Identification and Characterisation of Novel Tubulin-Binding Motifs

Journal of Neurochemistry, 2007, 101, 250–262 doi:10.1111/j.1471-4159.2006.04338.x

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 conserved in all known mammalian TRPV1 orthologues and Previously, we reported that TRPV1, the vanilloid receptor, partially conserved in some of the TRPV1 homologues. As interacts with soluble ab-tubulin dimers as well as microtubules these sequence stretches are not similar to any known tubulin- via its C-terminal cytoplasmic domain. The interacting region of binding sequences, we conclude that TRPV1 interacts with TRPV1, however, has not been deﬁned. We found that the tubulin and microtubule through two novel tubulin-binding TRPV1 C-terminus preferably interacts with b-tubulin and less motifs. with a-tubulin. Using a systematic deletion approach and bio- Keywords: cytoskeleton, motif sequence, pain, receptor, tinylated-peptides we identiﬁed two tubulin-binding sites pre- TRPV, tubulin. sent in TRPV1. These two sequence stretches are highly J. Neurochem. (2007) 101, 250–262.

Tubulin, the main constituent of microtubules is a cytoplas- Chen et al. 2003; Sarma et al. 2003; Popova and Rasenick mic protein. Nevertheless, it is often reported to be present 2004). The presence of tubulin was also identified in also in membrane preparations isolated from neuronal tissues complexes with voltage-dependent anion channel (VDAC) (Bhattacharyya and Wolff 1975; Walters and Matus 1975; (Carre et al. 2002), shaker channel (Moreno et al. 2002) Gozes and Littauer 1979; Zisapel et al. 1980; Babitch 1981; and with ion-pumps such as Na+-K+-ATPase (Vladimirova Strocchi et al. 1981; de Nećhaud et al. 1983; Hargreaves and et al. 2002). In many instances, tubulin/transmembrane Avila 1985). Although it is not an integral membrane protein, protein interactions are involved in complex signalling it can be enriched together with the membrane proteins after events such as neurite out growth, cell morphology and cell solubilising the membranes with the detergent Triton X-114 differentiation. Interaction of acetylated tubulin (a post- (Beltramo et al. 1994). Indeed, in recent years, a number of trsanslationally modified form of tubulin) with H+-ATPase transmembrane receptors have been shown to interact is reported to be important for the glucose uptake regulation specifically with either a-tubulin and/or b-tubulin and in yeast (Campetelli et al. 2005). thereby to account for the tubulin association with mem- Like other transient receptor potential (TRP) channels, branes. TRPV1 is a non-selective cation channel (Caterina et al. The interactions of tubulin with membrane proteins often 1997). Both N-terminal and C-terminal sequences of TRPV1 results in altered microtubule dynamics. Conversely, chan- form cytoplasmic domains. Previously, we identified ab- ges of microtubule dynamics alter receptor/channel func- tubulin as TRPV1 interacting partner (Goswami et al. 2004). tions. Tubulin interaction with a wide variety of membrane We demonstrated that the C-terminus of TRPV1 is sufficient proteins has been documented. For example, functional and interacts directly with microtubules (Goswami et al. significance of tubulin interaction has been shown for the isoforms of the metabotropic glutamate receptor mGluR1 Received July 23, 2006; revised manuscript received September 15, and mGluR7 (Ciruela et al. 1999; Ciruela and McIlhinney 2006; accepted October 3, 2006.

2001; Saugstad et al. 2002), the ionotropic GABAA Address correspondence and reprint requests to F. Hucho, Freie receptor (Item and Sieghart 1994), subunits of the NMDA Universita¨t Berlin, Institut fu¨r Chemie und Biochemie, Thielallee 63, 14195 Berlin, Germany. E-mail: [email protected] receptor (van Rossum et al. 1999), and for various Abbreviations used: VDAC, voltage-dependent anion channel; TRP, G-proteins (Wang et al. 1990; Popova et al. 1997; Roy- transient receptor potential; MBP, maltose-binding protein; MT, micro- chowdhury and Rasenick 1997; Roychowdhury et al. 1999; tubules; DMS, dimethyl suberimidate.

2007 The Authors 250 Journal Compilation 2007 International Society for Neurochemistry, J. Neurochem. (2007) 101, 250–262 Identiﬁcation and characterisation of tubulin-binding motifs 251

2004). It provides stability to microtubules both in vitro and Constructs and peptides pI in vivo (Goswami et al. 2004, 2006). Interestingly, tubulin 681 838 interaction is observed also for other members of the TRP Ct 9.20 681 800 super family. Interaction of b-tubulin with TRPC1 has been Ct-Δ1 9.67 reported recently (Bollimuntha et al. 2005). Two other 681 760 Ct-Δ2 9.34 members, namely TRPC5 and TRPC6, contain tubulin as 681 730 Ct-Δ3 constituent of its ‘signalplex’ (Goel et al. 2005). Very 10.13 761 838 recently, it has been shown that Polycystin-2 type TRP Ct-frag 6 8.21 channels are regulated by microtubular structures in primary 731 838 Ct-frag 7 6.08 cilia of renal epithelial cells (Li et al. 2006). This suggests Ct-frag 8 710 797 that tubulin interaction might be common for many of the 10.03 681 709 TRP ion channels. Ct-frag 1 6.35 710 730 In spite of the functional implication of the interaction of Ct-frag 2 11.17 tubulin with several transmembrane receptors and ion Ct-frag 3 731 769 channels, very little is known about the binding structure/s 4.03 770 797 that underlie these interactions. Therefore, we set out to Ct-frag 4 12.6 identify the exact tubulin-binding region of TRPV1 and Ct-frag 5 801 838 5.49 further characterised the interacting structures. Peptide 1 - Biotin 11.17

