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Truncation of human dopamine transporter by protease calpain
Neurochemistry International 52 (2008) 1436–1441
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Truncation of human dopamine transporter by protease calpain Veronika Franekova, Martina Baliova, Frantisek Jursky * Laboratory of Neurobiology, Institute of Molecular Biology, Slovak Academy of Sciences, Dubravska cesta 21, 842 51 Bratislava, Slovakia
A R T I C L E I N F O
A B S T R A C T
Article history: Received 28 August 2007 Received in revised form 17 February 2008 Accepted 1 April 2008 Available online 8 April 2008
It has been shown recently that the N-terminal domain of the dopamine transporter (DAT) plays a role in several transporter functions. Here we provide evidence for a possible cellular mechanism of how the Nterminus of dopamine transporter might be removed in vivo. We isolated a recombinant N-terminal protein region of human dopamine transporter and cleaved it with calpain protease. Peptide fragment analysis revealed the existence of two calpain cleavage sites at positions Thr43/Ser44 and Leu71/Ser72 of the DATN-terminus. We show that calpain activation in rat striatal synaptosomes leads to a rapid decrease of dopamine transporter N-terminal epitopes corresponding to the protein sequences removed by a calpain cleavage at Thr43/Ser44 and that the process is totally blocked by a calpain inhibitor. Calpain truncation of the DATN-terminus abolishes its interaction with the receptor of activated protein kinase C, RACK1 and removes protein sequences previously implicated in amphetamine-induced dopamine release, PKC-dependent endocytosis and the interaction of DAT with the dopamine D2 receptor. The above suggests that cleavage of DAT by calpain may significantly modify dopamine homeostasis under pathological or physiological conditions. ß 2008 Elsevier Ltd All rights reserved.
Keywords: Dopamine transporter Calpain Receptor of activated protein kinase C RACK1
1. Introduction The dopamine transporter (DAT), a member of the sodium dependent neurotransmitter transporter family (Kilty et al., 1991; Shimada et al., 1991; Usdin et al., 1991; Giros et al., 1991; Wu and Gu, 1999) is a plasma membrane transport protein expressed within a small subset of CNS neurons (Cerruti et al., 1993; Ciliax et al., 1995; Nirenberg et al., 1996) and in some peripheral locations such as nasal mucosa (Chemuturi et al., 2006), blood platelets, lymphocytes and the pineal gland (Frankhauser et al., 2006; Amenta et al., 2001; Phansuwan-Pujito et al., 2006). While the role of dopamine transporter outside the brain is currently not clear, in the brain, dopamine-mediated neurotransmission controls very important behavioral events (Schultz, 2007). Most recent studies are therefore focused on the potential roles of dopamine transporter in CNS disorders, including Parkinson disease, drug abuse, Tourette’s syndrome, schizophrenia and attention deficit hyperactivity disorder (ADHD) (Crocker et al., 2003; Trifiletti and Bandele, 2000; Breier et al., 1997; Winterer and Weinberger, 2004; Mazei-Robinson and Blakely, 2006). Release of dopamine in the brain and the time window of its effects on dopamine-responding receptors are precisely balanced.
* Corresponding author. Tel.: +421 2 5930 7437; fax: +421 2 5930 7416. E-mail address: [email protected] (F. Jursky). 0197-0186/$ – see front matter ß 2008 Elsevier Ltd All rights reserved. doi:10.1016/j.neuint.2008.04.001
After vesicular dopamine release, it is the dopamine transporter that is thought to play the major role in regulating the duration of dopamine action (Sotnikova et al., 2006). The crucial role of dopamine transporter in synaptic dopamine regulation was confirmed by antagonist studies as well as gene-knockout experiments (Giros et al., 1996). Variation of dopamine transporter protein level (Melikian and Buckley, 1999; Bauman et al., 2000; Mortensen and Amara, 2003; Sorkina et al., 2005) changes the lifetime of dopamine presence in some brain regions and allows its diffusion to remote receptors, causing so-called volume transmission (Herkenham, 1987). DAT currents seem to be sufficient to depolarize neurons (Ingram et al., 2002) and the channel mode of dopamine transporter has been described (Carvelli et al., 2004). In addition, the rate of dopamine uptake is decreased by drugs of abuse such us cocaine and amphetamines, which modulates the stimulatory and reward effects on behavior (Ritz et al., 1987; Schultz, 2007). On a long-term scale, dopamine transmission is regulated by the degradative enzymes monoamine oxidase (MAO) and catecholO-methyltransferase (COMT) (Shih, 2004; Gogos et al., 1998). The transporter core skeleton in the membrane is thought to play a major role in elementary transport events (Mabjeesh and Kanner, 1992). Recently, however, it has been increasingly recognized that deletion of the cytoplasmatic N-terminal transporter region influences intrinsic transporter properties, such as release (Khoshbouei et al., 2004), uncoupled currents (Binda et al.,
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2006), endocytosis (Miranda et al., 2007) and presynaptic dopamine signaling (Lee et al., 2007). In this article we report that the N-terminal region of the dopamine transporter may be removed in vivo by calpain protease, which is likely to cause changes in dopamine transporter regulation and local dopamine homeostasis. 2. Materials and methods 2.1. Materials [3H] dopamine (56.1 Ci/mmol) was supplied by American Radiochemicals Inc. 101 Arc Drive (St. Louis, MO, USA). Antibodies against human DATC-terminus (amino acids 568–620) were prepared by injecting rabbits with the pMAL-DATC fusion protein and affinity purified as described (Jursky and Nelson, 1995). Antibodies against the DATN-terminal region were obtained by injecting rabbits with DATN-GST fusion protein (for construction and isolation procedure, see below). Because of the existence of two calpain cleavage sites, we separated the epitopes against two different parts of the dopamine transporter N-terminus using differential affinity purification on fusion proteins coupled to Affigel 10 (Bio-Rad Laboratories, Hercules, CA, USA). Epitopes against the DATN peptide, encompassing amino acids 1–43, which were removed after calpain cleavage on site 1, were obtained by purification of antibodies on immobilized full length DATN-GST with subsequent adsorption of more internal epitopes on truncated DATN-GST fusion protein coupled to Affigel 10. DATN epitopes corresponding to amino acids 43–72 were recovered from serum by affinity purification on truncated DATN-GST fusion protein. The specificity of all antibodies was verified by cross absorption with corresponding fusion proteins. Peroxidase linked anti-rabbit antibodies were from Chemicon (Temecula, CA, USA). ECL reagents and Calpain inhibitor I were from Sigma (St. Loius, MO, USA). Oligonucleotides were synthesized by VBC Genomics Bioscience Research (Vienna, Austria). All other chemicals used were of the purest grade available. 