Identification and characterisation of novel tubulin-binding motifs located within the C-terminus of TRPV1.

Identification and characterisation of novel tubulin-binding motifs
located within the C-terminus of TRPV1
C. Goswami,*,� Tim B. Hucho� and F. Hucho*
*Freie Universita¨t Berlin, Institut fu¨r Chemie und Biochemie, Berlin, Germany
�Department of Human Molecular Genetics, Max Planck Institute for Molecular Genetics, Berlin, Germany
Abstract
Previously, we reported that TRPV1, the vanilloid receptor,
interacts with soluble ab-tubulin dimers as well as microtubules
via its C-terminal cytoplasmic domain. The interacting region of
TRPV1, however, has not been defined. We found that the
TRPV1 C-terminus preferably interacts with b-tubulin and less
with a-tubulin. Using a systematic deletion approach and bio-
tinylated-peptides we identified two tubulin-binding sites pre-
sent in TRPV1. These two sequence stretches are highly
conserved in all known mammalian TRPV1 orthologues and
partially conserved in some of the TRPV1 homologues. As
these sequence stretches are not similar to any known tubulin-
binding sequences, we conclude that TRPV1 interacts with
tubulin and microtubule through two novel tubulin-binding
motifs.
Keywords: cytoskeleton, motif sequence, pain, receptor,
TRPV, tubulin.
J. Neurochem. (2007) 101, 250–262.
Tubulin, the main constituent of microtubules is a cytoplas-
mic protein. Nevertheless, it is often reported to be present
also in membrane preparations isolated from neuronal tissues
(Bhattacharyya and Wolff 1975; Walters and Matus 1975;
Gozes and Littauer 1979; Zisapel et al. 1980; Babitch 1981;
Strocchi et al. 1981; de Ne´chaud et al. 1983; Hargreaves and
Avila 1985). Although it is not an integral membrane protein,
it can be enriched together with the membrane proteins after
solubilising the membranes with the detergent Triton X-114
(Beltramo et al. 1994). Indeed, in recent years, a number of
transmembrane receptors have been shown to interact
specifically with either a-tubulin and/or b-tubulin and
thereby to account for the tubulin association with mem-
branes.
The interactions of tubulin with membrane proteins often
results in altered microtubule dynamics. Conversely, chan-
ges of microtubule dynamics alter receptor/channel func-
tions. Tubulin interaction with a wide variety of membrane
proteins has been documented. For example, functional
significance of tubulin interaction has been shown for the
isoforms of the metabotropic glutamate receptor mGluR1
and mGluR7 (Ciruela et al. 1999; Ciruela and McIlhinney
2001; Saugstad et al. 2002), the ionotropic GABAA
receptor (Item and Sieghart 1994), subunits of the NMDA
receptor (van Rossum et al. 1999), and for various
G-proteins (Wang et al. 1990; Popova et al. 1997; Roy-
chowdhury and Rasenick 1997; Roychowdhury et al. 1999;
Chen et al. 2003; Sarma et al. 2003; Popova and Rasenick
2004). The presence of tubulin was also identified in
complexes with voltage-dependent anion channel (VDAC)
(Carre et al. 2002), shaker channel (Moreno et al. 2002)
and with ion-pumps such as Na+-K+-ATPase (Vladimirova
et al. 2002). In many instances, tubulin/transmembrane
protein interactions are involved in complex signalling
events such as neurite out growth, cell morphology and cell
differentiation. Interaction of acetylated tubulin (a post-
trsanslationally modified form of tubulin) with H+-ATPase
is reported to be important for the glucose uptake regulation
in yeast (Campetelli et al. 2005).
Like other transient receptor potential (TRP) channels,
TRPV1 is a non-selective cation channel (Caterina et al.
1997). Both N-terminal and C-terminal sequences of TRPV1
form cytoplasmic domains. Previously, we identified ab-
tubulin as TRPV1 interacting partner (Goswami et al. 2004).
We demonstrated that the C-terminus of TRPV1 is sufficient
and interacts directly with microtubules (Goswami et al.
Received July 23, 2006; revised manuscript received September 15,
2006; accepted October 3, 2006.