Peptide 2 - Biotin 12.48

Materials and methods Fig. 1 Constructs and peptides used to identify the tubulin-binding site located in the C-terminus of TRPV1. Schematic representation of Reagents and antibodies constructs prepared to express the deletion-proteins and fragments The microtubule stabilising drug Taxol (paclitaxel), the cross-linker corresponding to the different regions of C-terminus of TRPV1. Posi- DMS and purified actin, were purchased from Sigma–Aldrich tions of the amino acids are written in top. Different deleted and (Taufkirchen, Germany). Biotinylated-peptides (KSFLKCMRKA- fragmented parts of the C-terminal of TRPV1 are expressed as MBP- FRSGKLLQVGF-K-Biotin and KRTLSFSLRSGRVSGRNWKNF- fusion protein (MBP is at the N-terminus of each fusion constructs). K-Biotin) were synthesised at Biosynthan (Berlin, Germany). Mouse Biotinylated-peptides (biotin label is at the C-terminus) are indicated. monoclonal a-tubulin antibodies (clone DM1A), mouse monoclonal Dark background indicates the regions with higher pI (short basic b-tubulin antibodies (clone D66), mouse monoclonal tyrosinated stretches 1 and 2), whereas light background indicates the regions tubulin antibodies (clone TUB1A2), mouse monoclonal polyglutam- with lower pI. All theoretical isoelectric points of the deletion constructs ylated tubulin antibodies (clone B3), mouse monoclonal acetylated were calculated by using available software (http://www.expasy.org/ tubulin antibodies (clone 611-B-1), mouse monoclonal phosphoser- tools/pi_tool.html). 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) (New England Biolabs, Beverly, MA, USA). A stop codon was and rabbit polyclonal detyrosinated tubulin antibodies were pur- introduced in each construct at the C-terminus of the coding chased from Chemicon (Chandlers Ford, UK). Mouse monoclonal sequences. All expression constructs were verified by automated actin antibodies (clone JLA20) was purchased from Oncogene nucleotide sequencing. Escherichia coli (E. coli) strain BL21DE3 (Cambridge, MA, USA). Mouse monoclonal anti-maltose-binding was transformed by heat shock with the plasmid coding for the protein (MBP) antibodies and amylose resin were purchased from TRPV1 cytoplasmic domains and fragments fused with MBP New England Biolab (Beverly, MD, USA). Enriched neurofilament protein. E. coli cells were induced to express the proteins by fraction was a kind gift from O. Bogen (Bogen et al. 2005). isopropyl thiogalactoside (IPTG) for 2 h. The cells were lysed by Subtilisin-digested tubulin and control tubulin were kindly provided repeated freeze-thaw cycles in lysis buffer (20 mmol/L Tris–HCl, by Linda Amos (Cambridge, UK). For the detection of subtilisin- pH 7.4, 150 mmol/L NaCl, 0.1% Tween 20, lysozyme, benzonase digested tubulin and control tubulin by western-blot analysis, we used and protease inhibitor cocktail). The lysed extracts were cleared by mouse monoclonal anti-b-tubulin (clone D10, Santa Cruz Biotech- centrifugation (100 000 g in a TFT 45 rotor for 2 h). The cleared nology, Heidelberg, Germany). lysate was applied to amylose resin and washed thoroughly. Bound protein was eluted with 10 mmol/L maltose in elution buffer Expression and purification of TRPV1 fusion proteins (50 mmol/L PIPES, pH 6.8, 100 mmol/L NaCl, 1 mmol/L EGTA

Expression and purification of MBP-TRPV1-Nt (N-terminal cyto- and 0.2 mmol/L MgCl2). Protein concentration was determined plasmic domain of TRPV1 fused with MBP) and MBP-TRPV1-Ct according to method described by Bradford (1976). (C-terminal cytoplasmic domain of TRPV1 fused with MBP) were described in Goswami et al. (2004). The cDNA fragments of Purification of tubulin TRPV1-Ct (see Fig. 1) were amplified by PCR using specific ab-tubulin dimers were purified from porcine brain according to primers (Table 1). All amplified DNA fragments were subcloned Shelanski et al. (1973). In brief, two cycles of assembly from into the EcoR1 and Hind III restriction sites of the pMAL-c2x vector soluble brain extract in the presence of glycerol and GTP and

2007 The Authors Journal Compilation 2007 International Society for Neurochemistry, J. Neurochem. (2007) 101, 250–262 252 C. Goswami et al.

Table 1 Primers used for making the Primer Construct TRPV1-Ct deletions and fragments 1F:5¢ CCGGAATTCCTCATGGGTGAGACCGTCAAC 3¢ MBP-TRPV1-Ct-D1 R: 5¢ CCCAAGCTTTTAGCTTGCATCCCTCAGAAGGGG 3¢ 2F:5¢ CCGGAATTCCTCATGGGTGAGACCGTCAAC 3¢ MBP-TRPV1-Ct-D2 R: 5¢ CCCAAGCTTTTAGTTGATGATACCCACATTGGT 3¢ 3F:5¢ CCGGAATTCCTCATGGGTGAGACCGTCAAC 3¢ MBP-TRPV1-Ct-D3 R: 5¢ CCCAAGCTTTTAGAACCCCACCTGCAGCAGCTT 3¢ 4F:5¢ CCGGAATTCCTCATGGGTGAGACCGTCAAC 3¢ MBP-TRPV1-Ct-F1 R: 5¢ CCCAAGCTTTTACTCTGTATCCAGGATGGTGAT 3¢ 5F:5¢ CCGGAATTCAAGAGCTTCCTGAAGTGCATG 3¢ MBP-TRPV1-Ct-F2 R: 5¢ CCCAAGCTTTTAGAACCCCACCTGCAGCAGCTT 3¢ 6F:5¢ CCGGAATTCACTCCTGACGGCAAGGATGAC 3¢ MBP-TRPV1-Ct-F3 R: 5¢ CCCAAGCTT TTAGACGCCCTCACAGTTGCCTGG 3¢ 7F:5¢ CCGGAATTCAAGCGCACCCTGAGCTTCTCC 3¢ MBP-TRPV1-Ct-F4 R: 5¢ CCCAAGCTTTTACCTCAGAAGGGGAACCAGGGC 3¢ 8F:5¢ CCGGAATTCACTCGAGATAGACATGCCACC 3¢ MBP-TRPV1-Ct-F5 R: 5¢ CCCAAGCTTTTATTTCTCCCCTGGGACCATGGA 3¢ 9F: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. disassembly by cold temperature (ice-cold) were followed by thrice with PEM-S-T buffer and ﬁnally taken in to Laemmli sample chromatography on phosphocellulose. buffer and analysed by western-blot analysis for bound proteins.

Pull-down assay Cross-linking of proteins MBP-LacZ, MBP-TRPV1-Ct, different MBP-TRPV1-Ct fragments A protein mixture (1 mg/mL) of equal amounts of ab-tubulin dimer and deletion constructs (see Fig. 1) were expressed in E. coli, the and MBP-TRPV1-Ct, in PEM buffer was adjusted to 0.2 mol/L cleared cell lysates were applied to amylose resin (NEB), and triethanolamine (pH 8.1) buffer for cross-linking with dimethyl incubated for 1 h at 25C followed by washing. The amylose resin suberimidate (DMS, Sigma, 1 mg/mL). The reaction was carried out with bound proteins were re-suspended in PEM-S buffer (50 mmol/L at 25C for 1 min to 1 h and stopped by adding Tris–HCl (pH 6.8) PIPES, pH 6.8, 100 mmol/L NaCl, 1 mmol/L EGTA and 0.2 mmol/ to a ﬁnal concentration of 50 mmol/L. Samples were subjected to

