized MTs were incubated with 5 lg of purified MBP alone, MBP-

TRPV1-Ct, or of MBP-TRPV1-Nt, in the presence (+) or absence (–) of

Ca2+ (1 mM). Supernatant (S) and pellet (P) were separated by cen-

trifugation and analysed by 10% SDS–PAGE. (a) Distribution of the

proteins between MT pellet (P) and supernatant (S) as visualized by

silver staining of the proteins. MBP (left panel), MBP-TRPV1-

Ct (middle panel), or MBP-TRPV1-Nt (right panel) were assayed for

co-sedimentation with MT. Tubulin on the gel is indicated by an

asterisk (*), and the positions of the MBP and fusion proteins are

indicated by arrows. Only MBP-TRPV1-Ct co-sediments with MT.

(b) Western blot analysis of samples corresponding to those shown in

(a) using the anti-MBP antibody. Only when MBP-TRPV1-Ct was as-

sayed for co-sedimentation with MT, anti-MBP immunoreactivity ap-

peared in the pellet fractions (middle panel). In contrast, when MBP-

TRPV1-Nt or MBP alone were assayed for cosedimentation with MT,

anti MBP immunoreactivity was only detected in the supernatants.

(c) MBP-TRPV1-Ct never appeared in the pellet fractions in the ab-

sence of MT as confirmed by silver stain (left panel) and by anti-MBP

western blot (right panel).



occured in the supernatant (Fig. 7b, lane 3) and a pellet(Fig. 7b, lane 4) fraction after another round of centrifu-gation. We observed that, after the first cycle of polymer-ization, the amount of formed MT polymer pellet wasunaffected by the presence or absence of Ca2+. Awcomparable amount of MT polymers formed in thepresence of MBP-TRPV1-Ct with, in agreement with theexperiments shown above, a significant portion of theMBP-TRPV1-Ct was detectable in the MT pellet fraction(Fig. 7, middle panel). Strikingly, in the presence of Ca2+

and MBP-TRPV1-Ct, cold-induced MT depolymerizationwas dramatically reduced (Fig. 7, middle panel). Thepresence of MBP-TRPV1-Nt had no such effect. In fact,MBP-TRPV1-Nt was never observed in a MT polymerpellet, not even after the first round of MT polymerizationfrom tubulin dimers (Fig. 7, bottom panel). This suggestsa specific Ca2+-sensitive stabilizing effect of theTRPV1 C-terminus on MTs against cold-induced depo-lymerization.

In summary, there appears to be not only an interaction ofthe TRPV1 C-terminal portion with tubulin dimers and withpolymerized MTs but – at least under some experimentalconditions – also a Ca2+-sensitivity of this interaction.Furthermore, our data suggest that the C-terminus of TRPV1affects the oligomerization status of tubulin.

Discussion

This work is the first report on a direct interaction of thevanilloid receptor TRPV1 with the cytoskeleton. In ascreen for cytoplasmic interaction partners of the vanilloidreceptor TRPV1 by proteomic means, we identifiedb-tubulin as a protein which interacts with the TRPV1C-terminus in a Ca2+-sensitive manner. This findingsuggests that the MT cytoskeleton is a downstreameffector of TRPV1-mediated Ca2+-signalling. Furthermore,we observed effects of the TRPV1 C-terminal fragment,but not with the TRPV1 N-terminal portion, on the tubulin

Fig. 6 MBP-TRPV1-Ct, but not MBP-TRPV1-Nt, allows MT polymer-

ization in the presence of nocodazole: Comparison of tubulin distri-

bution between supernatant (indicated by S), and pellet

(corresponding to MT polymer), indicated by P, formed from ab-tubulin

dimer alone (upper panel), in the presence of MBP-TRPV1-Ct (middle

panel), and in the presence of MBP-TRPV1-Nt (bottom panel), in

buffer (lane 1), with nocodazole (lane 2), with Ca2+ (lane 3) and in

presence of both nocodazole and Ca2+ (lane 4). Proteins were visu-

alized by SDS–PAGE followed by silver staining of proteins in the

supernatants and pellets, and anti tubulin western blot of the pellets.

The asterisks (*) indicate the position of tubulin, MBP-TRPV1-Nt is

indicated by the dotted arrow, and MBP-TRPV1-Ct by the solid arrow.

