266
Brain Research, 584 (1992) 266-271 (('; 1992 Elsevier Science Publishers B.V. All rights reserved 0006-8993/92/$05.111~
BRES 17912
Microheterogeneity of dopamine transporters in rat striatum and nucleus accumbens Robert Lew *, Amrat Patel, Roxanne A. Vaughan, Alan Wilson
**
and Michael J. Kuhar
Neuroscience Branch, NIDA Addiction Research Center, Baltimore, MD 21224 (USA) (Accepted 25 February 1992)
Key words: Dopamine transporter; Striatum; Nucleus accumbens; [I25I]DEEP; Transporter heterogeneity
Previously we have shown that the [125I]DEEP-labeled dopamine transporter from the rat nucleus accumbens has a higher apparent molecular weight than that from striatum. The present study confirms and extends these observations 24. Experiments with nucleus accumbens showed [125II-DEEP to specifically bind to a protein with an apparent molecular weight of 76 kDa and with the pharmacological properties of the dopamine transporter. In exoglycosidase studies, treatment with neuraminidase, but not alpha-mannosidase, reduced the apparent molecular weight of the dopamine transporter from both the striatum and nucleus accumbens; however, a difference in the apparent molecular weight was still observed. N-Glycanase treatment, on the other hand, did reduce the apparent molecular weight of the dopamine transporters from the two regions to a similar value, approximately 56 kDa. In radioligand binding studies examining the effect of partial deglycosylation on striatal dopamine transporters, neuraminidase did not affect specific [3H]WIN 35,428 binding at 4 and 40 nM concentrations. In conclusion, the present study demonstrates that the difference in the apparent molecular weight of the dopamine transporter from these two regions is due to a difference in glycosylation and that the dopamine transporter from both regions contains similar amounts of sialic acid in their carbohydrate structure. Furthermore, the present data also indicate that the polypeptide portion of the dopamine transporter from both regions could be the same gene product.
INTRODUCTION The dopamine transporter is responsible for terminating the action of neuronally released dopamine by transporting it back from the synaptic cleft into presynaptic nerve terminals. During the last 2 decades, the biochemical and pharmacological characteristics of the t r a n s p o r t e r have b e e n extensively investigated 13-15'2°'2t'3°'34. In particular, recent studies show that the binding site for several selective radioligands for the dopamine transporter is the binding site for the reinforcing properties of cocaine in drug self-administration studies 2'31. Recently, some of the molecular characteristics of the dopamine transporter have been determined using irreversible photo-affinity probes 1A2'23'24'32. Studies show that the striatal dopamine transporter is an N* Present address: Department of Pharmacology and Physiology, University of Chicago, 947 East 58th Street, Chicago, IL 60637, USA. ** Present address: Clarke Institute of Psychiatry, 250 College Street, Toronto, Ont. M5T1R8, Canada. Correspondence: M.J. Kuhar, Neuroscience Branch, NIDA Addiction Research Center, PO Box 5180, Baltimore, MD 21224, USA. Fax: (1)(4101550-1645.
linked glycoprotein rich in sialic acid and N-acetylglucosamine 1'23'32, with an apparent molecular weight of between 60 kDa 12'32 and 80 kDa 1. More recent studies show that the apparent molecular weight of the transporter from the nucleus accumbens is slightly greater than that from striatum 23'24. This difference could be due to differences in the polypeptide portion of the transporter; thus there may be multiple dopamine transporters. Another possible explanation is that it may be due to post-translational modifications such as differences in glycosylation (microheterogeneity) of the transporter. Microheterogeneity has been observed in several systems including the flz-adrenoceptor 35, the corticotrophin releasing factor (CRF) receptorl°'lt and the dopamine D 1 and D 2 receptors t7'18. This difference in the apparent molecular weight of the transporter from the striatum and nucleus accumbens may thus have important functional ramifications. In order to address the question of microheterogeneity, the present study examines the effects of various exo- and endo-glycosidases on [125I]DEEP-labeled dopamine transporters from the striatum and nucleus accumbens, and also the effect of partial deglycosylation on [3H]WIN 35,428 binding.
267 MATERIALS AND METHODS Membrane preparation Male Sprague-Dawley rats (200-300 g) were sacrificed by decapitation and the brains rapidly excised on ice. Striata and nucleus accumbens were removed by microdissection, frozen on dry ice and stored at - 70°C until needed. On the day of experiment membrane homogenates of striatum and nucleus accumbens were prepared as previously described 12'23. Briefly, tissues were thawed on ice and homogenized with a Brinkmann Polytron (setting 6, 30 s) in incubation buffer consisting of 50 mM Tris-HCl, pH 7.4, 4°C, 120 mM NaC1, 5 mM KCI, 1 mM phenylmethylsulfonylfluoride (PMSF) and 1 m g / m l leupeptin. Homogenates were centrifuged at 40,000× g at 4°C for 10 min. After centrifugation the supernatants were discarded and the pellets resuspended and re-centrifuged as described above. Pellets were finally resuspended in the above incubation buffer to yield stock concentrations of 20 and 80 m g / m l original wet weight of striatum and nucleus accumbens, respectively.