2.2. DATN fusion proteins construction and determination of calpain cleavage sites For cleavage site determination and antibody production, the methionine initiation codon of glutathione-S-transferase (GST) was fused to the 30 end of the DAT cDNA sequences encoding the first 72 N-terminal amino acids. The DNA fragment coding for the DATN-terminus was obtained by PCR reactions on the DAT cDNA using the forward Nde primer 50 -cccagtgtgcatatgagtaagagcaaatgc-30 and the reverse BamHI primer 50 -gcaaagccaggatccgacaggagaaagtcg-30 . The PCR fragment was digested with restriction nucleases NdeI and BamHI. A BglII/SalI DNA fragment encoding GST was obtained from plasmid pDS473 (Forsburg and Sherman, 1997). Both fragments were then inserted into the NdeI/SalI sites of the expression vector pET-21a(+) (Novagen, Merck, Darmstadt, Germany) by double ligation to produce an in-frame fusion of the DATN-terminal sequence with GST. Fusion constructs containing the truncated DATN-terminal sequences were prepared identically, except that a different forward NdeI primer (50 -ggagtgcagcatatgagctccaccctcacc-30 was used. Constructs were transformed into BL21DE3 E. coli cells, which were grown in LB medium and induced for 2 h in the presence of 0.3 mM isopropyl-b-D thiogalactopyranoside. The fusion protein was bound to a glutathione-Sepharose column and eluted with 50 mM Tris–HCl, pH 7.5, containing 10 mM glutathione. An aliquot of the eluted fraction was supplemented with CaCl2 to a final concentration of 2 mM. After the addition of recombinant rat calpain (generous gift from Prof. J. Elce, Queen’s University, Ontario, Canada) isolated according to (Elce et al., 1995), the fusion protein was incubated for 1 min at 37 8C prior to the addition of SDS sample buffer and boiling for 3 min. Appropriate aliquots were separated by 12% SDS-PAGE. Resolved proteins were transferred onto a Fluorobind membrane (Serva, Heidelberg, Germany) and visualized by Coomassie Blue R250 staining. The first 10 amino acids of the truncated DATN-GST fusion proteins were determined by Edman degradation using N-terminal protein sequencing at the sequencing facility of the Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic. 2.3. Construction of intact and truncated human DAT transporters A human pCRMV-hDAT clone was obtained from Prof. Marc Caron (Duke University, USA). For further experiments the HindIII/XbaI fragment of hDAT was transferred to pEGFP N1 (Clontec, Palo Alto, CA, USA). This resulted in the deletion of GFP proteins from the plasmid. To introduce equal DNA sequences upstream, DNA corresponding to calpain truncated DAT with an initiating methionine were amplified by PCR using wild-type hDAT cDNA as a template and the forward HindIII primers 50 -actaagcttgccaccatgagtaagagcaaatgctccg-30 (wild type) 50 -ctcaagcttgccaccatgagctccaccctcaccaac-30 (calpain cleavage site 1), 50 -ctcaagcttgccaccatgtccgtcattggctttgctg-30 (second calpain cleavage site) and the internal DAT0 s reverse BamHI primer 50 -ttgttgcagtggatccaggggagc-30 . The resulting PCR fragments were inserted into the HindIII/BamHI digested pEGFP N1-hDAT plasmid. The forward primers were designed to provide a Kozak sequence (GCCACC) (Kozak, 1991)
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preceding the wild type DAT methionine and to introduce initiator methionines in the truncated DATs before serines 44 and 72. 2.4. HEK293T transfection, uptake of [3H] dopamine and surface biotinylation HEK293T cells (ATCC, Rockville, MD, USA) were maintained in MEM medium containing 10% (v/v) fetal calf serum (Gibco, Paisley, UK). Plasmid DNA was isolated using Qiagen DNA purification tips according to manufacturer’s instructions (Qiagen GmbH, Germany). DNA transfection was performed using a PEI ratio of 10 according to Boussif et al. (1995). For stable cell lines selections plasmids were linearized with ApaLI before transfection and G418 (MP Biomedicals, Irvine, CA, USA) was used for selection at a concentration 0.5–1 mg/ml. [3H] dopamine uptake assays with transiently or stably transfected HEK293T cells were performed for 5 min at 37 8C. Nonspecific uptake was determined in the presence of 0.4 mM dopamine. For determination of Vmax and Km values, increasing concentrations of dopamine from 0.5 to 20 mM were used. Results were fitted by nonlinear regression analysis using GraphPad Prism (GraphPad Software, San Diego, CA, USA). To compare the transport efficiency of intact and truncated DAT, the amount of both forms present on the surface of the HEK293T cells was estimated by measuring antiDATC immunoreactivity in the surface localized protein fractions isolated upon surface biotinylation on streptavidin agarose according to (Baliova and Jursky, 2005). Scanned immunoreactive ECL spots were quantified using UN-SCAN-IT software (Silk Scientific Inc. UT, USA). 2.5. Interaction of intact and truncated DAT with RACK1 The rat RACK1 gene fused in frame with maltose binding protein in plasmid pMAL-c (New England Biolabs) (clone obtained from Dr. Karen Hyde, Vanderbilt University, USA) was isolated according to manufacturer’s instructions on maltose resin. The fusion protein was then eluted with 20 mM Tris–HCl pH 7.5 supplemented with 10 mM maltose. Pmal - RACK1 fusion protein was incubated in an interaction buffer supplemented with 1% Triton X100 for 1 h at room temperature with intact and truncated DATN-GST fusion proteins immobilized on GST-Sepharose (Pharmacia). After the sample was washed 4 times with interaction buffer, the GST fusions were eluted with glutathione and resolved on 12% PAG. Samples were transferred to immobilon and probed with anti-maltose binding protein antibodies using peroxidase/ECL detection system. 2.6. Isolation of rat spinal striatal synaptosomes and calpain activation Female Sprague–Dawley rats 250–330 g were from Charles River Wiga, Silzfeld, Germany. The use of animals was carried out following the European Communities Council Directive (86/609/ECC) and experiments were approved by the Institutional Ethical Committee. Rats were killed by cervical dislocation and striatal tissue from two hemispheres was immediately homogenized in 2 mL of ice cold 0.32 M sucrose-10 mM HEPES, pH 7.4.with a glass/Teflon homogenizer. Homogenization was followed by centrifugation at 1000 g for 12 min at 4 8C. The supernatant was centrifuged at 12,500 g to isolate the synaptosomal P2 pellet. For endogenous calpain activation via hypoosmotic lysis, the aliquot of P2 synaptosomal pellet was suspended in ten times volume excess of solution containing 25 mM Tris–HCl pH 7.5, and either 5 mM EGTA, 5 mM CaCl2, or 5 mM CaCl2/calpain inhibitor I (50 mg/ ml). Samples were incubated for 15 min at 37 8C with shaking. After stopping the incubation on ice, aliquots were dissolved in SDS sample buffer, separated by 7.5% SDS-PAGE and immunoblotted with antibodies raised against DATN epitopes.