L MgCl2). Approximately, 50 lL of amylose resin with the bound SDS-PAGE separation and western-blot analysis. fusion protein was incubated with 50 lL of soluble tubulin (1 mg/mL protein) for 1 h at 25C either in the presence or absence of Ca2+ Western-blot analysis (2 mmol/L). This was followed by three washes with 200 mL each To perform western-blot analysis, the proteins were separated by time and constant buffer conditions. The proteins were eluted by SDS-PAGE, and transferred either to a nitrocellulose membrane or 10 mmol/L maltose in 100 lL solution. Eluted samples were PVDF (Millipore, Schwalbach, Germany) by semidry electro analysed by 10% sodium dodecyl sulphate polyacrylamide gel blotting. The membranes were blocked with 5% non-fat milk in electrophoresis (SDS-PAGE) according to Laemmli (1970). TBS-T (20 mmol/L Tris, 150 mmol/L NaCl. 0.1% Tween-20) buffer For experiments determining the binding of subtilisin-digested followed by incubation with the respective primary antibody for 1 h tubulin with TRPV1-Ct, MBP-TRPV1-Ct immobilised on amylose at 25C, washed thrice times with TBS-T buffer. Subsequently, the resin (20 lL) in PEM-S buffer were incubated with 2 lgof membranes were incubated with horseradish peroxidase-conjugated subtilisin-digested tubulin or same amount of control tubulin for 1 h secondary antibody for 1 h at 25C. For the detection of at 25C. After three washes in PEM-S buffer, the bound protein biotinylated-peptides, PVDF membranes containing peptide spots complexes were eluted and analysed further. were probed with HRP-conjugated avidin (Sigma–Aldrich, 1 : 1000 To identify if there is a direct interaction between tubulin and dilution). The membranes were washed thoroughly with TBST. The short peptides carrying TRPV1 sequences, biotinylated-peptides ECL detection system (Amersham Biosciences, Freiburg, Germany) were incubated with avidin agarose (Sigma–Aldrich) at 25C for was used for the visualisation of the immunoreactivity. 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% Co-sedimentation assay with taxol-stabilised microtubules (MT) Tween 20) buffer thrice, incubated with the soluble tubulin dimer Approximately 100 lg of puriﬁed ab-tubulin dimer in a total (40 lg in 100 lL) for 1 h. Avidin–agarose resins were washed volume of 100 lL were incubated in modiﬁed PEM buffer

2007 The Authors Journal Compilation 2007 International Society for Neurochemistry, J. Neurochem. (2007) 101, 250–262 Identiﬁcation and characterisation of tubulin-binding motifs 253

(20 mmol/L PIPES, pH 6.8, 0.2 mmol/L MgCl2 and 1 mmol/L (a) EGTA supplemented by 1 lmol/L taxol and 5 mmol/L GTP) for Input Tubulin Actin Neurofilament 30 min at 37C, to form MT. After MT formation, 5 lg of puriﬁed proteins representing different MBP-fusion proteins were incubated Ct LacZ Ct LacZ with taxol-stabilised MT for 40 min at 37C followed by centrifugal Ct LacZ 2+ Input Input Input separation of pellet (MT) and supernatant (free dimer) at 70 000 g/ Ca –+ –+ – – + – + – – + – + – kDa 30 min/37C. In a similar manner biotinylated-peptides (approxi- 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 mately 1 lg each in 100 lL) were ﬁrst centrifuged at 25 000 g for 205 5 min to remove all aggregates. Clear supernatant containing 114 soluble peptides were further used for microtubule co-sedimentation 97 assay. Corresponding pellet and supernatant fractions were further 67 spotted on a PVDF membrane (Amersham) by a dot-blot apparatus 45

(Bio-Rad, Munich, Germany) and analysed for bound peptides. For stain Silver MT formation under taxol-free conditions, 100 lg of tubulin dimer 30 were used in PEM buffer with 5 mmol/L GTP in the absence of taxol and incubated for 30 min at 37C. WB Blot overlay Tubulin Actin NF 200 kDa To carry out overlay experiments, either native tubulin dimer were (b) spotted directly or denatured and SDS-PAGE-separated proteins 1 2 (tubulin dimer) were transferred from gels to nitrocellulose or PVDF WB: membrane. Membranes were blocked for 1 h with 5% fat-free milk MBP 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 Acetylated tub in PBST buffer) for 1 h at 25C. In blot overlay experiment with biotinylated-peptides, PEM-S-T (50 mmol/L PIPES; pH 6.8, Tyrosinated tub 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 Detyrosinated tub concentration of approximately 5 ng/mL. After incubation, the membranes were washed thrice (each time for 10 min) and Polyglutamylated tub incubated with 0.1% formaldehyde for 30 min to cross-link the bound proteins. Finally, the membranes were quenched with Phospho (serine)tub 100 mmol/L glycine in TBS buffer and processed for western-blot analysis with MBP antibodies or HRP-labelled avidin to detect the β III tub bound proteins or peptides respectively.

Fig. 2 The C-terminus of TRPV1 specifically interacts with constit- Results 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 TRPV1 interacts with soluble tubulin, but neither with incubated with purified soluble tubulin dimer (left panel), or purified soluble actin nor with soluble neurofilaments soluble actin (middle panel) or enriched neurofilament fraction (right 2+ Previously, we observed that the C-terminus of TRPV1 binds panel) either in the presence (lanes 2 and 4) or absence of Ca (lanes to tubulin and stabilises microtubules (Goswami et al. 2004). 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- To understand if the C-terminus of TRPV1 can also interact PAGE. Lane 1 shows the input amount of soluble proteins. Proteins with cytoskeleton components other than tubulin, we were stained with silver stain (upper panel) and also probed for bound performed pull-down experiments with purified soluble actin tubulin, actin or neurofilaments by western-blot analysis (lower panel) and enriched soluble neurofilament preparations. The C- of the corresponding samples. A significant amount of tubulin binds to terminal cytoplasmic domain of TRPV1 fused with MBP the MBP-TRPV1-Ct but not to the MBP-LacZ. In contrast to tubulin, (MBP-TRPV1-Ct) was used as bait. However, we could not neither actin nor neurofilament binds to the MBP-TRPV1-Ct. (b) MBP- observe any significant direct interaction of actin or neuro- TRPV1-Ct (lane 1) but not MBP-LacZ (lane 2) pulls down different filaments with the MBP-TRPV1-Ct (Fig. 2a). As activation post-translationally modified tubulins and neuron-specific b-tubulin sub of TRPV1 results in influx of Ca2+, we tested if actin and type III. neurofilaments interact with MBP-TRPV1-Ct in the presence of high Ca2+. We observed no interaction even in the Ct. In agreement with our previous results (Goswami et al. presence of Ca2+. Under the same conditions a significant 2004), this interaction was slightly stronger in the presence amount of soluble tubulin interacts with the MBP-TRPV1- of Ca2+. In similar experiments, MBP-TRPV1-Ct, but not

2007 The Authors Journal Compilation 2007 International Society for Neurochemistry, J. Neurochem. (2007) 101, 250–262 254 C. Goswami et al.