Covalent photo-affinity binding with [125I]DEEP to membrane homogenates [ 125 I] 1-[2-(diphenylmethoxy)ethyl]-4-[2-(4-azido-3-iodophenyl)]piperazine (DEEP) was prepared as previously described 36. Aliquots (100/~l) of striatum or nucleus accumbens homogenates were added to tubes containing [12SI]DEEP (1-2 nM) and incubation buffer to give a total volume of 1 ml. Binding was allowed to proceed for 60 rain at 4°C in the dark. Non-specific binding was defined by including 10 /xM mazindol in some samples. Following incubation samples were transferred to petri dishes and irradiated with UV light (wavelength, 280 nM) for 40 s (path length, 3-4 mm) to covalently attach DEEP to the dopamine transporter. Afterwards the photolabeled samples were centrifuged at 12,000× g for 10 min and the supernatant discarded. Pellets were washed with incubation buffer (1 ml) and centrifuged as described above before processing for SDS-PAGE electrophoresis.
SDS-polyacrylamide electrophoresis [12SI]DEEP-labeled samples of striatum (2 mg original wet weight) and nucleus accumbens (8 mg original wet weight) were resuspended in 100 /zl of SDS-sampling buffer (50 mM Tris-HCl, 2% sodium dodecyl sulphate, 10% glycerol, 5%/3-mercaptoethanol and 0.005% Bromphenol blue), pH 6.8, and agitated for 60 min. Afterwards, samples were loaded onto a discontinuous slab gel (6% stacking and 10% running gel) and run overnight at a constant current of 10-20 mA 2z. Pre-stained molecular weight standards (Amersham rainbow markers) were included in each gel to determine the apparent molecular weight of specifically labeled proteins. Following electrophoresis, gels were dried on a Bio-Rad gel dryer and apposed to Kodak X-Omatic film and exposed for 3-5 h at -70°C for gels loaded with photolabeled crude membranes, or 3-5 days for gels loaded with electroeluted samples.
Electroelution of DEEP-labeled dopamine transportersfrom SDS-polyacrylamide gels Following autoradiography the dried gel was matched with the autoradiogram and regions corresponding to specifically DEEPlabeled dopamine transporters were excised. Gel pieces were hydrated and placed in a Bio-Rad electroeluter (model 422). Dopamine transporters were electroeluted at a constant current of 10 m A / t u b e for 4-6 h in a buffer consisting of 25 mM Tris, 192 mM glycine, and 0.1% SDS, pH 6.8. After electroelution the samples were measured for radioactivity, and recovery was estimated to be 70-85%. Electroeluted samples of the dopamine transporter from striatum and nucleus accumbens were then subjected to either exo- or endoglycosidase treatment.
Exoglycosidase treatment Electroeluted samples of the dopamine transporter from striatum (20/~1) and nucleus accumbens (40 ~1) were adjusted to contain 100 mM sodium acetate, pH 5.0, 0.1 mM PMSF and 0.21% CHAPS in
the absence and presence of neuraminidase (2 U / m l ) to give a total volume of 120 /zl. Incubation was performed at 37°C for 60 min. Following incubation SDS sample buffer (90/zl) was added t o e a c h sample and the mixture was subjected to gel electrophoresis as described above. In alpha-mannosidase studies electroeluted samples of the dopamine transporter from striatum (20/~1) and nucleus accumbens (40/.tl) were adjusted to contain 0.1 mM PMSF and 0.21% CHAPS in the absence and presence of alpha-mannosidase (12 U / m l ) to give a total volume of 120 ~1. Incubation was performed at 22-25°C for 16-18 h. After incubation SDS sample buffer (90/zl) was added to each sample and the mixture was subjected to gel electrophoresis as described above.
Endoglycosidase treatment In N-glycanase studies electroeluted samples from striatum (20 /~1) and nucleus accumbens (40/~1) were adjusted to contain 100 mM sodium phosphate, pH 8.1, 0.21% CHAPS and 25 mM EDTA in the absence and presence of N-glycanase (60 U / m l ) to give a total volume of 120 /zl. Samples were incubated for 16-18 h at 37°C. Following treatment, SDS sample buffer 90 /zl) was added to each sample and the mixture subjected to gel electrophoresis as described above.