3. Results 3.1. Localization of calpain cleavage sites in human DATN-terminus Our previous studies revealed that the N-terminal domains of glycine transporters are substrates of calpain protease (Baliova et al., 2004; Baliova and Jursky, 2005). Since calpain has been previously widely implicated in dopamine homeostasis, we decided to test the sensitivity of dopamine transporter (DAT) to a calcium dependent protease. In our search for epitopes suitable for detection of calpain proteolysis of DAT in vivo, we used a recombinant DATN-terminus. To determine potential calpain cleavage sites a GST fusion protein containing the entire Nterminal region of human dopamine transporter (hDAT) was overexpressed in bacteria and the isolated fusion protein was treated with recombinant calpain protease. Analysis of the cleaved fusion protein revealed the presence of two calpain cleavage products appearing with distinct velocities (Fig. 1A). The cleavage products were transferred to immobilon and the first five amino
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Fig. 1. Determination of calpain cleavage sites in the N-terminal region of human DAT and construction of recombinant truncated DAT variants. Purified DATN-terminus fused to GST was incubated with purified recombinant calpain protease. The resulting cleavage products (A) were transferred to immobilon and artificially truncated recombinant DATs were constructed according to the determined calpain cleavage sites. (B) Plasmids containing truncated DATs were transiently transfected into HEK293T and their uptake efficiency was determined in the presence of 2 mM 3H-labeled dopamine for 5 min at 37 8C (C).
acids (SSTLT and SDLHRG) were determined for each fragment using Edman protein sequencing. Results suggested that the higher affinity calpain cleavage site is located at position T43/S44 while the second site, exhibiting about five-fold lower affinity, resides between L71/S72 (Fig. 1B). The second protein sequence SDLHRG obtained by Edman degradation cannot be seen in the DATNterminus because only the first residue corresponding to S72 is from the hDAT sequence, the other five residues are from the fusion protein GST boundary sequence.
of DAT immunoreactive bands in the surface localized protein fraction isolated on streptavidin agarose upon surface biotinylation (Fig. 2A). Surface biotinylation studies showed that the amount of truncated DAT was approximately equal to wild type nonmodified DAT. Immunoreactive bands with higher molecular weight represents aggregated dimers of DAT. Similarly, uptake efficiency (DATWT Vmax = 79.10 4.60 pmol/mg/min, Km = 1.33 0.35 mM; DATD1 Vmax = 86.26 7.4 pmol/mg/min, Km = 1.27 0.47 mM) did not change significantly (Fig. 2B). Results were confirmed by three independent experiments.
3.2. Comparison of intact and truncated DATs in HEK293T cells 3.3. Verification of accessibility of calpain cleavage sites in situ To investigate the potential consequences of dopamine transporter N-terminal region removal on DAT properties, we deleted the DNA sequences coding for amino acids that are removed by calpain using recombinant DNA techniques, with exception of the initiating methionine. Wild type DAT and truncated DAT were then transiently transfected into HEK293T cells and dopamine transport efficiency was estimated for both normal and truncated DATs. Deletion of DATN-terminal sequences corresponding to the coding region for amino acids 1–43 (first calpain cleavage site) did not significantly change DAT uptake activity, but the deletion corresponding to the potential second cleavage site (amino acids 1–72) totally abolished DAT activity (Fig. 1C). For this reason, in further experiments HEK293T stable cell lines were selected in the presence of 0.5–1 mg/G418 per ml of MEM expressing wild type DAT and DAT truncated at the first calpain cleavage site. Relative amounts of uptake competent surface localized transporters were determined by quantification
In order to verify that cleavage sites in DATN-terminus are accessible to calpain protease in situ, we prepared rat striatal synaptosomes and increased calcium in the vicinity of synaptosomal DAT by hypoosmotic shock in a low osmolarity buffer containing either EGTA, high calcium or high calcium in the presence of calpain inhibitor I. We expected that under these conditions, endogenous calpain protease will be activated and part of the dopamine transporter N-terminus will be removed. After 15 min of incubation at 37 8C, we observed substantial loss of DATN-terminal immunoreactivity corresponding to N-terminal epitopes 1–43 in synaptosomal DAT in the sample where calcium was included (Fig. 3). This process was totally blocked by the addition of calpain inhibitor I. Because of the weak affinity of purified epitopes corresponding to amino acids 44–72 against rat striatal synaptosomes, we were not able to verify the existence of the second cleavage site in DATN terminus in situ.
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Fig. 4. Truncation of DATN abolishes its interaction with RACK1. GST fusion proteins containing intact and truncated DATN-terminus were immobilized on GSTSepharose and incubated with a solution containing whole RACK1 sequence fused to maltose binding protein. After washing, GST fusion proteins were eluted with glutathione, resolved on 12% PAG and immunobloted with antibodies against maltose binding protein.
4. Discussion
Fig. 2. Comparison of uptake efficiency and surface expression of DATWT and DATD1 in HEK293T cells. Intact DATWT and truncated DATD1 expressed on the surface of HEK293T stable cell lines were labeled with biotin. After cell lysis, biotin labeled surface localized transporters were recovered on streptavidin agarose from total protein extracts and loaded on 7.5% PAG. Resolved proteins were transferred to immobilon and probed with antibodies against the C-terminal region of dopamine transporter (A). In the same sample sets, efficiency of uptake was determined in the presence of various concentrations of 3H-labeled dopamine for 5 min at 37 8C (B).
3.4. Binding of RACK1 to intact and truncated DAT It has been shown previously that the DATN-terminal region interacts with RACK1 (Lee et al., 2004), but the precise site of interaction was not mapped. To see if calpain truncation eliminates interaction with RACK1, we incubated immobilized GST fusion proteins containing intact and truncated DATN-terminal regions with pMAL fusion protein containing full-length RACK1. As shown in Fig. 4, N-terminal truncation of DAT with calpain abolishes its interaction with RACK1.