MBP-LacZ pulls down neuron-specific beta III tubulin as complex after only 1 min of cross-linking. All b-tubulin in well as other post-translationally modified tubulins, i.e. the reaction mixture also appeared in the same high- acetylated tubulin, polyglutamylated tubulin, phosphotubulin molecular weight complex. Also the a-tubulin was found in (phosphoserine), tyrosinated tubulin and de-tyrosinated tub- that complex, but approximately 50% of the a-tubulin did ulin (Fig. 2b). These results indicate that the C-terminus of not react. Even after 60 min of reaction, the non-cross- TRPV1 interacts specifically with the constituents of both linked a-tubulin population remained at its monomeric dynamic and stable microtubules. molecular weight on the SDS-PAGE. The high-molecular weight complex was not observed when we used purified b-tubulin, but not a-tubulin preferentially interacts with MBP instead of MBP-TRPV1-Ct for cross-linking experi- TRPV1 ments with ab-tubulin dimer (data not shown). From these Tubulin preparations from brain tissue consist predomin- data we conclude that the MBP-TRPV1-Ct interacts with antly of ab-tubulin heterodimers. In order to analyse, which the tubulin dimer predominantly via b-tubulin. subunit of the tubulin dimer interacts with the C-terminal domain of TRPV1, we performed a cross-linking experi- The C-terminus of TRPV1 interacts with blotted ment (Fig. 3a). A mixture of ab-tubulin dimer and MBP- denatured tubulin TRPV1-Ct was cross-linked by using dimethyl suberimidate Three-dimensional crystal structures and EM pictures reveal (DMS), a homobifunctional cross-linking agent, which that the C-terminal tail of the tubulin dimer does not integrate reacts with amino groups. The cross-linked products of into the core of the microtubule filaments, but remains outside tubulin dimers and MBP-TRPV1-Ct were subsequently (Nogales et al. 1999; Lowe et al. 2001). These exposed C- analysed by gel electrophoresis and western-blot analysis terminal over-hanging regions of both a-tubulin and b-tubulin with the appropriate antibodies. We observed that cross- are strongly negatively charged and unstructured too (Lowe linking occurred fast and the entire amount of MBP- et al. 2001; Nogales 2001). Most of the known microtubule- TRPV1-Ct appeared on the gel as a high-molecular weight binding proteins interact with microtubules and tubulins through these regions (see Fig. 4a, see also Discussion). (a) (b) 1 2 3 1 2 3 1 2 3 1 2 kDa 205 (a) α 205 114 Porcine -tubulin VE--GEGEEEGEEY-- 114 97 Porcine β-tubulin ADEQGEFEEEGEEDEA 97 67 67 (b) KSFL KCMRKAFRSGKLL KRTLSFSL RSGRVSG RN 45 45

30 30

WB: α tub β tub MBP WB: MBP

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 Stretch 1 Stretch 2 separated by SDS-PAGE (4–10% gradient) and transferred to a nitrocellulose membrane. Blots were probed with the anti-a-tubulin Fig. 4 Positively charged amino acids of stretch sequence 1 and 2 antibody (left), the anti-b-tubulin antibody (middle) and the anti-MBP are clustered in one side of the helix. (a) C-terminal tail of a-tubulin and antibody (right). The arrow indicates the high-molecular-weight cross- b-tubulin contain highly negatively charged amino acids. These neg- link product. After cross-linking, all the MBP-TRPV1-Ct and all the atively charged amino acids (indicated in bold letter) are conserved in b-tubulin shows up in a high-molecular weight complex, whereas a almost all species, and most of the microtubule-binding proteins significant amount of the a-tubulin failed to react and remains in the interact via these sequences. (b) Amino acids of stretch sequences 1 monomeric state. (b) MBP-TRPV1-Ct but not MBP alone detects and stretch sequence 2 (in each case 17 amino acid-long, indicated in purified tubulin dimers in blot overlay experiment. Equal amount of top) of the C-terminus of TRPV1 (rat) are plotted for helical distribution. purified tubulin dimer separated by SDS-PAGE and transferred into Top-view of the helix is shown. Programme available at http://kael.net/ the nitrocellulose membrane were overlayed with MBP-TRPV1-Ct helical.htm site is used to draw the helical wheel. Positively charged (lane 1) and with MBP only (lane 2). Stripes were probed with anti- amino acids (bold letter) are marked with asterisk sign. Note the dis- MBP antibody. Anti-MBP immunoreactivity appeared in the position of tribution of positively charged residues (asterisk) in one side of the tubulin at lane 1 (indicated by arrow). wheel.

2007 The Authors Journal Compilation 2007 International Society for Neurochemistry, J. Neurochem. (2007) 101, 250–262 Identiﬁcation and characterisation of tubulin-binding motifs 255

To analyse if the TRPV1 C-terminus also interacts with As expected, no tubulin was observed in pull-down samples tubulin via this acidic unstructured region, we separated where an unrelated construct (MBP-LacZ) was used as bait purified ab-tubulin dimer on SDS-PAGE and performed a (data not shown, a similar experiment is shown in Fig. 2). blot overlay on the denatured tubulin with purified MBP- However, the amount of tubulin pulled down by different TRPV1-Ct. We subsequently probed for bound protein by deletion-proteins varies, indicating that different regions of western-blot analysis with MBP antibodies. MBP immuno- the C-terminus of TRPV1 influence the tubulin interaction reactivity was detected at 55 kDa (the position of tubulin) on to a different extent (Fig. 5). Tubulin binding with MBP- the membrane, but only if the blot overlay was performed TRPV1-D1 was slightly higher when compared with MBP- with MBP-TRPV1-Ct and not with MBP alone (Fig. 3b). TRPV1-Ct. This indicates that the amino acid sequence This suggests that the C-terminus of TRPV1 interacts even 800–838 is less important for the interaction. Deletion of with unstructured tubulin polypeptides, possibly at the this region may even enhance the tubulin interaction. C-terminal negatively charged overhanging region. Similarly, a stronger amount of tubulin binding was observed with MBP-TRPV1-D3, which contains amino AA 681-730 is sufficient for tubulin binding acids 681–730 of TRPV1. Significantly less tubulin binding To identify the tubulin-binding region in the C-terminus of was observed with MBP-TRPV1-CtD2 (amino acid residues TRPV1, we undertook a systematic deletion approach. We 681–760). designed three MBP-fused C-terminal deletion constructs These results indicate that a stretch of 50 amino acids along with the complete C-terminus, namely MBP-TRPV1- (position 681–730) is sufficient for the interaction with Ct-D1 (aa 681–800) and MBP-TRPV1-Ct-D2 (aa 681–760) tubulin. This also suggests that the sequences amino acids as well as MBP-TRPV1-Ct-D3 (aa 681–730), each shorter by 731–760 and 800–838 of TRPV1 reduce tubulin binding, few amino acids at the C-terminal end respectively (Fig. 1). while the sequence residues 760–800 enhance it. All these fusion proteins were expressed in E.coli, purified and confirmed by western-blot analysis (data not shown). Two small basic sequences located in the C-terminal MBP-pull-down experiments were performed with these cytoplasmic domain of TRPV1 have potentiality to deletion-proteins. Pulled-down eluate samples were first modulate the interaction analysed by SDS-PAGE and silver staining and then probed Many tubulin-binding proteins and microtubule-binding for bound tubulin by western-blot analysis. proteins contain more than one short basic-repeat sequences We observed that all three MBP-TRPV1-Ct deletion which mediates the interaction with tubulin and/or micro- proteins pulled down tubulin when used as baits (Fig. 5). tubules (see Discussion). To explore, if the C-terminal cytoplasmic domain of TRPV1 also contains basic-sequence Ct Ct- 1Ct-2 Ct- 3 stretches for tubulin binding, the theoretical pI values of the 1 2 3 1 2 3 1 2 3 1 2 3 entire C-terminus of TRPV1 as well as of short sequence Tub + – + + – + + – + + – + + stretches were calculated (see Fig. 1). Two short sequence 2+ – + – – + – – + – – + – Ca kDa stretches that contain basic amino-acid residues and thus 67 * have a higher pI were identified at residues 710–730 * * * 45 (calculated pI; 11.17) and 770–797 (calculated pI; 12.6). Silver stain Silver These two sequences contain a number of positively charged 67