Effect of neuraminidase treatment on [ 3H]WIN 35,428 binding Tissue preparation. To assess the role of glycosylation on [3H]WlN 35,428 binding rat striatal homogenates were prepared as described above using 100 mM sodium acetate buffer, pH 7.4, 25°C and then treated in the absence (control) and presence of neuraminidase (2 U / m l ) for 30 min, 37°C. Control and treated striatal homogenates were then washed twice by centrifugation (40,000× g for 10 min, 4°C) using 0.32 M sucrose/10 mM Na2HPO 4 buffer, pH 7.4, 4°C, before finally resuspending control and treated pellets to a tissue concentration of 10 m g / m i original wet weight. [3H]WIN 35,428 binding assay. [3H]WlN 35,428 binding was performed as previously described 5. Aliquots (100 /.tl) of control or treated striatal homogenates were added to tubes containing [3H]WlN 35,428 (4 or 40 nM) and incubation buffer (0.32 M sucrose/10 mM Na2HPO4, pH 7.4, 4°C) to give a total volume of 0.5 ml. Binding was allowed to proceed for 120 min at 4°C. To some tubes 30/xM (-)cocaine was added to determine non-specific binding. Incubation was terminated by filtration through Whatmann G F / B filters previously soaked in 0.05% PEI (polyethylenimine) followed by 3 × 5 /xl washes with the above incubation buffer. Radioactivity was determined using a Beckman LS 3801 scintillation counter (45-50% efficiency).
Materials [125I]DEEP (spec. act. 1400-1800 Ci/mmol) was prepared as previously described (Wilson et al.36). [3H]WIN 35,428 (spec. act 82.7 Ci/mmol) was obtained from New England Nuclear (Boston, MA). Mazindol was obtained from Sandoz (NJ). Neuraminidase and alpha-mannosidase were obtained from Sigma (St. Louis, MO) and N-glycanase was from Genzyme (Boston, MA). Rainbow molecular weight markers were from Amersham (Arlington Hgts, IL).
RESULTS
We have previously demonstrated that specific [~25I]DEEP binding in rat striatum has the pharmacological properties of binding to membrane preparations of the dopamine transporter 12. In Fig. 1, specific [~2SI]DEEP binding in the rat nucleus accumbens was also observed to have the pharmacological properties of the dopamine transporter. Mazindol, GBR 12909, (-)cocaine and nomifensine (at 10 /xM concentra-
268 tions) blocked [125I]DEEP binding to a protein with an apparent molecular weight of about 76,000 Da. Desipramine (norepinephrine uptake inhibitor) and citalopram (serotonin uptake inhibitor) did not significantly inhibit [125I]DEEP incorporation to this protein. Furthermore, the (+)isomer of cocaine (10 /xM), did not inhibit DEEP binding to this 76,000 Da protein, thus demonstrating stereoselectivity. Consequently, no pharmacological differences were observed between the dopamine transporters from the striatum and nucleus accumbens. It can be seen in Fig. 1 that [t25I]DEEP labels multiple proteins in rat striatum and nucleus accumbens, although only the band at 70,000-80,000 Da displays the appropriate pharmacology. Because these non-specifically labeled bands could interfere with subsequent analytical techniques transporter samples were subjected to electroelution after SDS-PAGE. When re-electrophoresed on SDS gels electroeluted transporters from each brain region co-migrated with the same apparent M r as transporters from SDS-solubilized membranes i.e. electroelution had no apparent effect on the molecular weight of transporters from either brain region (not shown). Electroeluted samples were thus free of radioactive contaminants and could be analyzed with a greater degree of confidence. In order to determine if differential glycosylation of dopamine transporters was the source of the molecular
Fig. 1. Pharmacological characterization of [lzSI]DEEP binding to membranes from the nucleus accumbens. Experiments were carried out as described in Materials and Methods. Mazindol, GBR 12909, nomifensine and ( - )cocaine, which block dopamine transport, inhibited [125I]DEEP binding to a protein with an apparent molecular weight of 76,000 Da, while citalopram, desipramine and (+)cocaine, which do not inhibit dopamine transport, did not prevent [125I]DEEP attachment to this protein.
Mr l 200
97 68
45
30
STR
NA
STR
NA
Fig. 2. Effect of neuraminidase (NEURAMIN) treatment on the [lasI]DEEP-labeled dopamine transporter from rat striatum (STR) and nucleus accumbens (NA). Treatment with neuraminidase reduced the apparent molecular weight of the transporter from both brain regions but did not eliminate the difference in molecular weight between the two brain regions thus suggesting that the transporter from the two regions have the similar sialic acid contents. Experiments were repeated four times with the same results.