Address correspondence and reprint requests to F. Hucho, Freie
Universita¨t Berlin, Institut fu¨r Chemie und Biochemie, Thielallee 63,
14195 Berlin, Germany. E-mail: hucho@chemie.fu-berlin.de
Abbreviations used: VDAC, voltage-dependent anion channel; TRP,
transient receptor potential; MBP, maltose-binding protein; MT, micro-
tubules; DMS, dimethyl suberimidate.
Journal of Neurochemistry, 2007, 101, 250–262 doi:10.1111/j.1471-4159.2006.04338.x
250 Journal Compilation � 2007 International Society for Neurochemistry, J. Neurochem. (2007) 101, 250–262
� 2007 The Authors

2004). It provides stability to microtubules both in vitro and
in vivo (Goswami et al. 2004, 2006). Interestingly, tubulin
interaction is observed also for other members of the TRP
super family. Interaction of b-tubulin with TRPC1 has been
reported recently (Bollimuntha et al. 2005). Two other
members, namely TRPC5 and TRPC6, contain tubulin as
constituent of its ‘signalplex’ (Goel et al. 2005). Very
recently, it has been shown that Polycystin-2 type TRP
channels are regulated by microtubular structures in primary
cilia of renal epithelial cells (Li et al. 2006). This suggests
that tubulin interaction might be common for many of the
TRP ion channels.
In spite of the functional implication of the interaction of
tubulin with several transmembrane receptors and ion
channels, very little is known about the binding structure/s
that underlie these interactions. Therefore, we set out to
identify the exact tubulin-binding region of TRPV1 and
further characterised the interacting structures.
Materials and methods
Reagents and antibodies
The microtubule stabilising drug Taxol� (paclitaxel), the cross-linker
DMS and purified actin, were purchased from Sigma–Aldrich
(Taufkirchen, Germany). Biotinylated-peptides (KSFLKCMRKA-
FRSGKLLQVGF-K-Biotin and KRTLSFSLRSGRVSGRNWKNF-
K-Biotin) were synthesised at Biosynthan (Berlin, Germany). Mouse
monoclonal a-tubulin antibodies (clone DM1A), mouse monoclonal
b-tubulin antibodies (clone D66), mouse monoclonal tyrosinated
tubulin antibodies (clone TUB1A2), mouse monoclonal polyglutam-
ylated tubulin antibodies (clone B3), mouse monoclonal acetylated
tubulin antibodies (clone 611-B-1), mouse monoclonal phosphoser-
ine antibodies (Clone PSR-45) and mouse monoclonal anti-b-tubulin
sub type III (clone SDL.3D10) were purchased from Sigma–Aldrich.
Mouse monoclonal neurofilament 200 kDa antibodies (clone RT97)
and rabbit polyclonal detyrosinated tubulin antibodies were pur-
chased from Chemicon (Chandlers Ford, UK). Mouse monoclonal
actin antibodies (clone JLA20) was purchased from Oncogene
(Cambridge, MA, USA). Mouse monoclonal anti-maltose-binding
protein (MBP) antibodies and amylose resin were purchased from
New England Biolab (Beverly, MD, USA). Enriched neurofilament
fraction was a kind gift from O. Bogen (Bogen et al. 2005).
Subtilisin-digested tubulin and control tubulin were kindly provided
by Linda Amos (Cambridge, UK). For the detection of subtilisin-
digested tubulin and control tubulin by western-blot analysis, we used
mouse monoclonal anti-b-tubulin (clone D10, Santa Cruz Biotech-
nology, Heidelberg, Germany).