Fig. 3. Truncation of DAT in rat striatal synaptosomes. Crude rat striatal synaptosomes were lysed in 25 mM Tris–HCl pH 7.5 containing either 5 mM EGTA 5 mM CaCl2 or 5 mM CaCl2 in the presence of calpain inhibitor I. After 15 min incubation at 37 8C, equal aliquots of the synaptosomes were mixed with 2 SDS buffer, boiled, and the proteins were resolved on 7.5% PAG and immunobloted with anti-DATN (1–43).
Even though calpain degrades many cellular and cytoskeletal proteins and processes many hormones and enzymes (Carafoli and Molinari, 1998), its physiological function is largely unknown and complex. Increased calpain activation is, except for its normal cellular function, thought to be involved in the pathophysiology of degenerative diseases (Dufty et al., 2007; Higuchi et al., 2005). In brain disorders such as Parkinson’s disease or Tourette’s syndrome, dopamine homeostasis is significantly altered. There are several evidences that calpain plays a role in the modulation or deterioration of brain dopaminergic function (Yurko-Mauro and Friedman, 1996; Trifiletti and Bandele, 2000; Kuhn et al., 2003; Crocker et al., 2003; Merali and Park, 2003). Calpain cleavage site specificity is coded by a high-ordered structure and thus it is not well conserved at the amino acid sequence level. The size of the adjacent region influencing calpain cleavage has been studied previously (Tompa et al., 2004). In the recombinant N-terminal cytoplasmic domain of DAT fused to GST, we found two calpain cleavage sites with significantly different affinities. The first calpain cleavage site, located close to the middle of the DATN-terminal sequence, likely contains enough DATN amino acid residues adjacent to both sides of the cleaved peptide bond to create calpain cleavage site specificity identical to that existing in vivo. The second cleavage site is located between the third and fourth amino acids of the first transmembrane domain and exhibits much lower affinity in the fusion protein. This cleavage site is, however, very close to the DATN-GST fusion boundary and part of the potential in vivo calpain recognition site has been replaced by GST residues. This means that the second cleavage site could be either artificially created or its affinity could have been reduced by replacement of the reciprocal half of the calpain recognition sequence by the GST protein sequence. In order to verify if both calpain cleavage sites are accessible to calpain cleavage in vivo, we tried to separate affinity purified polyclonal antibody epitopes corresponding to amino acids 1–43 and 44–72, recognizing the DATN region between two calpain cleavage sites. Later epitopes were however not strong enough to give satisfactory results and at present we cannot determine if the second calpain cleavage site in DATN-terminus is accessible for calpain in vivo. Rat striatal synaptosomes lysed in the presence of calcium however exhibited a clear decrease of immunoreactivity epitopes 1–43, which indicates that under conditions of hypoosmotic high calcium lysis, this calpain cleavage site is accessible. Transporters are distributed between the inner cellular compartment and the membrane. Only membrane localized transporters are transport competent. To correlate measured uptake of wild type and truncated dopamine transporters with the membrane localized transporters in HEK293T cells, we estimated the relative amount of
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DATs on cell membrane using surface biotinylation labeling. In biotinylated fraction, we observed significant amount of DAT immunoreactivity in the position corresponding to DAT dimers. Relative abundance of these dimers is the same in wild type as well as truncated DAT samples. Since these dimers are resistant to boiling in SDS sample buffer, we consider it as unspecific aggregation. On average, its truncation results in about the same level of surface expression of DAT with a similar maximal velocity of transport and affinity constant in HEK293T cells. On the other hand, HEK293T cells do not express all components of the DAT signaling machinery. Further detailed studies will be necessary in a natural dopaminergic cellular environment. The N-terminal region of DAT removed by calpain contains the cluster of three lysines previously implicated in dramatic inhibition of PKC-dependent ubiquitylation and endocytosis of DAT, resulting in a severe delay in DAT degradation (Miranda et al., 2007). DAT in PKC activated cells was less efficiently sorted to the lysosomal degradation pathway and was instead recycled back to the cell surface. In this case calpain truncation would have a profound effect on DAT trafficking. In our preliminary experiments in HEK293T cells, however, we observed a similar extent of PKC induced internalization of both wild type and truncated DAT when stimulated with PMA. These results are in agreement with a previous report (Sorkina et al., 2005) indicating that acute PMA induced endocytosis is not abolished even if the entire DATNterminal region is replaced with GFP. Recently, the direct association of DATN-terminal sequences with the presynaptic dopamine D2 receptor was described (Lee et al., 2007). Since calpain cleavage of the DATN-terminus also removes residues responsible for this interaction, regulation of DAT via presynaptic D2 receptor could also be altered by Nterminal truncation of DAT. So far we do not know if specific pathways exist, which could selectively activate calpain in dopaminergic terminals close to DAT, leading to its N-terminal truncation. However one situation in which such a process is quite possible is calcium overload and calpain activation under pathological conditions. Recently, Liu et al. (2007) showed that calcium influx through L-type channels in dopamine ventral tegmental neurons activates a calcium-dependent protease that cleaves PKC to generate constitutively active and labile PKM resulting in burst firing of dopamine cells, a pathway that is involved in glutamatergic or cholinergic modulation of the central dopamine system. It has also been previously shown that prolonged treatment of cultured hippocampal slices with selected ampakines leads to spectrin degradation mediated by activation of the calciumdependent protease calpain (Jourdi et al., 2005). Similarly, methamphetamine induces spectrin proteolysis in the rat striatum (Staszewski and Yamamoto, 2006). Methamphetamine increased calpain-mediated spectrin proteolysis in the rat striatum 5 and 7 days after METH administration without affecting caspase 3dependent spectrin breakdown. This effect was completely blocked by an AMPA receptor antagonist, but not by an NMDA receptor antagonist. However, antibodies used to monitor changes of DAT were directed against the C-terminal transporter region and could not have detected possible calpain mediated truncation of the N-terminal region of DAT. Interestingly, Trifiletti and Bandele (2000) investigated a group of patients with Tics, Tourette’s syndrome and obsessive– compulsive disorder associated with streptococcal infection. They have found that in eighty percent of patient’s, molecular mimicry of streptococcal antigens induced antibody epitopes directed against calpain and calpastatin. This interesting coincidence indicates that calpain-calpastatin play important roles in balancing striatal dopamine homeostasis and that the potential calpain
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Neurochemistry International journal homepage: www.elsevier.com/locate/neuint
Truncation of human dopamine transporter by protease calpain Veronika Franekova, Martina Baliova, Frantisek Jursky * Laboratory of Neurobiology, Institute of Molecular Biology, Slovak Academy of Sciences, Dubravska cesta 21, 842 51 Bratislava, Slovakia
A R T I C L E I N F O
A B S T R A C T
Article history: Received 28 August 2007 Received in revised form 17 February 2008 Accepted 1 April 2008 Available online 8 April 2008
It has been shown recently that the N-terminal domain of the dopamine transporter (DAT) plays a role in several transporter functions. Here we provide evidence for a possible cellular mechanism of how the Nterminus of dopamine transporter might be removed in vivo. We isolated a recombinant N-terminal protein region of human dopamine transporter and cleaved it with calpain protease. Peptide fragment analysis revealed the existence of two calpain cleavage sites at positions Thr43/Ser44 and Leu71/Ser72 of the DATN-terminus. We show that calpain activation in rat striatal synaptosomes leads to a rapid decrease of dopamine transporter N-terminal epitopes corresponding to the protein sequences removed by a calpain cleavage at Thr43/Ser44 and that the process is totally blocked by a calpain inhibitor. Calpain truncation of the DATN-terminus abolishes its interaction with the receptor of activated protein kinase C, RACK1 and removes protein sequences previously implicated in amphetamine-induced dopamine release, PKC-dependent endocytosis and the interaction of DAT with the dopamine D2 receptor. The above suggests that cleavage of DAT by calpain may significantly modify dopamine homeostasis under pathological or physiological conditions. ß 2008 Elsevier Ltd All rights reserved.