weight heterogeneity three deglycosylases with differing specificities were used. Figure 2 shows the effect of neuraminidase, which cleaves terminal sialic acid residues 35. In control samples (no enzyme added) the molecular weight difference between the striatal transporter and the nucleus accumbens transporter is observed (Fig. 2, lanes 1 and 2). Treatment with neuraminidase reduced the apparent molecular weight of the dopamine transporter from both brain regions by approximately 8 kDa (Fig. 2, lanes 3 and 4). However, the apparent molecular weight of the dopamine transporter from the nucleus accumbens after neuraminidase treatment was still greater than that from the striatum. Thus, it appears that the dopamine transporter from the nucleus accumbens and striatum have simliar sialic acid content. In order to determine if the neuraminidase reaction had gone to completion further experiments were performed in which samples were given additional neuraminidase (2 U / m l ) after the first incubation. This additional treatment had no further effect on the apparent molecular weight change of the transporter from either region (data not shown); thus deglycosylation by neuraminidase had gone to completion in the first reaction. Previous studies have demonstrated that the dopamine transporter from rat striatum is devoid of mannose sugar residues 23'32. The possibility of mannose as a component of the carbohydrate structure of the dopamine transporter from the nucleus accumbens
269 could explain the apparent molecular weight difference. However as shown in Fig. 3, treatment with alpha-manosidase (12 U / m l ) did not have any effect on the apparent molecular weight of the dopamine transporter from either brain region, indicating that mannose is not present to a significant degree in the transporter from the nucleus accumbens or striatum as previously observed 23'32. T r e a t m e n t of the [125I]DEEP-labeled dopamine transporter from both brain regions with N-glycanase (which cleaves the whole carbohydrate structure from the polypeptide at N groups on asparagine residues 8'28) substantially reduced the apparent molecular weight of the transporter from both brain regions to about the same value of 56 kDa (Fig. 4). Thus, the difference in molecular weight between the two brain regions was apparently eliminated by N-glycanase treatment. This result was observed in each of four separate experiments. When samples were given an additional amount of N-glycanase at the end of the incubation period there was no further reduction in molecular weight and the results were the same as described above (data not shown). Effect o f neuraminidase treatment on [ 3 H ] W I N 35,428 binding in rat striatum To determine the effect of partial deglycosylation on [3H]WlN 35,428 binding to the dopamine transporter rat striatal homogenates were p r e p a r e d and treated with neuraminidase (2 U / m l ) as described in Materials and Methods. [3H]WlN 35,428 binding to predomi-
MI
TABLE I Effect of neuraminidase (2 U / ml) on [3H]WIN 35,428 binding to rat striatal membranes Rat striatal membranes (2 mg wet weight) in 0.1 /zl of 0.1 M sodium acetate buffer, pH 7.4, and 0.1 mM PMSF were incubated at 37°C in the presence or absence of neuraminidase (2 U/ml) for 30 min. Membranes were collected and resuspended in 0.32 M sucrose/10 mM Na2HPO4 buffer, pH 7.4, at 10 mg/ml. [3H]WIN 35,428 (4 and 40 nM) binding was conducted on ice for 2 h in triplicate in the absence and presence of 30/xM ( - )cocaine to determine non-specific binding. Values are mean + S.E.M.
Control Neuraminidase
[ 3H]WIN 35, 428 specifically bound (pmol / m g protein) 4 nM 40 nM 0.28 + 0.02 1.29 + 0.09 0.26 + 0.02 1.22 + 0.05
nantly high or low affinity binding sites on the dopamine transporter 5'25 was performed by incubating control and treated tissues with [3H]WlN 35,428 at 4 or 40 nM concentrations respectively. Table I shows that at both concentrations specific binding (as expressed in p m o l / m g protein) was not affected by neuraminidase treatment compared to controls. To ensure that neuraminidase treatment under the above conditions was complete, control and treated striatal homogenates were photo-labeled with [125I]DEEP and subjected to S D S - P A G E electrophoresis and autoradiography. Autoradiograms (not shown) revealed that neuraminidase treatment under the present conditions reduced the apparent molecular weight of the striatal dopamine transporter to the same apparent molecular size as previously observed 23. Thus it would appear that at least sialic acids are not required for [3H]WlN 35,428 to bind to the dopamine transporter. DISCUSSION
STR N A STR N A
Fig. 3. Effect of alpha-mannosidase treatment on [l~i]DEEP.labeled dopamine transporter. Alpha-mannosidase had no effect on the molecular weight of the transporter from either brain region. Experiments were carried out four times and yielded the same results.