Expression and purification of TRPV1 fusion proteins
Expression and purification of MBP-TRPV1-Nt (N-terminal cyto-
plasmic domain of TRPV1 fused with MBP) and MBP-TRPV1-Ct
(C-terminal cytoplasmic domain of TRPV1 fused with MBP) were
described in Goswami et al. (2004). The cDNA fragments of
TRPV1-Ct (see Fig. 1) were amplified by PCR using specific
primers (Table 1). All amplified DNA fragments were subcloned
into the EcoR1 and Hind III restriction sites of the pMAL-c2x vector
(New England Biolabs, Beverly, MA, USA). A stop codon was
introduced in each construct at the C-terminus of the coding
sequences. All expression constructs were verified by automated
nucleotide sequencing. Escherichia coli (E. coli) strain BL21DE3
was transformed by heat shock with the plasmid coding for the
TRPV1 cytoplasmic domains and fragments fused with MBP
protein. E. coli cells were induced to express the proteins by
isopropyl thiogalactoside (IPTG) for 2 h. The cells were lysed by
repeated freeze-thaw cycles in lysis buffer (20 mmol/L Tris–HCl,
pH 7.4, 150 mmol/L NaCl, 0.1% Tween 20, lysozyme, benzonase
and protease inhibitor cocktail). The lysed extracts were cleared by
centrifugation (100 000 g in a TFT 45 rotor for 2 h). The cleared
lysate was applied to amylose resin and washed thoroughly. Bound
protein was eluted with 10 mmol/L maltose in elution buffer
(50 mmol/L PIPES, pH 6.8, 100 mmol/L NaCl, 1 mmol/L EGTA
and 0.2 mmol/L MgCl2). Protein concentration was determined
according to method described by 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
Constructs and peptides
681 838
681 800
681 760
681 730
761 838
731 838
Ct
Ct-Δ1
Ct-Δ2
Ct-Δ3
Ct-frag 6
Ct-frag 7
Ct-frag 8 710 797
801 838
770 797
731 769
681 709
Ct-frag 1
Ct-frag 2
Ct-frag 3
Ct-frag 4
Ct-frag 5
710 730
9.20
9.67
9.34
10.13
8.21
6.08
10.03
6.35
11.17
4.03
12.6
5.49
pI
Peptide 1
Peptide 2
- Biotin
- Biotin
11.17
12.48
Fig. 1 Constructs and peptides used to identify the tubulin-binding
site located in the C-terminus of TRPV1. Schematic representation of
constructs prepared to express the deletion-proteins and fragments
corresponding to the different regions of C-terminus of TRPV1. Posi-
tions of the amino acids are written in top. Different deleted and
fragmented parts of the C-terminal of TRPV1 are expressed as MBP-
fusion protein (MBP is at the N-terminus of each fusion constructs).
Biotinylated-peptides (biotin label is at the C-terminus) are indicated.
Dark background indicates the regions with higher pI (short basic
stretches 1 and 2), whereas light background indicates the regions
with lower pI. All theoretical isoelectric points of the deletion constructs
were calculated by using available software (http://www.expasy.org/
tools/pi_tool.html).
Identification and characterisation of tubulin-binding motifs 251
� 2007 The Authors
Journal Compilation � 2007 International Society for Neurochemistry, J. Neurochem. (2007) 101, 250–262

disassembly by cold temperature (ice-cold) were followed by
chromatography on phosphocellulose.
Pull-down assay
MBP-LacZ, MBP-TRPV1-Ct, different MBP-TRPV1-Ct fragments
and deletion constructs (see Fig. 1) were expressed in E. coli, the
cleared cell lysates were applied to amylose resin (NEB), and
incubated for 1 h at 25�C followed by washing. The amylose resin
with bound proteins were re-suspended in PEM-S buffer (50 mmol/L
PIPES, pH 6.8, 100 mmol/L NaCl, 1 mmol/L EGTA and 0.2 mmol/
L MgCl2). Approximately, 50 lL of amylose resin with the bound
fusion protein was incubated with 50 lL of soluble tubulin (1 mg/mL
protein) for 1 h at 25�C either in the presence or absence of Ca2+
(2 mmol/L). This was followed by three washes with 200 mL each
time and constant buffer conditions. The proteins were eluted by
10 mmol/L maltose in 100 lL solution. Eluted samples were
analysed by 10% sodium dodecyl sulphate polyacrylamide gel
electrophoresis (SDS-PAGE) according to Laemmli (1970).
For experiments determining the binding of subtilisin-digested
tubulin with TRPV1-Ct, MBP-TRPV1-Ct immobilised on amylose
resin (20 lL) in PEM-S buffer were incubated with 2 lg of
subtilisin-digested tubulin or same amount of control tubulin for 1 h
at 25�C. After three washes in PEM-S buffer, the bound protein
complexes were eluted and analysed further.