Note that the amount of MT polymer formed in the presence of no-

codazol or nocodazol/Ca2+ is significantly increased in the presence of

MBP-TRPV1-Ct as compared to the ‘tubulin-only ‘control. In contrast,

the presence of the same amount of MBP-TRPV1-Nt has no effect.



oligomerization status. Remarkably, there was a Ca2+-sensitive stabilizing effect of the TRPV1 C-terminalfragment on MTs towards cold-induced depolymerizationand a Ca2+-independent stabilization of MTs with respectto depolymerization by nocodazole.

How do these observations fit the current knowledge oninteractions and functions of TRPV1? Though knownsequence motifs are absent from the primary structure ofthe cytoplasmic C-terminal tail of TRPV1, there is a body ofexperimental evidence that attribute important functionalproperties to this region of the protein. The C-terminalsequence of TRPV1 was demonstrated to contain a bindingsite for the phosphoinositide PIP2, which negatively regu-lates the ion channel activity (Prescott and Julius 2003).Moreover, the C-terminal tail contains phosphorylation sitesused by protein kinase C (Numazaki et al. 2002) and Ca2+/calmodulin-dependent protein kinase II, and usage of this siteaffects the TRPV1 desensitization characteristics in thepresence of Ca2+ (Jung et al. 2004). Furthermore, Numazakiet al. (2003) detected a short sequence critical for receptordesensitization within the C-terminal tail, and this wassuggested to carry a binding site for calmodulin. Vlachova

et al. (2003) demonstrated that the TRPV1 C-terminal tailalso carries structural determinants that take part in renderingthe receptor sensitive to heat and capsaicin. Notably, severalfuntionally relevant structural determinants were all mappedto a certain sequence of the TRPV1 C-terminal tail, namelythe range within the sequence stretch of amino acid residues760–800 (based on the rat TRPV1 primary structure), whichmay represent a ‘hot spot’ for TRPV1 regulation. Accordingto the structural model for the TRPV1 C-terminus suggestedby Vlachova et al. (2003) this sequence stretch contains twobeta strands. However, up to now we do not know whetherthe tubulin binding site is localized within this sequencestretch as well.

Interestingly, in the same article (Vlachova et al. 2003),a structural similarity between the TRPV1 C-terminal tailand the fragile histidine triad protein (FHIT), a tumoursuppressor gene product, was reported. This was based onsequence alignments and molecular modelling of theputative 3D structure of the TRPV1 C-terminal tail basedon the FHIT crystal structure. Remarkably, FHIT wasreported to be a tubulin-binding protein (Chaudhuri et al.1999).

However, with respect to the calmodulin binding, in ourhands there was no detectable interaction between purifiedcalmodulin and the C-terminal TRPV1-MBP fusion protein,

Tubulin diner

Polymerisation by GTP at 37°C

Depolymerisation by cold buffer at 4°C

Centrifugal separation

Centrifugal separation

Supernatant (S, lane 1)

Supernatant (S, lane 3)

Pellet (P, lane 2)

Pellet (P, lane 4)

Fig. 7 MBP-TRPV1-Ct provides stability to the MT towards depoly-

merization at low temperatures. (a) Flow chart of the experiment: First,

MT were allowed to form from tubulin dimers, followed by separation of

MT polymers from remaining dimers by centrifugation. Then, the MT

polymer pellet was subjected to cold-temperature-induced depolym-

erization on ice. Subsequently, cold-stable MT polymers were separ-

ated from disassembled tubulin by another round of centrifugation.

This sequential polymerization/depolymerization was performed with

tubulin alone, in the presence of MBP-TRPV1-Ct, or MBP-TRPV1-Nt,

in the presence or absence of Ca2+, respectively. The distribution of

tubulin and added proteins between supernatant and pellet fractions

was monitored. (b) ab-Tubulin dimer alone (top panel) or in the pres-

ence of MBP-TRPV1-Ct (middle panel), or in the presence of MBP-

TRPV1-Nt (bottom panel), were subjected to polymerization either in

the absence of Ca2+ (–, left side) or presence of Ca2+ (+, right side).