Results of this study confirm earlier observations that the transporter from the striatum is a glycoprotein with an N-linked carbohydrate enriched in sialic acid a n d / o r N-acetylglucosamine but not in mannose 1,a3,3z. Additionally, this study shows for the first time that the transporter from the nucleus accumbens is also a glycoprotein with similar properties. Studies by Missale et al. a6 have reported that dopamine uptake in striatum is different from that in the nucleus accumbens. However, in the present study no pharmacological differences were observed between the dopamine transporters from striatum and nucleus accumbens, at least in so far as [125I]DEEP binding shows. This latter result is in agreement with several other studies which show that the dopamine transporters in the striatum and nucleus accumbens have
270 similar [3H]cocaine binding characteristics and [3H] dopamine uptake properties 6'1~. A striking finding was that the difference in apparent molecular weight of the transporter in the two brain regions was eliminated by treatment with Nglycanase but not with neuraminidase or alpha-mannosidase. This suggests that the carbohydrate structure is the major source of the apparent size heterogeneity and that the sialic acid component of the transporters in both regions are similar. The exact nature of the difference of the carbohydrate moieties is yet to be determined. Furthermore, it is therefore possible that the two proteins are the same gene product. Recent studies indicate that a cDNA for a single protein from rat midbrain libraries can confer dopamine uptake, as well as cocaine binding, in transfected cells 7'19'33. This protein has several potential glycosylation sites on a proposed extracellular loop between transmembrane regions three and four. The results shown here and elsewhere 12'23'32, that glycosylation of dopamine transporters occurs in brain, are consistent with results of these recent cloning experiments. Furthermore, data in this communication indicate that glycosylation patterns for the dopamine transporter are different in different brain regions. Data from the cloning experiments suggest a M r of about 69,000 for the deglycosylated transporter, which is higher than is suggested here (Fig. 4, approximately 56,000). This discrepancy may be at least partly acMr
STR
N A STR
NA
Fig. 4. Effect of N-glycanase on [125I]DEEP-labeled dopamine transporter from striatum (Str) and nucleus accumbens (Na). Treatment with N-glycanase reduced the apparent molecular weight of the transporter from both brain regions (compared to control samples incubated in the absence of N-glycanase) and eliminated the difference in apparent molecular weight. Experiments were carried out four times with essentially the same results.
counted for by the behavior of this protein in SDSP A G E gels. The higher the acrylamide content of the gels, the higher the apparent molecular weight of the transporter (R. Vaughan, unpublished observations with glycosylated protein). In this study, an acrylamide content of 10% was selected because it reliably resolved the apparent M r difference between the two brain regions. There are many known cases of different glycosylation patterns exhibited by identical gene products 29. In particular, Stiles et alY, have reported /32-adrenoce ptors in hamster lung and rat erythrocytes are either high mannose- or complex-type glycoproteins. Furthermore, the CRF receptor in rat brain and anterior pituitary have apparent molecular weights of 58 and 75 kDa, respectively, and this difference is due to different glycosylation patterns 1°'11. Similar observations have also been reported for the DI and D 2 dopamine receptors~7, TM. A variety of functions have been described for the carbohydrate portion of glycoproteins 27. Several studies show glycosylation to be essential for protein translocation. The non-glycosylated form of the G-protein in vesicular stomatitis virus is expressed in tunicamycin-treated cells but is not transported to the cell surface compared to the glycosylated form 9. Glycosylation may also be involved in the functionality of cell surface receptors. In tunicamycin-treated A431 ceils the non-glycosylated /32-adrenoceptor exhibits similar binding affinity and number of sites compared to untreated cells. However, there is a loss in adenylate cyclase activity, which is due to a coupling defect at the receptor level, since other components of the signal transmission chain remained functional 4. In contrast, the non-glycosylated /3t-adrenoceptor from turkey erythrocytes exhibits enhanced adenylate cyclase activity compared to glycosylated /3~-adrenoceptor 3. In the present study removal of sialic acids from striatal dopamine transporters by neuraminidase treatment did not affect [3H]WIN 35,428 binding to the dopamine transporter at either 4 or 40 nM, respectively. This would suggest that sialic acids are not involved in cocaine binding to the dopamine transporter. Zaleska and Erecinska 3v have reported neuraminidase treatment of rat brain synaptosomes to reduce dopamine uptake (Vmax) without affecting the affinity (K m) of dopamine for the transporter. Since affinity was not affected this would support the present observation that sialic acids are not involved in the binding of dopamine to the transporter. However, since dopamine uptake (Vmax) was reduced by removal of sialic acid, it is possible that sialic acids are involved in the chain of events that occurs following binding of dopamine to
271 the transporter. Indeed, in several transport systems it has been suggested that terminal sialic acids may either form part of the binding site for Na ÷ or they are involved in the active conformation of the carrier protein that binds Na + 38. In summary, the present study demonstrates that the apparent heterogeniety of the dopamine transporter in rat striatum and nucleus accumbens is due to differences in glycosylation patterns and that the polypeptide portion of the dopamine transporter in both regions have similar apparent molecular weights.