To identify if there is a direct interaction between tubulin and
short peptides carrying TRPV1 sequences, biotinylated-peptides
were incubated with avidin agarose (Sigma–Aldrich) at 25�C for
1 h, washed extensively with PEM-S-T (50 mmol/L PIPES, pH 6.8,
1 mmol/L EGTA, 0.2 mmol/L MgCl2, 150 mmol/L NaCl and 0.1%
Tween 20) buffer thrice, incubated with the soluble tubulin dimer
(40 lg in 100 lL) for 1 h. Avidin–agarose resins were washed
thrice with PEM-S-T buffer and finally taken in to Laemmli sample
buffer and analysed by western-blot analysis for bound proteins.
Cross-linking of proteins
A protein mixture (1 mg/mL) of equal amounts of ab-tubulin dimer
and MBP-TRPV1-Ct, in PEM buffer was adjusted to 0.2 mol/L
triethanolamine (pH 8.1) buffer for cross-linking with dimethyl
suberimidate (DMS, Sigma, 1 mg/mL). The reaction was carried out
at 25�C for 1 min to 1 h and stopped by adding Tris–HCl (pH 6.8)
to a final concentration of 50 mmol/L. Samples were subjected to
SDS-PAGE separation and western-blot analysis.
Western-blot analysis
To perform western-blot analysis, the proteins were separated by
SDS-PAGE, and transferred either to a nitrocellulose membrane or
PVDF (Millipore, Schwalbach, Germany) by semidry electro
blotting. The membranes were blocked with 5% non-fat milk in
TBS-T (20 mmol/L Tris, 150 mmol/L NaCl. 0.1% Tween-20) buffer
followed by incubation with the respective primary antibody for 1 h
at 25�C, washed thrice times with TBS-T buffer. Subsequently, the
membranes were incubated with horseradish peroxidase-conjugated
secondary antibody for 1 h at 25�C. For the detection of
biotinylated-peptides, PVDF membranes containing peptide spots
were probed with HRP-conjugated avidin (Sigma–Aldrich, 1 : 1000
dilution). The membranes were washed thoroughly with TBST. The
ECL detection system (Amersham Biosciences, Freiburg, Germany)
was used for the visualisation of the immunoreactivity.
Co-sedimentation assay with taxol-stabilised microtubules (MT)
Approximately 100 lg of purified ab-tubulin dimer in a total
volume of 100 lL were incubated in modified PEM buffer
Table 1 Primers used for making the
TRPV1-Ct deletions and fragmentsPrimer Construct
1 F: 5¢ CCGGAATTCCTCATGGGTGAGACCGTCAAC 3¢ MBP-TRPV1-Ct-D1
R: 5¢ CCCAAGCTTTTAGCTTGCATCCCTCAGAAGGGG 3¢
2 F: 5¢ CCGGAATTCCTCATGGGTGAGACCGTCAAC 3¢ MBP-TRPV1-Ct-D2
R: 5¢ CCCAAGCTTTTAGTTGATGATACCCACATTGGT 3¢
3 F: 5¢ CCGGAATTCCTCATGGGTGAGACCGTCAAC 3¢ MBP-TRPV1-Ct-D3
R: 5¢ CCCAAGCTTTTAGAACCCCACCTGCAGCAGCTT 3¢
4 F: 5¢ CCGGAATTCCTCATGGGTGAGACCGTCAAC 3¢ MBP-TRPV1-Ct-F1
R: 5¢ CCCAAGCTTTTACTCTGTATCCAGGATGGTGAT 3¢
5 F: 5¢ CCGGAATTCAAGAGCTTCCTGAAGTGCATG 3¢ MBP-TRPV1-Ct-F2
R: 5¢ CCCAAGCTTTTAGAACCCCACCTGCAGCAGCTT 3¢
6 F: 5¢ CCGGAATTCACTCCTGACGGCAAGGATGAC 3¢ MBP-TRPV1-Ct-F3
R: 5¢ CCCAAGCTT TTAGACGCCCTCACAGTTGCCTGG 3¢
7 F: 5¢ CCGGAATTCAAGCGCACCCTGAGCTTCTCC 3¢ MBP-TRPV1-Ct-F4
R: 5¢ CCCAAGCTTTTACCTCAGAAGGGGAACCAGGGC 3¢
8 F: 5¢ CCGGAATTCACTCGAGATAGACATGCCACC 3¢ MBP-TRPV1-Ct-F5