Keywords: Dopamine transporter Calpain Receptor of activated protein kinase C RACK1
1. Introduction The dopamine transporter (DAT), a member of the sodium dependent neurotransmitter transporter family (Kilty et al., 1991; Shimada et al., 1991; Usdin et al., 1991; Giros et al., 1991; Wu and Gu, 1999) is a plasma membrane transport protein expressed within a small subset of CNS neurons (Cerruti et al., 1993; Ciliax et al., 1995; Nirenberg et al., 1996) and in some peripheral locations such as nasal mucosa (Chemuturi et al., 2006), blood platelets, lymphocytes and the pineal gland (Frankhauser et al., 2006; Amenta et al., 2001; Phansuwan-Pujito et al., 2006). While the role of dopamine transporter outside the brain is currently not clear, in the brain, dopamine-mediated neurotransmission controls very important behavioral events (Schultz, 2007). Most recent studies are therefore focused on the potential roles of dopamine transporter in CNS disorders, including Parkinson disease, drug abuse, Tourette’s syndrome, schizophrenia and attention deficit hyperactivity disorder (ADHD) (Crocker et al., 2003; Trifiletti and Bandele, 2000; Breier et al., 1997; Winterer and Weinberger, 2004; Mazei-Robinson and Blakely, 2006). Release of dopamine in the brain and the time window of its effects on dopamine-responding receptors are precisely balanced.
* Corresponding author. Tel.: +421 2 5930 7437; fax: +421 2 5930 7416. E-mail address: [email protected] (F. Jursky). 0197-0186/$ – see front matter ß 2008 Elsevier Ltd All rights reserved. doi:10.1016/j.neuint.2008.04.001
After vesicular dopamine release, it is the dopamine transporter that is thought to play the major role in regulating the duration of dopamine action (Sotnikova et al., 2006). The crucial role of dopamine transporter in synaptic dopamine regulation was confirmed by antagonist studies as well as gene-knockout experiments (Giros et al., 1996). Variation of dopamine transporter protein level (Melikian and Buckley, 1999; Bauman et al., 2000; Mortensen and Amara, 2003; Sorkina et al., 2005) changes the lifetime of dopamine presence in some brain regions and allows its diffusion to remote receptors, causing so-called volume transmission (Herkenham, 1987). DAT currents seem to be sufficient to depolarize neurons (Ingram et al., 2002) and the channel mode of dopamine transporter has been described (Carvelli et al., 2004). In addition, the rate of dopamine uptake is decreased by drugs of abuse such us cocaine and amphetamines, which modulates the stimulatory and reward effects on behavior (Ritz et al., 1987; Schultz, 2007). On a long-term scale, dopamine transmission is regulated by the degradative enzymes monoamine oxidase (MAO) and catecholO-methyltransferase (COMT) (Shih, 2004; Gogos et al., 1998). The transporter core skeleton in the membrane is thought to play a major role in elementary transport events (Mabjeesh and Kanner, 1992). Recently, however, it has been increasingly recognized that deletion of the cytoplasmatic N-terminal transporter region influences intrinsic transporter properties, such as release (Khoshbouei et al., 2004), uncoupled currents (Binda et al.,
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2006), endocytosis (Miranda et al., 2007) and presynaptic dopamine signaling (Lee et al., 2007). In this article we report that the N-terminal region of the dopamine transporter may be removed in vivo by calpain protease, which is likely to cause changes in dopamine transporter regulation and local dopamine homeostasis. 2. Materials and methods 2.1. Materials [3H] dopamine (56.1 Ci/mmol) was supplied by American Radiochemicals Inc. 101 Arc Drive (St. Louis, MO, USA). Antibodies against human DATC-terminus (amino acids 568–620) were prepared by injecting rabbits with the pMAL-DATC fusion protein and affinity purified as described (Jursky and Nelson, 1995). Antibodies against the DATN-terminal region were obtained by injecting rabbits with DATN-GST fusion protein (for construction and isolation procedure, see below). Because of the existence of two calpain cleavage sites, we separated the epitopes against two different parts of the dopamine transporter N-terminus using differential affinity purification on fusion proteins coupled to Affigel 10 (Bio-Rad Laboratories, Hercules, CA, USA). Epitopes against the DATN peptide, encompassing amino acids 1–43, which were removed after calpain cleavage on site 1, were obtained by purification of antibodies on immobilized full length DATN-GST with subsequent adsorption of more internal epitopes on truncated DATN-GST fusion protein coupled to Affigel 10. DATN epitopes corresponding to amino acids 43–72 were recovered from serum by affinity purification on truncated DATN-GST fusion protein. The specificity of all antibodies was verified by cross absorption with corresponding fusion proteins. Peroxidase linked anti-rabbit antibodies were from Chemicon (Temecula, CA, USA). ECL reagents and Calpain inhibitor I were from Sigma (St. Loius, MO, USA). Oligonucleotides were synthesized by VBC Genomics Bioscience Research (Vienna, Austria). All other chemicals used were of the purest grade available. 