17
18 19 20 21
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striatum, nucleus accumbens, olfactory tubercle and medial prefrontal cortex, Brain Res., 520 (1990) 303-309. Jarvie, K.R., Niznik, H.B. and Seeman, P., Dopamine D2~receptor binding subunits of M r = 140,000 and 94,000 in brain: deglycosylation yields a common unit of M r = 44,000, Mol. Pharmacol., 34 (1988) 91-97. Jarvie, K.R., Booth, G., Brown, E.M. and Niznik, H.B., Glycoprorein nature of dopamine D I receptors in the brain and parathyroid gland, Mol. Pharmacol., 36 (1989) 566-574. Kilty, J.E., Lorang, D. and Amara, S.G., Cloning and expression of a cocaine-sensitive rat dopamine transporter, Science, 254 (1991) 578-579. Kuhar, M.J., Neurotransmitter uptake: a tool in identifying neurotransmitter-specific pathways, Life Sci., 13 (1973) 1623-1634. Kuhar, M.J., Ritz, M.C. and Boja, J.W., The dopamine hypothesis of the reinforcing properties of cocaine, Trends Neurosci., 14 (1991) 299-302. Laemmli, U.K., Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature, 227 (1970) 680-685. Lew, R., Grigoriadis, D.E., Wilson, A., Boja, J.W., Simantov, R. and Kuhar, M.J., Dopamine transporter: deglycosylation with exo- and endoglycosidases, Brain Res., 539 (1991) 239-246. Lew, R., Vaughan, R., Simantov, R., Wilson, A. and Kuhar, M.J., Dopamine transporters in the nucleus accumbens and the striatum have different apparent molecular weights, Synapse, 8 (1991) 152-153. Madras, B.K., Spealman, R.D., Fahey, M.A., Neumeyer, J.L., Saha, J.K. and Milius, R.A., Cocaine receptors labelled by [3H]/3-carbomethoxy-3/3-(4-flurophenyl)tropane, Mol. Pharmacol., 36 (1989) 518-524. Missale, C., Castelletti, L., Govoni, S., Spano, P.F., Trabuchi, M. and Hanbauer, I., Dopamine uptake is differentially regulated in striatum and nucleus accumbens, J. Neurochem., 45 (1985) 51-56. Paulson, J.C., Glycoproteins: what are the sugars for? Trends Biol. Sci., 14 (1989) 272-276. Plummer, T.H., Elder, J.H., Alexander, S., Phelan, A.W. and Tarentino, A.L., Demonstration of peptide: N-glycosidase F activity in endo-/3-N-acetylglucosaminidase F preparation, J. Biol. Chem., 259 (1984) 10700-10704. Rademacher, T.W., Parekh, R.B. and Dwek, R.A., Glycobiology, Annu. Rev. Biochem., 57 (1988) 785-835. Raiteri, M., Cerrito, F., Cervoni, A.M., Del Carmino, R., Ribera, M.T. and Levi, G., Studies on dopamine uptake and release in synaptosomes, Adv. Biochem. Psychopharmacol., 19 (1978) 35-56. Ritz, M.C., Lamb, R.J., Goidberg, S.R: and Kuhar, M.J., Cocaine receptors on dopamine transporters are related to self-administration of cocaine, Science, 237 (1987) 1219-1223. Sallee, F.R., Fogel, E.L., Schwartz, E., Choi, S.M., Curran, DIP. and Niznik, H.B., Photoaffinity labeling of the mammalian dopamine transporter, FEBS Lett., 256 (1989) 219-224. Shimada, S., Kitayama, S., Lin, C-L., Patel, A., Nanthakumar, E., Gregor, P., Kuhar, M. and Uhl, G., Cloning and expression of a cocaine-sensitive dopamine transporter complementary DNA, Science, 254 (1991) 576-578. Snyder, S., Putative neurotransmitter in the brain: selective neuronal uptake, subcellular localization and interactions with centrally acting drugs, Biol. Psychiatry, 2 (1970) 367-389. Stiles, G.L., Benovic, J.L. Caron, M.G. and Lefkowitz, R.J., Mammalian/3-adrenergic receptors: distinct glycoprotein populations containing high mannose- or complex-type carbohydrate chains, J. Biol. Chem., 259 (1984) 8655-8663. Wilson, A.A., Grigoriadis, D.E., Dannals, R.F., Ravert, H.T. and Wagner Jr., H.N., A 1-pot radiosynthesis of [125I]iodazido photoaffinity label, J. Labelled Comp. Radiopharm., 27 (1989) 12991305. Zaleska, M.M. and Erecinska, M., Involvement of sialic acid in high-affinity uptake of dopamine by synaptosomes from rat brain, Neurosci. Lett., 82 (1987) 107-112. Zaleska, M.M. and Erecinska, M., Role of sialic acid in synaptosomal transport of amino acid transmitters, Proc. Natl. Acad. Sci. USA, 84 (1987) 1709-1712.