After the first cycle of polymerization, free dimers and unbound pro-

teins (which remained in the supernatant, S, lane 1) were separated

from MT polymers and associated proteins (pellet, P, lane 2) by

centrifugation. The MT polymers were subsequently depolymerized by

ice-cold buffer, followed by the centrifugal separation of disassembled

tubulin dimers (S, lane 3) and cold-stable MTs (P, lane 4). Samples

were anaylsed by 10% SDS–PAGE followed by silver staining. The

position of tubulin is indicated by an asterisk (*), MBP-TRPV1-Ct is

indicated by a solid arrow, and MBP-TRPV1-Nt by a dotted arrow. In

contrast to MBP-TRPV1-Nt, which remained in the supernatant in step

1 (lanes 1, bottom panel), MBP-TRPV1-Ct co-sedimented with poly-

merized MT both in the presence or absence of Ca2+ (lanes 2, middle

panel). Strikingly, the association of MBP-TRPV1-Ct with MT in the

presence of Ca2+ results in an increased amount of cold-stable MT

polymer (middle panel, right side, lanes 3 and 4).



regardless of the presence or absence of tubulin and/or Ca2+.At the present stage, we cannot explain the reason for thisdiscrepancy to what has been reported by others (Numazakiet al. 2003). However, there was a recent report that ratherthe N-terminus than the C-terminus of TRPV1 might beinvolved in the interaction with calmodulin (Rosenbaumet al. 2004).

Tubulin carries a number of negetively charged amino acidresidues on its surface, and most of the known MT-associatedproteins contain many basic amino acid residues (Nogales2001). Interestingly, fitting into this scheme, the C-terminalsequence of TRPV1 also contains several basic amino acidresidues, and the calculated pI of the entire C-terminalsequences is as high as 9.2.

With regard to the identification of b-tubulin as a TRPV1-interactive partner in a Ca2+-sensitive screen, it is importantto mention that tubulin has been shown to bind twomolecules of Ca2+ at its C-terminal region (Hayashi andMatsumura 1975; Solomon 1977; Serrano et al. 1986), and aCa2+-dependent conformational change of tubulin has beendetected (Soto et al. 1996).

A number of membrane receptors have been shown tointeract specifically with either a- or b-tubulin, such asisoforms of the metabotropic glutamate receptors mGluR1and mGluR7 (Ciruela et al. 1999; Ciruela and McIlhinney2001; Saugstad et al. 2002), or subunits of the NMDAreceptor (van Rossum et al. 1999). Those isoforms that bindb-tubulin were reported to inhibit MT formation. A numberof studies established a wide range of interactions betweentubulin and different G proteins and pointed to functionalimplications of these interactions (Popova et al. 1997;Roychowdhury and Rasenick 1997; Roychowdhury et al.1999; Sarma et al. 2003).

In this work we showed that the C-terminal sequence ofTRPV1 not only interacts with the soluble tubulin dimer, butalso enhances the resistance of the MT polymers to nocodaz-ole, a potent drug often used for MT depolymerization.Interestingly, the polymers formed in presence of MBP-TRPV1-Ct show an enhanced stability against cold-induceddepolymerization, and this process is Ca2+-dependent. This, ingeneral terms, suggests that the MT cytoskeleton close to theplasmamembrane is subject to signal-induced plastic changes.Within the context of nociception, there is emerging interest inthe cytoskeleton because it has been demonstrated that certainforms of hyperalgesia depend on cytoskeleton integrity (Dinaet al. 2003). Whether potential TRPV1-mediated effects onthe cytoskeleton contribute to the modulation of nociceptivesignalling, remains to be determined.

Acknowledgements

Technical assistance by Doris Kruck and Hermann Bayer is

gratefully acknowledged. This work was supported by grant no.

01 GG 9818/0, Molecular Pain Research; by the Deutsche

Forschungsgemeinschaft, Sfb 515; and by the Fonds der

Chemischen Industrie.

Supplementary material

The following material is available from:http://www.blackwellpublishing.com/products/journals/suppmat/JNC/JNC2795/JNC2795sm.htm

Figure S1. Expression and purification of MBP-TRPV1-Ctand MBP-TRPV1-Nt.Figure S2. Peptide mass fingerprint as obtained fromMALDI-MS analysis of the peptide mixture derived fromthe protein corresponding to the 55 kDa band observed as aCa2+-sensitive TRPV1 interaction partner. Peptides matching-tubulin are indicated.Figures S3. Assessment of the specificity of the antiN-terminal and anti C-terminal TRPV1 antibodies.Figure S4. Co-localisation of TRPV1 and tubulin in F11cells.

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Identification and characterization of a Ca2+-sensitive interaction of the vanilloid receptor TRPV1...

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