amino acids, but are devoid of any negative charges. WB 45 However, the two sequences are flanked by sequence stretches that consist of negatively charged amino acids. Fig. 5 AA 681–730 is sufficient for tubulin binding. MBP-TRPV1-Ct, Therefore, these motifs (short basic sequences flanked by MBP-TRPV1-Ct-D1, MBP-TRPV1-Ct-D2 and MBP-TRPV1-Ct-D3, all acidic amino acids) may act as tubulin-binding structures. immobilised on amylose resin (lane 1), were incubated with purified Interestingly, a helical plot of these two short sequences tubulin dimer either in the presence (lane 2) or absence of Ca2+ (lane shows that all the basic amino acids are located on one side 3) to analyse the relative affinity of the deletion fragments for tubulin. of the a-helix (discussed later, see Fig. 4b). This may The proteins were eluted from the amylose resin with 10 mmol/L indicate that these basic amino acids are engaged in the maltose and resolved by SDS-PAGE. Proteins were stained with silver interaction with negatively charged sequences, either of stain (upper panel). Western-blot analysis (lower panel) of corres- neighbouring sequences of the same polypeptide (with ponding samples with anti-tubulin antibody shows the differential negatively charged residues) or of sequences from other presence of tubulin (arrow) in the different pull-down elutes. A significant amount of tubulin binds to the MBP-TRPV1-Ct, MBP-TRPV1-Ct- interacting proteins, like the C-terminal overhanging acidic D1 and MBP-TRPV1-Ct-D3. MBP-TRPV1-Ct-D2 in contrast binds region of tubulin. The latter explanation is supported by our much less tubulin. The input amount of tubulin is shown by silver tubulin pull-down experiments. Beside the MBP-TRPV1-Ct, stained SDS-PAGE (left, upper panel) and by western-blot analysis only MBP-TRPV1-CtD1 and MBP-TRPV1-CtD3, but not (left, lower panel). MBP-TRPV1-CtD2 binds tubulin.

2007 The Authors Journal Compilation 2007 International Society for Neurochemistry, J. Neurochem. (2007) 101, 250–262 256 C. Goswami et al.

TRPV1-Ct amino acid sequences 710–730 and 770–797 inﬂuence TRPV1/tubulin interaction

To further narrow down the interacting regions, which Ct-Fr 1 Ct-Fr 2 Ct-Fr 3 Ct-Fr 4 Ct-Fr 5 mediate the tubulin interaction, the C-terminus of TRPV1 Tubulin -+ -+ -+ -+ -+ was subdivided into five short segments. These short kDa 1 2 1 2 1 2 1 2 1 2 fragments expressed as N-terminal MBP fusion proteins 205 and referred as MBP-TRPV1-Ct-fr1 to MBP-TRPV1-Ct-fr5 114 (see Fig. 1). Fragments 1, 3 and 5 contain negatively charged 97 amino acids (with low pI), while fragments 2 and 4 represent 67 basic stretch 1 and basic stretch 2 respectively. Two more WB: fragments were prepared, in which the basic stretch 2 has an Tubulin acidic stretch to its right side (named MBP-TRPV1-Ct-Fr6, 45 see Fig. 1) or with acidic sequences on both sides (referred to as MBP-TRPV1-Ct-Fr7, see Fig. 1). One further fragment 30 was prepared which contained both the basic sequence stretches connected by the acidic stretch in between. This fragment is referred to as MBP-TRPV1-Ctfr8 (see Fig. 1). Silver All these deletion constructs and fragments of the stain C-terminus of TRPV1 were created as fusion proteins with Fig. 6 Differential binding of tubulin to different fragments of TRPV1- an N-terminal MBP and expressed in E.coli. The whole set Ct. MBP-TRPV1-Ct-Fr1, MBP-TRPV1-Ct- Fr2, MBP-TRPV1-Ct- Fr3, covers most of all possible combinations, i.e. either two short MBP-TRPV1-Ct-Fr4 and MBP-TRPV1-Ct- Fr5, all immobilised on basic sequences are present in combination, or alone, or are amylose resin (lane 1) were incubated with purified tubulin dimer (lane excluded completely. The expressed fusion proteins were 2) to analyse the relative affinity of the deletion fragments for tubulin. used for pull-down assays and also for microtubule-binding The fusion proteins along with the bound tubulin were eluted from the assays. These fusion proteins were additionally tested for amylose resin with 10 mmol/L maltose and resolved by SDS-PAGE. their binding to SDS-PAGE-separated denatured tubulin Western-blot analysis (upper panel) of corresponding samples with dimer or to native tubulin dimers spotted on membranes in a anti-tubulin antibody shows the differential presence of tubulin (indi- blot-overlay experiment (data not shown). cated by arrow) in the different pull-down elutes. A significant amount of tubulin binds to the MBP-TRPV1-Ct-Fr2 and MBP-TRPV1-Ct-Fr4. In the MBP-pull-down assay, it was observed that MBP- The lower panel reveal the presence of equal amount of fusion pro- TRPV1-Ct-fr 2 and MBP-TRPV1-Ct-fr 4 bind tubulin teins in each lane as visualised by silver staining. strongly (Fig. 6). Notably, these two fragments represent basic stretch sequence 1 and 2 respectively. In contrast, MBP-TRPV1-Ct-fr1 and MBP-TRPV1-Ct-fr 5 reveal no stretches. We observed that the two peptides co-sediment binding (Fig. 6). We observed some weak binding for MBP- with taxol-stabilised microtubules (Fig. 8a). Avidin–agarose TRPV1-Ct-fr3. Comparison of tubulin-binding and microtu- coupled to either of these two peptides pulled down a bule-binding to MBP-TRPV1-Ct-fr6, MBP-TRPV1-Ct-fr7 significant part of the purified soluble tubulin (Fig. 8b). In and MBP-TRPV1-Ct-fr8 reveals that MBP-TRPV1-Ct-fr7 contrast, avidin–agarose beads alone did not pull down any has the lowest affinity to both soluble tubulin and polymer- tubulin. We observed that these two peptides can detect SDS- ised microtubules while MBP-TRPV1-Ct-fr8 showed the PAGE-separated denatured tubulin on membranes in the blot strongest binding for both. MBP-TRPV1-Ct-fr6 also reveals overlay assay in nanomolar concentration (Fig. 8c). This not significant binding to both tubulin and microtubules (Fig. 7). only confirms that these two peptides can interact with Similar results were obtained, when these fragments were tubulin and microtubules specifically, but also shows that the used in a blot-overlay experiment with SDS-PAGE-separated interaction does not need a native tubulin structure. tubulin (data not show). These results support a positive modulation of tubulin binding by the presence of the two The C-terminal overhanging region of tubulin is short basic amino acid stretches. important for TRPV1-Ct binding As the C-terminal overhanging regions of both a-tubulin and Biotinylated-peptides corresponding to these two short b-tubulin are very acidic and involved in the interaction with basic stretches bind to soluble tubulin and co-sediment most of the microtubule-binding proteins (see Discussion), with the polymerised microtubules we tested if this is the region where the C-terminal To confirm these findings by a different method, we cytoplasmic domain of TRPV1 binds. To test this, we synthesised biotinylated-peptides (each peptides containing analysed if subtilisin-digested tubulin (see Fig. S1) binds 21 amino acid, amino acid 710–730 and 770–790). There- with MBP-TRPV1-Ct. We performed MBP-pull-down assay fore, these two peptides represent the basic amino acid with control tubulin and subtilisin-digested tubulin using