Brain Research, 584 (1992) 266-271 (('; 1992 Elsevier Science Publishers B.V. All rights reserved 0006-8993/92/$05.111~
BRES 17912
Microheterogeneity of dopamine transporters in rat striatum and nucleus accumbens Robert Lew *, Amrat Patel, Roxanne A. Vaughan, Alan Wilson
**
and Michael J. Kuhar
Neuroscience Branch, NIDA Addiction Research Center, Baltimore, MD 21224 (USA) (Accepted 25 February 1992)
Key words: Dopamine transporter; Striatum; Nucleus accumbens; [I25I]DEEP; Transporter heterogeneity
Previously we have shown that the [125I]DEEP-labeled dopamine transporter from the rat nucleus accumbens has a higher apparent molecular weight than that from striatum. The present study confirms and extends these observations 24. Experiments with nucleus accumbens showed [125II-DEEP to specifically bind to a protein with an apparent molecular weight of 76 kDa and with the pharmacological properties of the dopamine transporter. In exoglycosidase studies, treatment with neuraminidase, but not alpha-mannosidase, reduced the apparent molecular weight of the dopamine transporter from both the striatum and nucleus accumbens; however, a difference in the apparent molecular weight was still observed. N-Glycanase treatment, on the other hand, did reduce the apparent molecular weight of the dopamine transporters from the two regions to a similar value, approximately 56 kDa. In radioligand binding studies examining the effect of partial deglycosylation on striatal dopamine transporters, neuraminidase did not affect specific [3H]WIN 35,428 binding at 4 and 40 nM concentrations. In conclusion, the present study demonstrates that the difference in the apparent molecular weight of the dopamine transporter from these two regions is due to a difference in glycosylation and that the dopamine transporter from both regions contains similar amounts of sialic acid in their carbohydrate structure. Furthermore, the present data also indicate that the polypeptide portion of the dopamine transporter from both regions could be the same gene product.
INTRODUCTION The dopamine transporter is responsible for terminating the action of neuronally released dopamine by transporting it back from the synaptic cleft into presynaptic nerve terminals. During the last 2 decades, the biochemical and pharmacological characteristics of the t r a n s p o r t e r have b e e n extensively investigated 13-15'2°'2t'3°'34. In particular, recent studies show that the binding site for several selective radioligands for the dopamine transporter is the binding site for the reinforcing properties of cocaine in drug self-administration studies 2'31. Recently, some of the molecular characteristics of the dopamine transporter have been determined using irreversible photo-affinity probes 1A2'23'24'32. Studies show that the striatal dopamine transporter is an N* Present address: Department of Pharmacology and Physiology, University of Chicago, 947 East 58th Street, Chicago, IL 60637, USA. ** Present address: Clarke Institute of Psychiatry, 250 College Street, Toronto, Ont. M5T1R8, Canada. Correspondence: M.J. Kuhar, Neuroscience Branch, NIDA Addiction Research Center, PO Box 5180, Baltimore, MD 21224, USA. Fax: (1)(4101550-1645.
linked glycoprotein rich in sialic acid and N-acetylglucosamine 1'23'32, with an apparent molecular weight of between 60 kDa 12'32 and 80 kDa 1. More recent studies show that the apparent molecular weight of the transporter from the nucleus accumbens is slightly greater than that from striatum 23'24. This difference could be due to differences in the polypeptide portion of the transporter; thus there may be multiple dopamine transporters. Another possible explanation is that it may be due to post-translational modifications such as differences in glycosylation (microheterogeneity) of the transporter. Microheterogeneity has been observed in several systems including the flz-adrenoceptor 35, the corticotrophin releasing factor (CRF) receptorl°'lt and the dopamine D 1 and D 2 receptors t7'18. This difference in the apparent molecular weight of the transporter from the striatum and nucleus accumbens may thus have important functional ramifications. In order to address the question of microheterogeneity, the present study examines the effects of various exo- and endo-glycosidases on [125I]DEEP-labeled dopamine transporters from the striatum and nucleus accumbens, and also the effect of partial deglycosylation on [3H]WIN 35,428 binding.
267 MATERIALS AND METHODS Membrane preparation Male Sprague-Dawley rats (200-300 g) were sacrificed by decapitation and the brains rapidly excised on ice. Striata and nucleus accumbens were removed by microdissection, frozen on dry ice and stored at - 70°C until needed. On the day of experiment membrane homogenates of striatum and nucleus accumbens were prepared as previously described 12'23. Briefly, tissues were thawed on ice and homogenized with a Brinkmann Polytron (setting 6, 30 s) in incubation buffer consisting of 50 mM Tris-HCl, pH 7.4, 4°C, 120 mM NaC1, 5 mM KCI, 1 mM phenylmethylsulfonylfluoride (PMSF) and 1 m g / m l leupeptin. Homogenates were centrifuged at 40,000× g at 4°C for 10 min. After centrifugation the supernatants were discarded and the pellets resuspended and re-centrifuged as described above. Pellets were finally resuspended in the above incubation buffer to yield stock concentrations of 20 and 80 m g / m l original wet weight of striatum and nucleus accumbens, respectively.