2.2. DATN fusion proteins construction and determination of calpain cleavage sites For cleavage site determination and antibody production, the methionine initiation codon of glutathione-S-transferase (GST) was fused to the 30 end of the DAT cDNA sequences encoding the first 72 N-terminal amino acids. The DNA fragment coding for the DATN-terminus was obtained by PCR reactions on the DAT cDNA using the forward Nde primer 50 -cccagtgtgcatatgagtaagagcaaatgc-30 and the reverse BamHI primer 50 -gcaaagccaggatccgacaggagaaagtcg-30 . The PCR fragment was digested with restriction nucleases NdeI and BamHI. A BglII/SalI DNA fragment encoding GST was obtained from plasmid pDS473 (Forsburg and Sherman, 1997). Both fragments were then inserted into the NdeI/SalI sites of the expression vector pET-21a(+) (Novagen, Merck, Darmstadt, Germany) by double ligation to produce an in-frame fusion of the DATN-terminal sequence with GST. Fusion constructs containing the truncated DATN-terminal sequences were prepared identically, except that a different forward NdeI primer (50 -ggagtgcagcatatgagctccaccctcacc-30 was used. Constructs were transformed into BL21DE3 E. coli cells, which were grown in LB medium and induced for 2 h in the presence of 0.3 mM isopropyl-b-D thiogalactopyranoside. The fusion protein was bound to a glutathione-Sepharose column and eluted with 50 mM Tris–HCl, pH 7.5, containing 10 mM glutathione. An aliquot of the eluted fraction was supplemented with CaCl2 to a final concentration of 2 mM. After the addition of recombinant rat calpain (generous gift from Prof. J. Elce, Queen’s University, Ontario, Canada) isolated according to (Elce et al., 1995), the fusion protein was incubated for 1 min at 37 8C prior to the addition of SDS sample buffer and boiling for 3 min. Appropriate aliquots were separated by 12% SDS-PAGE. Resolved proteins were transferred onto a Fluorobind membrane (Serva, Heidelberg, Germany) and visualized by Coomassie Blue R250 staining. The first 10 amino acids of the truncated DATN-GST fusion proteins were determined by Edman degradation using N-terminal protein sequencing at the sequencing facility of the Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic. 2.3. Construction of intact and truncated human DAT transporters A human pCRMV-hDAT clone was obtained from Prof. Marc Caron (Duke University, USA). For further experiments the HindIII/XbaI fragment of hDAT was transferred to pEGFP N1 (Clontec, Palo Alto, CA, USA). This resulted in the deletion of GFP proteins from the plasmid. To introduce equal DNA sequences upstream, DNA corresponding to calpain truncated DAT with an initiating methionine were amplified by PCR using wild-type hDAT cDNA as a template and the forward HindIII primers 50 -actaagcttgccaccatgagtaagagcaaatgctccg-30 (wild type) 50 -ctcaagcttgccaccatgagctccaccctcaccaac-30 (calpain cleavage site 1), 50 -ctcaagcttgccaccatgtccgtcattggctttgctg-30 (second calpain cleavage site) and the internal DAT0 s reverse BamHI primer 50 -ttgttgcagtggatccaggggagc-30 . The resulting PCR fragments were inserted into the HindIII/BamHI digested pEGFP N1-hDAT plasmid. The forward primers were designed to provide a Kozak sequence (GCCACC) (Kozak, 1991)
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preceding the wild type DAT methionine and to introduce initiator methionines in the truncated DATs before serines 44 and 72. 2.4. HEK293T transfection, uptake of [3H] dopamine and surface biotinylation HEK293T cells (ATCC, Rockville, MD, USA) were maintained in MEM medium containing 10% (v/v) fetal calf serum (Gibco, Paisley, UK). Plasmid DNA was isolated using Qiagen DNA purification tips according to manufacturer’s instructions (Qiagen GmbH, Germany). DNA transfection was performed using a PEI ratio of 10 according to Boussif et al. (1995). For stable cell lines selections plasmids were linearized with ApaLI before transfection and G418 (MP Biomedicals, Irvine, CA, USA) was used for selection at a concentration 0.5–1 mg/ml. [3H] dopamine uptake assays with transiently or stably transfected HEK293T cells were performed for 5 min at 37 8C. Nonspecific uptake was determined in the presence of 0.4 mM dopamine. For determination of Vmax and Km values, increasing concentrations of dopamine from 0.5 to 20 mM were used. Results were fitted by nonlinear regression analysis using GraphPad Prism (GraphPad Software, San Diego, CA, USA). To compare the transport efficiency of intact and truncated DAT, the amount of both forms present on the surface of the HEK293T cells was estimated by measuring antiDATC immunoreactivity in the surface localized protein fractions isolated upon surface biotinylation on streptavidin agarose according to (Baliova and Jursky, 2005). Scanned immunoreactive ECL spots were quantified using UN-SCAN-IT software (Silk Scientific Inc. UT, USA). 2.5. Interaction of intact and truncated DAT with RACK1 The rat RACK1 gene fused in frame with maltose binding protein in plasmid pMAL-c (New England Biolabs) (clone obtained from Dr. Karen Hyde, Vanderbilt University, USA) was isolated according to manufacturer’s instructions on maltose resin. The fusion protein was then eluted with 20 mM Tris–HCl pH 7.5 supplemented with 10 mM maltose. Pmal - RACK1 fusion protein was incubated in an interaction buffer supplemented with 1% Triton X100 for 1 h at room temperature with intact and truncated DATN-GST fusion proteins immobilized on GST-Sepharose (Pharmacia). After the sample was washed 4 times with interaction buffer, the GST fusions were eluted with glutathione and resolved on 12% PAG. Samples were transferred to immobilon and probed with anti-maltose binding protein antibodies using peroxidase/ECL detection system. 2.6. Isolation of rat spinal striatal synaptosomes and calpain activation Female Sprague–Dawley rats 250–330 g were from Charles River Wiga, Silzfeld, Germany. The use of animals was carried out following the European Communities Council Directive (86/609/ECC) and experiments were approved by the Institutional Ethical Committee. Rats were killed by cervical dislocation and striatal tissue from two hemispheres was immediately homogenized in 2 mL of ice cold 0.32 M sucrose-10 mM HEPES, pH 7.4.with a glass/Teflon homogenizer. Homogenization was followed by centrifugation at 1000 g for 12 min at 4 8C. The supernatant was centrifuged at 12,500 g to isolate the synaptosomal P2 pellet. For endogenous calpain activation via hypoosmotic lysis, the aliquot of P2 synaptosomal pellet was suspended in ten times volume excess of solution containing 25 mM Tris–HCl pH 7.5, and either 5 mM EGTA, 5 mM CaCl2, or 5 mM CaCl2/calpain inhibitor I (50 mg/ ml). Samples were incubated for 15 min at 37 8C with shaking. After stopping the incubation on ice, aliquots were dissolved in SDS sample buffer, separated by 7.5% SDS-PAGE and immunoblotted with antibodies raised against DATN epitopes.