R: 5¢ CCCAAGCTTTTATTTCTCCCCTGGGACCATGGA 3¢
9 F: 5¢ CCGGAATTCGAGGACCCAGGCAACTGTGAG 3¢ MBP-TRPV1-Ct-F6
R: 5¢ CCCAAGCTTTTATTTCTCCCCTGGGACCATGGA 3¢
10 F: 5¢ CCGGAATTCACTCCTGACGGCAAGGATGAC 3¢ MBP-TRPV1-Ct-F7
R: 5¢ CCCAAGCTTTTATTTCTCCCCTGGGACCATGGA 3¢
11 F: 5¢ CCGGAATTCAAGAGCTTCCTGAAGTGCATG 3¢ MBP-TRPV1-Ct-F8
R: 5¢ CCCAAGCTTTTACCTCAGAAGGGGAACCAGGGC 3¢
F, forward primer; R, reverse primer; Underlines indicate the presence of stop codon.
252 C. Goswami et al.
Journal Compilation � 2007 International Society for Neurochemistry, J. Neurochem. (2007) 101, 250–262
� 2007 The Authors

(20 mmol/L PIPES, pH 6.8, 0.2 mmol/L MgCl2 and 1 mmol/L
EGTA supplemented by 1 lmol/L taxol and 5 mmol/L GTP) for
30 min at 37�C, to form MT. After MT formation, 5 lg of purified
proteins representing different MBP-fusion proteins were incubated
with taxol-stabilised MT for 40 min at 37�C followed by centrifugal
separation of pellet (MT) and supernatant (free dimer) at 70 000 g/
30 min/37�C. In a similar manner biotinylated-peptides (approxi-
mately 1 lg each in 100 lL) were first centrifuged at 25 000 g for
5 min to remove all aggregates. Clear supernatant containing
soluble peptides were further used for microtubule co-sedimentation
assay. Corresponding pellet and supernatant fractions were further
spotted on a PVDF membrane (Amersham) by a dot-blot apparatus
(Bio-Rad, Munich, Germany) and analysed for bound peptides. For
MT formation under taxol-free conditions, 100 lg of tubulin dimer
were used in PEM buffer with 5 mmol/L GTP in the absence of
taxol and incubated for 30 min at 37�C.
Blot overlay
To carry out overlay experiments, either native tubulin dimer were
spotted directly or denatured and SDS-PAGE-separated proteins
(tubulin dimer) were transferred from gels to nitrocellulose or PVDF
membrane. Membranes were blocked for 1 h with 5% fat-free milk
in PBST buffer. Subsequently, the membranes were washed thrice.
Membranes were air-dried and incubated with MBP-TRPV1-Ct or
MBP alone (protein concentration 0.2 lg/mL, with 5% fat-free milk
in PBST buffer) for 1 h at 25�C. In blot overlay experiment with
biotinylated-peptides, PEM-S-T (50 mmol/L PIPES; pH 6.8,
1 mmol/L EGTA, 0.2 mmol/L MgCl2, 150 mmol/L NaCl and
0.1% Tween 20) buffer was used. Peptides were used at a
concentration of approximately 5 ng/mL. After incubation, the
membranes were washed thrice (each time for 10 min) and
incubated with 0.1% formaldehyde for 30 min to cross-link the
bound proteins. Finally, the membranes were quenched with
100 mmol/L glycine in TBS buffer and processed for western-blot
analysis with MBP antibodies or HRP-labelled avidin to detect the
bound proteins or peptides respectively.
Results
TRPV1 interacts with soluble tubulin, but neither with
soluble actin nor with soluble neurofilaments
Previously, we observed that the C-terminus of TRPV1 binds
to tubulin and stabilises microtubules (Goswami et al. 2004).