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(b) F6 F7 F8 digested tubulin (Fig. 9a). This indicates that the C-terminal acidic region of tubulin is important for TRPV1-Ct binding. (a) 123 PSPSPS

Discussion Recently, we demonstrated that the C-terminus of TRPV1 Silver not only interacts but also stabilises microtubules in vitro (Goswami et al. 2004), and when over-expressed, this results stain in the formation of more stable and bundled microtubules in vivo (Goswami et al. 2006). In a reverse manner, activation of TRPV1 results in rapid disassembly of dynamic microtubules (Goswami et al. 2006). This demonstrates that TRPV1 is physically linked to microtbules, and also suggests that the microtubule cytoskeleton is a downstream effector of WB: TRPV1 activation. However, the exact tubulin-binding sequence was not determined. In this study, we characterised Anti-tubulin Anti-MBP the TRPV1/tubulin interaction and also demonstrate that the C-terminus of TRPV1 neither interacts with soluble actin nor Fig. 7 The presence of short basic sequence stretches influences with soluble neurofilaments, but specifically interacts with the interaction of TRPV1 with tubulin dimers and with microtubules. (a) the components of microtubule cytoskeleton. MBP-TRPV1-Ct-fr6 (lane 1), MBP-TRPV1-Ct-fr7 (lane 2) and MBP- We demonstrate that the C-terminus of TRPV1 preferably TRPV1-Ct-fr8 (lane 3), all immobilised on amylose resin, were incu- interacts with b-tubulin rather than a-tubulin. In a similar bated with purified tubulin to analyse the relative affinity of the deletion manner, two groups independently reported that b-tubulin fragments for tubulin. Bound proteins were eluted from the amylose interacts with members of TRPC channels (Bollimuntha resin with 10 mmol/L maltose and resolved by SDS-PAGE. Proteins et al. 2005; Goel et al. 2005). Microtubule plus ends were visualised by silver stain (upper panel). Western-blot analysis terminate their protofilaments with a b-tubulin at the end. (lower panel) of corresponding samples with anti-tubulin antibody shows the differential presence of tubulin in the pull-down elutes. A The higher preference of the TRPV1-Ct for b-tubulin and the significant amount of tubulin binds to MBP-TRPV1-Ct-fr6 and MBP- ability to stabilise microtubules therefore indicates that the TRPV1-Ct-fr8. Much less tubulin binds to MBP-TRPV1-Ct-fr7 (Note observed interaction may exert its effect at the microtubule the excess amount of MBP-TRPV1-Ct-Fr7 in the silver stain). Arrows plus end rather than at the minus end. This accords well with indicate the position of tubulin. (b) MBP-TRPV1-Ct-fr8 co-sediments a recent observation that Xenopus TRPN1, another member with polymerised microtubules. Purified ab-tubulin dimers were incu- of the TRP channel super family is localised at the plus ends bated with taxol and GTP to form microtubules. Taxol-stabilised MTs of the microtubule-based cilia structures (Shin et al. 2005). were incubated with 6 lg of purified MBP-TRPV1-Ct-fr6, MBP- In contrast to the C-terminus, the N-terminus of TRPV1 TRPV1-Ct-fr7 and MBP-TRPV1-Ct-fr8 respectively. Microtubules and neither interacts with soluble tubulin nor with polymerised bound proteins were separated by centrifugation and analysed by 10% microtubules (Goswami et al. 2004). Similarly, the N- SDS-PAGE. The distribution of the proteins between microtubule terminus of TRPV1 fails to modify the properties of pellet (P) and supernatant (S) fractions was visualised by silver staining of the proteins (upper panel) and western-blot analysis of the microtubules in vitro (Goswami et al. 2004). The N-terminal same samples with anti-MBP antibody (lower panel). A significant domain of TRPV1 does not form any specific high-molecular amount of MBP-TRPV1-Ct-fr8 and a small amount of MBP-TRPV1-Ct- weight complex when cross-linked with tubulin dimers (data fr6 co-sediment with microtubules. The majority of MBP-TRPV1-Ct-fr6 not shown). From all these observations it seems that the N- and MBP-TRPV1-Ct-fr7 remain in the supernatant. terminus of TRPV1 is not involved with the tubulin interaction. MBP-TRPV1-Ct, and analysed the eluates by SDS-PAGE. Because of the fact that both a-tubulin and b-tubulin are Although undigested control tubulin binds significantly to coded by several genes, and are subjected to different post- the MBP-TRPV1-Ct, we could not detect any binding of translational modifications, much heterogeneity among the subtilisin-digested tubulin with MBP-TRPV1-Ct (Fig. 9a). tubulin monomers as well as dimers exists. The C-terminal To confirm this result by a more sensitive method, we used sequence of both a-tubulin and b-tubulin is unstructured and mouse monoclonal anti-b-tubulin antibody, clone D10 (Santa contains highly negatively charged residues (Nogales et al. Cruz). This antibody detects control tubulin as well as 1998; Lowe et al. 2001). Despite great heterogeneity within subtilisin-digested tubulin, both in SDS-PAGE-separated the C-terminal tails of different tubulins, the negatively denatured and in native form (Fig. 9b). By western-blot charged residues are highly conserved in both a-tubulin and analysis, we could detect a significant binding of control b-tubulin (Fig. 4a). Due to the presence of these highly tubulin, but we failed to find any binding of subtilisin- negatively charged residues, the calculated and measured

2007 The Authors Journal Compilation 2007 International Society for Neurochemistry, J. Neurochem. (2007) 101, 250–262 258 C. Goswami et al.

(a) (b) (c) Co-sedimentation Tubulin pull-down Blot overlay with MT

ds Cont +PT1 +PT2 Input PT 1 PT 2 Bea kDa PT 1 PT 2 Cont kDa S P S P S P 1 2 3 4 1 2 3 kDa 205 205 205 114 114 114 97 97 97 67 67 67 45 45 45 Coomassie Coomassiee 30 30 30 α

