Identification and Characterisation of Novel Tubulin-Binding Motifs
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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 Ne´chaud 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. Ó 2007 The Authors 250 Journal Compilation Ó 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 Ò 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 Ó 2007 The Authors Journal Compilation Ó 2007 International Society for Neurochemistry, J. Neurochem. (2007) 101, 250–262 252 C.