To understand if the C-terminus of TRPV1 can also interact
with cytoskeleton components other than tubulin, we
performed pull-down experiments with purified soluble actin
and enriched soluble neurofilament preparations. The C-
terminal cytoplasmic domain of TRPV1 fused with MBP
(MBP-TRPV1-Ct) was used as bait. However, we could not
observe any significant direct interaction of actin or neuro-
filaments with the MBP-TRPV1-Ct (Fig. 2a). As activation
of TRPV1 results in influx of Ca2+, we tested if actin and
neurofilaments interact with MBP-TRPV1-Ct in the presence
of high Ca2+. We observed no interaction even in the
presence of Ca2+. Under the same conditions a significant
amount of soluble tubulin interacts with the MBP-TRPV1-
Ct. In agreement with our previous results (Goswami et al.
2004), this interaction was slightly stronger in the presence
of Ca2+. In similar experiments, MBP-TRPV1-Ct, but not
– + – + –
Si
lv
er
s
ta
in
WB
1 2 3 4 5 1 2 3 4 5
Tubulin Actin NF 200 kDa
1 2 3 4 5
Ca2+
– + – + – – + – + –
C
t
La
cZ
Input Tubulin Actin Neurofilament
In
pu
t
C
t
La
cZ
In
pu
t
C
t
La
cZ
In
pu
t
205
kDa
β III tub
1 2
MBP
Acetylated tub
Tyrosinated tub
Detyrosinated tub
Polyglutamylated tub
Phospho (serine)tub
114
97
67
45
30
WB:
(a)
(b)
Fig. 2 The C-terminus of TRPV1 specifically interacts with constit-
uents of microtubule cytoskeleton. (a) MBP-TRPV1-Ct (lanes 2 and 3)
and MBP-LacZ (lanes 4 and 5), all immobilised on amylose resin were
incubated with purified soluble tubulin dimer (left panel), or purified
soluble actin (middle panel) or enriched neurofilament fraction (right
panel) either in the presence (lanes 2 and 4) or absence of Ca2+ (lanes
3 and 5) to analyse the specific binding. The proteins were eluted from
the amylose resin with 10 mmol/L maltose and resolved by SDS-
PAGE. Lane 1 shows the input amount of soluble proteins. Proteins
were stained with silver stain (upper panel) and also probed for bound
tubulin, actin or neurofilaments by western-blot analysis (lower panel)
of the corresponding samples. A significant amount of tubulin binds to
the MBP-TRPV1-Ct but not to the MBP-LacZ. In contrast to tubulin,
neither actin nor neurofilament binds to the MBP-TRPV1-Ct. (b) MBP-
TRPV1-Ct (lane 1) but not MBP-LacZ (lane 2) pulls down different
post-translationally modified tubulins and neuron-specific b-tubulin sub
type III.
Identification and characterisation of tubulin-binding motifs 253
� 2007 The Authors
Journal Compilation � 2007 International Society for Neurochemistry, J. Neurochem. (2007) 101, 250–262

MBP-LacZ pulls down neuron-specific beta III tubulin as
well as other post-translationally modified tubulins, i.e.
acetylated tubulin, polyglutamylated tubulin, phosphotubulin
(phosphoserine), tyrosinated tubulin and de-tyrosinated tub-
ulin (Fig. 2b). These results indicate that the C-terminus of
TRPV1 interacts specifically with the constituents of both
dynamic and stable microtubules.