3. Results 3.1. Localization of calpain cleavage sites in human DATN-terminus Our previous studies revealed that the N-terminal domains of glycine transporters are substrates of calpain protease (Baliova et al., 2004; Baliova and Jursky, 2005). Since calpain has been previously widely implicated in dopamine homeostasis, we decided to test the sensitivity of dopamine transporter (DAT) to a calcium dependent protease. In our search for epitopes suitable for detection of calpain proteolysis of DAT in vivo, we used a recombinant DATN-terminus. To determine potential calpain cleavage sites a GST fusion protein containing the entire Nterminal region of human dopamine transporter (hDAT) was overexpressed in bacteria and the isolated fusion protein was treated with recombinant calpain protease. Analysis of the cleaved fusion protein revealed the presence of two calpain cleavage products appearing with distinct velocities (Fig. 1A). The cleavage products were transferred to immobilon and the first five amino
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Fig. 1. Determination of calpain cleavage sites in the N-terminal region of human DAT and construction of recombinant truncated DAT variants. Purified DATN-terminus fused to GST was incubated with purified recombinant calpain protease. The resulting cleavage products (A) were transferred to immobilon and artificially truncated recombinant DATs were constructed according to the determined calpain cleavage sites. (B) Plasmids containing truncated DATs were transiently transfected into HEK293T and their uptake efficiency was determined in the presence of 2 mM 3H-labeled dopamine for 5 min at 37 8C (C).
acids (SSTLT and SDLHRG) were determined for each fragment using Edman protein sequencing. Results suggested that the higher affinity calpain cleavage site is located at position T43/S44 while the second site, exhibiting about five-fold lower affinity, resides between L71/S72 (Fig. 1B). The second protein sequence SDLHRG obtained by Edman degradation cannot be seen in the DATNterminus because only the first residue corresponding to S72 is from the hDAT sequence, the other five residues are from the fusion protein GST boundary sequence.
of DAT immunoreactive bands in the surface localized protein fraction isolated on streptavidin agarose upon surface biotinylation (Fig. 2A). Surface biotinylation studies showed that the amount of truncated DAT was approximately equal to wild type nonmodified DAT. Immunoreactive bands with higher molecular weight represents aggregated dimers of DAT. Similarly, uptake efficiency (DATWT Vmax = 79.10 4.60 pmol/mg/min, Km = 1.33 0.35 mM; DATD1 Vmax = 86.26 7.4 pmol/mg/min, Km = 1.27 0.47 mM) did not change significantly (Fig. 2B). Results were confirmed by three independent experiments.
3.2. Comparison of intact and truncated DATs in HEK293T cells 3.3. Verification of accessibility of calpain cleavage sites in situ To investigate the potential consequences of dopamine transporter N-terminal region removal on DAT properties, we deleted the DNA sequences coding for amino acids that are removed by calpain using recombinant DNA techniques, with exception of the initiating methionine. Wild type DAT and truncated DAT were then transiently transfected into HEK293T cells and dopamine transport efficiency was estimated for both normal and truncated DATs. Deletion of DATN-terminal sequences corresponding to the coding region for amino acids 1–43 (first calpain cleavage site) did not significantly change DAT uptake activity, but the deletion corresponding to the potential second cleavage site (amino acids 1–72) totally abolished DAT activity (Fig. 1C). For this reason, in further experiments HEK293T stable cell lines were selected in the presence of 0.5–1 mg/G418 per ml of MEM expressing wild type DAT and DAT truncated at the first calpain cleavage site. Relative amounts of uptake competent surface localized transporters were determined by quantification
In order to verify that cleavage sites in DATN-terminus are accessible to calpain protease in situ, we prepared rat striatal synaptosomes and increased calcium in the vicinity of synaptosomal DAT by hypoosmotic shock in a low osmolarity buffer containing either EGTA, high calcium or high calcium in the presence of calpain inhibitor I. We expected that under these conditions, endogenous calpain protease will be activated and part of the dopamine transporter N-terminus will be removed. After 15 min of incubation at 37 8C, we observed substantial loss of DATN-terminal immunoreactivity corresponding to N-terminal epitopes 1–43 in synaptosomal DAT in the sample where calcium was included (Fig. 3). This process was totally blocked by the addition of calpain inhibitor I. Because of the weak affinity of purified epitopes corresponding to amino acids 44–72 against rat striatal synaptosomes, we were not able to verify the existence of the second cleavage site in DATN terminus in situ.
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Fig. 4. Truncation of DATN abolishes its interaction with RACK1. GST fusion proteins containing intact and truncated DATN-terminus were immobilized on GSTSepharose and incubated with a solution containing whole RACK1 sequence fused to maltose binding protein. After washing, GST fusion proteins were eluted with glutathione, resolved on 12% PAG and immunobloted with antibodies against maltose binding protein.
4. Discussion
Fig. 2. Comparison of uptake efficiency and surface expression of DATWT and DATD1 in HEK293T cells. Intact DATWT and truncated DATD1 expressed on the surface of HEK293T stable cell lines were labeled with biotin. After cell lysis, biotin labeled surface localized transporters were recovered on streptavidin agarose from total protein extracts and loaded on 7.5% PAG. Resolved proteins were transferred to immobilon and probed with antibodies against the C-terminal region of dopamine transporter (A). In the same sample sets, efficiency of uptake was determined in the presence of various concentrations of 3H-labeled dopamine for 5 min at 37 8C (B).
3.4. Binding of RACK1 to intact and truncated DAT It has been shown previously that the DATN-terminal region interacts with RACK1 (Lee et al., 2004), but the precise site of interaction was not mapped. To see if calpain truncation eliminates interaction with RACK1, we incubated immobilized GST fusion proteins containing intact and truncated DATN-terminal regions with pMAL fusion protein containing full-length RACK1. As shown in Fig. 4, N-terminal truncation of DAT with calpain abolishes its interaction with RACK1.