S P T S P T S P -tub HRP Avidin WB β-tub Avidin HRP

Fig. 8 Two 21-mer amino acid sequences are sufficient for interac- were eluted from the avidin–agarose beads with Laemmli buffer and tion with polymerised microtubules and tubulins. (a) Two short pep- resolved by SDS-PAGE. Proteins were visualised by Coomassie stain tides co-sediment with taxol-stabilised microtubules. Taxol-stabilised (upper panel). Western-blot analysis (lower panel) of corresponding polymerised microtubules (Cont) were incubated with peptide-1 (PT-1) samples with anti-a-tubulin antibody and anti-b-tubulin antibody reveal or peptide-2 (PT-2) at 37C for 30 min. Polymerised microtubules and the specific presence of tubulin in the pull-down elutes from peptide- associated peptides were subsequently separated from soluble un- coupled beads, but not in only beads control. (c) Two short peptides bound tubulin/peptide by centrifugal separation of pellet fractions (P) interact and thus detect SDS-PAGE-separated tubulin on membrane. from supernatant (S). Corresponding pellets and supernatants were Equal amounts of soluble tubulin dimer were separated by SDS-PAGE further analysed by SDS-PAGE (Coomassie staining) and also by dot- and transferred on PVDF membrane. Membrane strips were incuba- blot analysis with HRP-avidin. A significant portion of both peptides is ted with (approximately 5 ng/mL) biotinylated-peptide-1 (PT-1, lane 1), observed to be present in pellet fraction. Total amount of peptide is biotinylated-peptide-2 (PT-2 lane 2), or only with buffer for 1 h. Sub- indicated by (T). (b) Two short peptides pull-down soluble tubulin. sequently, the membrane strips were washed and cross-linked with Purified soluble tubulin dimer (lane 1, 1/4th amount of input is shown) formaldehyde, and probed with HRP-labelled avidin. Significant reac- were added to avidin–agarose coupled with peptide-1 (PT-1, lane 2), tivity was detected at around 55 kDa (at the position of tubulin, indi- or peptide-2 (PT-2, lane 3) or to avidin–agarose beads only (lane 4), cated by arrow) in the lane 1 and lane 2, but not in lane 3. incubated for 1 h at 25C, and washed subsequently. Bound proteins isoelectric points of different a-tubulin and b-tubulin mono- sequences (Himmler et al. 1989; Goode et al. 2000). Dou- mers are very low and range from 4.8 to 5.2 (Towbin et al. blecortin (Dcx), another microtubule-binding protein, con- 2001; Stracke et al. 2002; Verdier-Pinard et al. 2003). The tains two repeat sequences with high pI (9.7 and 9.9) by measured pI around 4.2 for the ab-tubulin dimer (the which the microtubule interaction is mediated (Taylor et al. predominant form in which both monomers exist) is even 2000). Several microtubule-associated proteins (MAPs) also lower than the pI of the monomers alone (Stracke et al. contain repeat sequences with basic pI important for 2002). microtubule binding (Lewis et al. 1988; Noble et al. 1989; The majority of the microtubule-binding proteins interact Al-Bassam et al. 2002). Microtubule interaction of the with microtubules and tubulin via these acidic C-terminal tail kinesin motor domains is attributed to the basic amino acids sequences. Our study points at two small sequence stretches located at the surface (Woehlke et al. 1997). A monomeric within the C-terminus of TRPV1 (amino acid 710–730 for kinesin (KIF1A) has much higher affinity for microtubules stretch 1 and amino acid 770–797 for stretch 2, respectively), and an enhanced processivity along the microtubules due to each of which can modulate the tubulin interaction. These the presence of six extra lysine residues within the ‘K-loop’ sequences have a basic pI value of 11.17 and 12.6, of the motor domain (Okada and Hirokawa 1999). The respectively. The binding capability of smaller- and dele- microtubule-binding domain of Stu2p contains highly basic tion-fragments of TRPV1-Ct correlates well with two factors: amino acids with a predicted pI of 10.7 (Wang and Huffaker the presence or absence of these two short basic stretches and 1997). Notably, the microtubule-binding domain of Stu2p the overall pI of the protein. This is in agreement with many contains two imperfect repeat sequences. A short peptide observations showing that the majority of tubulin interacting (KKKKKSKTKCVIM) with multiple lysine residues repre- proteins contain short imperfect repeat sequences composed senting the C-terminus of K-Ras has been reported to interact of basic amino acids. For example, isoforms of microtubule- with microtubules (Thissen et al. 1997; Chen et al. 2000). binding protein tau contain three to four 18-amino acid long Our conclusion that these two short basic sequences are imperfect repeats separated by 13–14-amino acid inter-repeat important for tubulin binding is furter strengthened by the

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(a) (b) many tubulin (dimer/monomer?) binds per receptor. It is MBP-TRPV1-Ct + + + + + + possible that these two sequences bind tubulin independent αβ – + – – + – + – + – α β of each other and there is a cooperative binding. It is also s s ––+ ––+ –+ –+ 1 2 3 1 2 3 1 2 1 2 kDa possible that in native condition, the tubulin binding by the 205 two short stretches are masked by the presence of short 114 97 neighbouring sequences or other interacting proteins that 67 contain negative charges. From our study it is also not clear 45 whether all the positively charged residues present within 30 each stretch are important for tubulin binding. But our experiments show that subtilisin-digested tubulin does not bind to the MBP-TRPV1-Ct. As subtilisin-digested tubulin retain its core structure but loose the C-terminal over-hanging Coomassie WB: Tub Coomassie WB: Tub acidic region (see Fig. S1), it indicates that the TRPV1-Ct- Fig. 9 The C-terminal acidic region of tubulins are important for binding ability of tubulin is most likely located within the C- TRPV1-Ct binding. (a) Subtilisin-digested tubulin does not bind to terminal overhanging regions. TRPV1-Ct. MBP-TRPV1-Ct immobilised on amylose resin (lane 1) The C-terminal sequence of TRPV1 is shown to be was incubated with an equal amount of control tubulin (ab) (lane 2) or involved in several other interactions and functions and may subtilisin-digested tubulin (asbs) (lane 3). Bound proteins were eluted represent a ‘hot spot’ for TRPV1 regulation (Fig. 10). For with maltose and analysed by 10% SDS-PAGE. Proteins were visu- example, the region comprising amino acid residues 684–721 alised by Coomassie (left side) and also by western-blot analysis for has been shown to be important for tetramerisation (Garcıá- bound tubulin (right side). The arrow and the arrowhead indicate the Sanz et al. 2004). Amino acid 761 has been shown to be position of ab tubulin and asbs tubulin respectively. (b) SDS-PAGE and western-blot analysis of the control tubulin and subtilisin-digested important for the capsaicin-sensitivity (Jung et al. 2002). tubulin. Control ab-tubulin (lane 1) and subtilisin-digested asbs-tubulin Vlachova´ et al. demonstrated that the C-terminal tail of (lane 2) were separated by 10% SDS-PAGE and stained by Coo- TRPV1 carries structural determinants rendering the receptor massie (left side) or visualised by western-blot analysis with anti-b- sensitive to heat and capsaicin. They could demonstrate that tubulin antibody. Note that the antibody detects both control tubulin deletion of the distal 72 amino acids results in a decline of and subtilisin-digested tubulin in native form too (right side bottom). the capsaicin-, pH-, and heat-sensitivity of the receptor For more information, see Fig. S1. (Vlachova´ et al. 2003). Moreover, the C-terminal tail contains phosphorylation sites used by protein kinase C fact that positively charged amino acids located within these (Numazaki et al. 2002) and Ca2+/calmodulin-dependent two stretches are distributed on one side of a putative helix protein kinase II. Phosphorylation of these sites affects the (Fig. 4). More interestingly, the stretch 1 contains several TRPV1 desensitisation in the presence of Ca2+ (Jung et al. hydrophobic amino acids distributed on this putative helix 2004). Two serine residues (at position 774 and 820) located just opposite to the charged basic amino acids (Fig. 4). within the C-terminus of TRPV1 are phosphorylated by PKA Therefore, it is possible that this portion of the C-terminus is in vitro (Bhave et al. 2003; Mohapatra and Nau 2003). partially embedded in the plasma membrane while the other exposed surface is involved in tubulin binding. It is important Calmodulin to mention that the a-tubulin-binding site of mGluR7 also Tetramerization Binding region domain forms a putative helix with positively charged residues PKCε phosphorylation located towards one direction (Saugstad et al. 2002). site (800) In absence of X-ray or NMR data, the structure of the C- 681 838 terminal cytoplasmic domain of TRPV1 has been modelled * * by two groups (Vlachova´ et al. 2003; Garcıá-Sanz et al. 2004). According to the model proposed by Garcıá-Sanz CamKII PIP2 binding et al., the first stretch sequence is exposed and therefore phosphorylation region (778–819) easily accessible to interact (Garcıá-Sanz et al. 2004). In this site (704) Required for RTX study, we did not measure the ‘Kd’ value of the interaction, activation (761) but the binding of two different biotinylated-peptides to the Fig. 10 The C-terminus of TRPV1 contains structurally and func- tubulin at nanomolar concentration and observed interaction tionally important residues. Arrows and brackets indicate different between MBP-TRPV1-Ct with tubulin even at the high salt important domains and residues located within the C-terminus. Num- condition (0.5 mol/L, data not shown) indicates for a stable bers of amino acids are written in green. Blue areas indicate regions of and strong binding. Though our results strongly suggest that tubulin binding. The exact binding site for Eferin is not known. Putative the C-terminal cytoplasmic domain of TRPV1 contains two PKA and PKC phosphorylation sites (S774 and S820) are indicated by tubulin-binding sites, from our study, it is not clear how asterisk (*) sign. The drawing is not exactly to scale.