b-tubulin, but not a-tubulin preferentially interacts with
TRPV1
Tubulin preparations from brain tissue consist predomin-
antly of ab-tubulin heterodimers. In order to analyse, which
subunit of the tubulin dimer interacts with the C-terminal
domain of TRPV1, we performed a cross-linking experi-
ment (Fig. 3a). A mixture of ab-tubulin dimer and MBP-
TRPV1-Ct was cross-linked by using dimethyl suberimidate
(DMS), a homobifunctional cross-linking agent, which
reacts with amino groups. The cross-linked products of
tubulin dimers and MBP-TRPV1-Ct were subsequently
analysed by gel electrophoresis and western-blot analysis
with the appropriate antibodies. We observed that cross-
linking occurred fast and the entire amount of MBP-
TRPV1-Ct appeared on the gel as a high-molecular weight
complex after only 1 min of cross-linking. All b-tubulin in
the reaction mixture also appeared in the same high-
molecular weight complex. Also the a-tubulin was found in
that complex, but approximately 50% of the a-tubulin did
not react. Even after 60 min of reaction, the non-cross-
linked a-tubulin population remained at its monomeric
molecular weight on the SDS-PAGE. The high-molecular
weight complex was not observed when we used purified
MBP instead of MBP-TRPV1-Ct for cross-linking experi-
ments with ab-tubulin dimer (data not shown). From these
data we conclude that the MBP-TRPV1-Ct interacts with
the tubulin dimer predominantly via b-tubulin.
The C-terminus of TRPV1 interacts with blotted
denatured tubulin
Three-dimensional crystal structures and EM pictures reveal
that the C-terminal tail of the tubulin dimer does not integrate
into the core of the microtubule filaments, but remains outside
(Nogales et al. 1999; Lowe et al. 2001). These exposed C-
terminal over-hanging regions of both a-tubulin and b-tubulin
are strongly negatively charged and unstructured too (Lowe
et al. 2001; Nogales 2001). Most of the known microtubule-
binding proteins interact with microtubules and tubulins
through these regions (see Fig. 4a, see also Discussion).
1 2 3 1 2 3 1 2 3
205
114
97
67
45
30
kDa
WB: α tub β tub MBP
205
114
97
67
45
30
1 2
WB: MBP
(a) (b)
Fig. 3 MBP-TRPV1-Ct interacts directly with b-tubulin. (a) MBP-
TRPV1-Ct and ab-tubulin dimer mixture was cross-linked with DMS
cross-linker. Protein mixtures before cross-linking (lane 1), 1 min after
cross-linking (lane 2) and 60 min after cross-linking (lane 3) were
separated by SDS-PAGE (4–10% gradient) and transferred to a
nitrocellulose membrane. Blots were probed with the anti-a-tubulin
antibody (left), the anti-b-tubulin antibody (middle) and the anti-MBP
antibody (right). The arrow indicates the high-molecular-weight cross-
link product. After cross-linking, all the MBP-TRPV1-Ct and all the
b-tubulin shows up in a high-molecular weight complex, whereas a
significant amount of the a-tubulin failed to react and remains in the
monomeric state. (b) MBP-TRPV1-Ct but not MBP alone detects
purified tubulin dimers in blot overlay experiment. Equal amount of
purified tubulin dimer separated by SDS-PAGE and transferred into
the nitrocellulose membrane were overlayed with MBP-TRPV1-Ct
(lane 1) and with MBP only (lane 2). Stripes were probed with anti-
MBP antibody. Anti-MBP immunoreactivity appeared in the position of
tubulin at lane 1 (indicated by arrow).
KSFLKCMRKAFRSGKLL KRTLSFSL RSGRVSGRN
Stretch 1 Stretch 2
VE--GEGEEEGEEY--
ADEQGEFEEEGEEDEA
Porcine α-tubulin
Porcine β-tubulin
(a)
(b)
Fig. 4 Positively charged amino acids of stretch sequence 1 and 2
are clustered in one side of the helix. (a) C-terminal tail of a-tubulin and
b-tubulin contain highly negatively charged amino acids. These neg-
atively charged amino acids (indicated in bold letter) are conserved in
almost all species, and most of the microtubule-binding proteins
interact via these sequences. (b) Amino acids of stretch sequences 1
and stretch sequence 2 (in each case 17 amino acid-long, indicated in
top) of the C-terminus of TRPV1 (rat) are plotted for helical distribution.
Top-view of the helix is shown. Programme available at http://kael.net/
helical.htm site is used to draw the helical wheel. Positively charged
amino acids (bold letter) are marked with asterisk sign. Note the dis-
tribution of positively charged residues (asterisk) in one side of the
wheel.
254 C. Goswami et al.
Journal Compilation � 2007 International Society for Neurochemistry, J. Neurochem. (2007) 101, 250–262
� 2007 The Authors