Fig. 3. Truncation of DAT in rat striatal synaptosomes. Crude rat striatal synaptosomes were lysed in 25 mM Tris–HCl pH 7.5 containing either 5 mM EGTA 5 mM CaCl2 or 5 mM CaCl2 in the presence of calpain inhibitor I. After 15 min incubation at 37 8C, equal aliquots of the synaptosomes were mixed with 2 SDS buffer, boiled, and the proteins were resolved on 7.5% PAG and immunobloted with anti-DATN (1–43).
Even though calpain degrades many cellular and cytoskeletal proteins and processes many hormones and enzymes (Carafoli and Molinari, 1998), its physiological function is largely unknown and complex. Increased calpain activation is, except for its normal cellular function, thought to be involved in the pathophysiology of degenerative diseases (Dufty et al., 2007; Higuchi et al., 2005). In brain disorders such as Parkinson’s disease or Tourette’s syndrome, dopamine homeostasis is significantly altered. There are several evidences that calpain plays a role in the modulation or deterioration of brain dopaminergic function (Yurko-Mauro and Friedman, 1996; Trifiletti and Bandele, 2000; Kuhn et al., 2003; Crocker et al., 2003; Merali and Park, 2003). Calpain cleavage site specificity is coded by a high-ordered structure and thus it is not well conserved at the amino acid sequence level. The size of the adjacent region influencing calpain cleavage has been studied previously (Tompa et al., 2004). In the recombinant N-terminal cytoplasmic domain of DAT fused to GST, we found two calpain cleavage sites with significantly different affinities. The first calpain cleavage site, located close to the middle of the DATN-terminal sequence, likely contains enough DATN amino acid residues adjacent to both sides of the cleaved peptide bond to create calpain cleavage site specificity identical to that existing in vivo. The second cleavage site is located between the third and fourth amino acids of the first transmembrane domain and exhibits much lower affinity in the fusion protein. This cleavage site is, however, very close to the DATN-GST fusion boundary and part of the potential in vivo calpain recognition site has been replaced by GST residues. This means that the second cleavage site could be either artificially created or its affinity could have been reduced by replacement of the reciprocal half of the calpain recognition sequence by the GST protein sequence. In order to verify if both calpain cleavage sites are accessible to calpain cleavage in vivo, we tried to separate affinity purified polyclonal antibody epitopes corresponding to amino acids 1–43 and 44–72, recognizing the DATN region between two calpain cleavage sites. Later epitopes were however not strong enough to give satisfactory results and at present we cannot determine if the second calpain cleavage site in DATN-terminus is accessible for calpain in vivo. Rat striatal synaptosomes lysed in the presence of calcium however exhibited a clear decrease of immunoreactivity epitopes 1–43, which indicates that under conditions of hypoosmotic high calcium lysis, this calpain cleavage site is accessible. Transporters are distributed between the inner cellular compartment and the membrane. Only membrane localized transporters are transport competent. To correlate measured uptake of wild type and truncated dopamine transporters with the membrane localized transporters in HEK293T cells, we estimated the relative amount of
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DATs on cell membrane using surface biotinylation labeling. In biotinylated fraction, we observed significant amount of DAT immunoreactivity in the position corresponding to DAT dimers. Relative abundance of these dimers is the same in wild type as well as truncated DAT samples. Since these dimers are resistant to boiling in SDS sample buffer, we consider it as unspecific aggregation. On average, its truncation results in about the same level of surface expression of DAT with a similar maximal velocity of transport and affinity constant in HEK293T cells. On the other hand, HEK293T cells do not express all components of the DAT signaling machinery. Further detailed studies will be necessary in a natural dopaminergic cellular environment. The N-terminal region of DAT removed by calpain contains the cluster of three lysines previously implicated in dramatic inhibition of PKC-dependent ubiquitylation and endocytosis of DAT, resulting in a severe delay in DAT degradation (Miranda et al., 2007). DAT in PKC activated cells was less efficiently sorted to the lysosomal degradation pathway and was instead recycled back to the cell surface. In this case calpain truncation would have a profound effect on DAT trafficking. In our preliminary experiments in HEK293T cells, however, we observed a similar extent of PKC induced internalization of both wild type and truncated DAT when stimulated with PMA. These results are in agreement with a previous report (Sorkina et al., 2005) indicating that acute PMA induced endocytosis is not abolished even if the entire DATNterminal region is replaced with GFP. Recently, the direct association of DATN-terminal sequences with the presynaptic dopamine D2 receptor was described (Lee et al., 2007). Since calpain cleavage of the DATN-terminus also removes residues responsible for this interaction, regulation of DAT via presynaptic D2 receptor could also be altered by Nterminal truncation of DAT. So far we do not know if specific pathways exist, which could selectively activate calpain in dopaminergic terminals close to DAT, leading to its N-terminal truncation. However one situation in which such a process is quite possible is calcium overload and calpain activation under pathological conditions. Recently, Liu et al. (2007) showed that calcium influx through L-type channels in dopamine ventral tegmental neurons activates a calcium-dependent protease that cleaves PKC to generate constitutively active and labile PKM resulting in burst firing of dopamine cells, a pathway that is involved in glutamatergic or cholinergic modulation of the central dopamine system. It has also been previously shown that prolonged treatment of cultured hippocampal slices with selected ampakines leads to spectrin degradation mediated by activation of the calciumdependent protease calpain (Jourdi et al., 2005). Similarly, methamphetamine induces spectrin proteolysis in the rat striatum (Staszewski and Yamamoto, 2006). Methamphetamine increased calpain-mediated spectrin proteolysis in the rat striatum 5 and 7 days after METH administration without affecting caspase 3dependent spectrin breakdown. This effect was completely blocked by an AMPA receptor antagonist, but not by an NMDA receptor antagonist. However, antibodies used to monitor changes of DAT were directed against the C-terminal transporter region and could not have detected possible calpain mediated truncation of the N-terminal region of DAT. Interestingly, Trifiletti and Bandele (2000) investigated a group of patients with Tics, Tourette’s syndrome and obsessive– compulsive disorder associated with streptococcal infection. They have found that in eighty percent of patient’s, molecular mimicry of streptococcal antigens induced antibody epitopes directed against calpain and calpastatin. This interesting coincidence indicates that calpain-calpastatin play important roles in balancing striatal dopamine homeostasis and that the potential calpain
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