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(a) Stretch 1 Stretch 2 * * ** * * ** * * * * * Rat KSFLKCMRKAFRSGKLLQVGF / KRTLSFSLRSGRVSGRNWKNFALVPLLR Mice KSFLKCMRKAFRSGKLLQVGF / KRTLSFSLRSGRVSGRNWKNFALVPLLR Dog KSFLKCMRKAFRSGKLLQVGY / KRTLSFSLRSGRVSGRNWKNFSLVPLLR Human KSFLKCMRKAFRSGKLLQVGY / KRTLSFSLRSSRVSGRHWKNFALVPLLR Monkey KSFLKCMRKAFRSGKLLQVGY / KRTLSFSLRSSRVSARHWKNFALVPLLR Guinepig KSFLKCMRKAFRSGKLLQVGY / KRTLSFSLRSGRVSGRNWKNFALVPLLR Rabbit KGFLKCMRKAFRSGKLLQVGY / KRTLSFSLRSGRVSGRNWKNFALVPLLR Porcine KSFLKCMRKVFRSGKLLQVGY / KRTLSFSLRSSRVAGRNWKNFALVPLLR Chicken SY-LNCLRRSFRSGKRVLVGI / KRNPSYCIKPGRVSGKNWK--TLVPLLR (b) Stretch 1 Stretch 2 TRPV1 KSFLKCMRKAFRSGKLLQVGF / KRTLSFSLRSGRVSGRNWKN---FALVPLLR TRPV2 NGYWWCRRKKHREGRLLKVGT / ------SGPGITGNKKN---PTSKPGKN TRPV4 RSFPVFLRKAFRSGEMVTVGK / YQYYGFSHTMGRLRRDRWSSVVPRVVELNKN TRPV3 KMLPEWLRSRFRMGELCKVAD / ------GPIRRTADSN---KIQDSSRS TRPV5 RKMPRFLWP--RSG----ICG / QEQLSEKQPSGTETGTLARGSVVLQTPPLSR TRPV6 RKLPRCLWP--RSG----ICG / EKDSGEKLEMARPFGAYLS----FPTPSVSR

Fig. 11 Two short sequence stretches located within the C-terminus amino acids within the ﬁrst stretch is conserved to certain extent in few of TRPV1 are conserved. (a) Shown is the sequence alignment based other TRPV family members. Shown is the sequence alignment of on TRPV1 sequences from different species. NCBI accession num- different TRPV members (based on sequences from rat species only). bers are indicated. Rat (AF029310), Mice (CAF05661), Dog Using clustlaw software available in the expasy site does alignment. (AAT71314), Human (NP_542437), Rhesus monkey of Indian origin NCBI accession numbers are indicated. TRPV1 (AF029310), TRPV2 (XP_001117609), Guinea pig (AAU43730), Rabbit (AAR34458), (AAH89215), TRPV3 (NP-001020928), TRPV4 (NP-076460), TRPV5 Chicken (NP_989903). Porcine TRPV1 sequence is described in Ohta (AAV31121) and TRPV6 (Q9R186). Basic amino acids are written in et al. (2005). Identical amino acids are shown in blue colour. Basic blue colour. amino acids are indicated by asterisk (*). (b) The distribution of basic

Furthermore, Numazaki et al. detected a short sequence in mechanical hyperalgesia induced by taxol, a microtubule within the C-terminal tail that is critical for receptor cytoskeleton-regulating drug (Alessandri-Haber et al. 2004). desensitisation (Numazaki et al. 2003). They could also In agreement with that notion, the involvement of an intact prove that this sequence contains a binding site for calmo- microtubule cytoskeleton in the second messenger signalling dulin. Eferin, an EF-hands-containing Rab11/25-interacting for inflammatory pain has also been reported (Bhave and protein was reported to bind at the C-terminal cytoplasmic Gereau 2003; Dina et al. 2003). domain of TRPV1 (Lee 2005). Furthermore, the C-terminal In summary, our work identifies two short basic-amino sequence of TRPV1 contains a PIP2 binding site, which acid stretches within the C-terminus of TRPV1 that interacts negatively regulates the ion channel activity (Prescott and with tubulin. These stretches represent novel tubulin-binding Julius 2003). Remarkably tubulin-binding sites detected in motifs that are also conserved to a certain extent in some this study are located close to the regions that are important other members of the TRPV subfamily. for receptor tetramerisation, PIP2 binding or PKC phosphorylation (Fig. 10). Hence it is possible that tubulin Acknowledgements binding to the C-terminus of TRPV1 may alters these interactions. We thank Mark Hartman, Linda Stewani and Shu Liu for preparing The two short basic stretches present in TRPV1 are highly the TRPV1 constructs. We thank Oliver Bogen for providing conserved in most of the reported mammalian orthologous enriched neurofilament preparation. We are thankful to Dr Linda sequences (Fig. 11). Among the members of the TRPV Amos (Cambridge, UK) for providing subtilisin-digested tubulin subfamily, similar sequences including positively charged and related control reagents. We acknowledge the kind support provided by Prof H.H. Ropers. Financial support by Max Plank amino-acids within the first sequence stretch are conserved to Institute of Molecular Genetics (Berlin), BMBF, Deutsche Fors- a certain extent, especially among TRPV1, TRPV2, TRPV4, chungsgemeinschaft, Sfb 515, and Fonds der Chemischen Industrie and TRPV3 (not so much in TRPV5 and TRPV6) (Fig. 11). is acknowledged. The second sequence stretch described above is much less conserved (Fig. 11). In agreement with the significance of these short sequences, a small sequence of the C-terminal Supplementary material cytoplasmic domain of TRPV4 has been shown to interact The following material is available for this paper online. with MAP7, a microtubule binding protein (Suzuki et al. Fig. S1 Characterisation of subtilisin-digested tubulin. 2003). Additionally, TRPV4 has been shown to be important

2007 The Authors Journal Compilation 2007 International Society for Neurochemistry, J. Neurochem. (2007) 101, 250–262 Identiﬁcation and characterisation of tubulin-binding motifs 261

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