Covalent photo-affinity binding with [125I]DEEP to membrane homogenates [ 125 I] 1-[2-(diphenylmethoxy)ethyl]-4-[2-(4-azido-3-iodophenyl)]piperazine (DEEP) was prepared as previously described 36. Aliquots (100/~l) of striatum or nucleus accumbens homogenates were added to tubes containing [12SI]DEEP (1-2 nM) and incubation buffer to give a total volume of 1 ml. Binding was allowed to proceed for 60 rain at 4°C in the dark. Non-specific binding was defined by including 10 /xM mazindol in some samples. Following incubation samples were transferred to petri dishes and irradiated with UV light (wavelength, 280 nM) for 40 s (path length, 3-4 mm) to covalently attach DEEP to the dopamine transporter. Afterwards the photolabeled samples were centrifuged at 12,000× g for 10 min and the supernatant discarded. Pellets were washed with incubation buffer (1 ml) and centrifuged as described above before processing for SDS-PAGE electrophoresis.
SDS-polyacrylamide electrophoresis [12SI]DEEP-labeled samples of striatum (2 mg original wet weight) and nucleus accumbens (8 mg original wet weight) were resuspended in 100 /zl of SDS-sampling buffer (50 mM Tris-HCl, 2% sodium dodecyl sulphate, 10% glycerol, 5%/3-mercaptoethanol and 0.005% Bromphenol blue), pH 6.8, and agitated for 60 min. Afterwards, samples were loaded onto a discontinuous slab gel (6% stacking and 10% running gel) and run overnight at a constant current of 10-20 mA 2z. Pre-stained molecular weight standards (Amersham rainbow markers) were included in each gel to determine the apparent molecular weight of specifically labeled proteins. Following electrophoresis, gels were dried on a Bio-Rad gel dryer and apposed to Kodak X-Omatic film and exposed for 3-5 h at -70°C for gels loaded with photolabeled crude membranes, or 3-5 days for gels loaded with electroeluted samples.
Electroelution of DEEP-labeled dopamine transportersfrom SDS-polyacrylamide gels Following autoradiography the dried gel was matched with the autoradiogram and regions corresponding to specifically DEEPlabeled dopamine transporters were excised. Gel pieces were hydrated and placed in a Bio-Rad electroeluter (model 422). Dopamine transporters were electroeluted at a constant current of 10 m A / t u b e for 4-6 h in a buffer consisting of 25 mM Tris, 192 mM glycine, and 0.1% SDS, pH 6.8. After electroelution the samples were measured for radioactivity, and recovery was estimated to be 70-85%. Electroeluted samples of the dopamine transporter from striatum and nucleus accumbens were then subjected to either exo- or endoglycosidase treatment.
Exoglycosidase treatment Electroeluted samples of the dopamine transporter from striatum (20/~1) and nucleus accumbens (40 ~1) were adjusted to contain 100 mM sodium acetate, pH 5.0, 0.1 mM PMSF and 0.21% CHAPS in
the absence and presence of neuraminidase (2 U / m l ) to give a total volume of 120 /zl. Incubation was performed at 37°C for 60 min. Following incubation SDS sample buffer (90/zl) was added t o e a c h sample and the mixture was subjected to gel electrophoresis as described above. In alpha-mannosidase studies electroeluted samples of the dopamine transporter from striatum (20/~1) and nucleus accumbens (40/.tl) were adjusted to contain 0.1 mM PMSF and 0.21% CHAPS in the absence and presence of alpha-mannosidase (12 U / m l ) to give a total volume of 120 ~1. Incubation was performed at 22-25°C for 16-18 h. After incubation SDS sample buffer (90/zl) was added to each sample and the mixture was subjected to gel electrophoresis as described above.
Endoglycosidase treatment In N-glycanase studies electroeluted samples from striatum (20 /~1) and nucleus accumbens (40/~1) were adjusted to contain 100 mM sodium phosphate, pH 8.1, 0.21% CHAPS and 25 mM EDTA in the absence and presence of N-glycanase (60 U / m l ) to give a total volume of 120 /zl. Samples were incubated for 16-18 h at 37°C. Following treatment, SDS sample buffer 90 /zl) was added to each sample and the mixture subjected to gel electrophoresis as described above.
Effect of neuraminidase treatment on [ 3H]WIN 35,428 binding Tissue preparation. To assess the role of glycosylation on [3H]WlN 35,428 binding rat striatal homogenates were prepared as described above using 100 mM sodium acetate buffer, pH 7.4, 25°C and then treated in the absence (control) and presence of neuraminidase (2 U / m l ) for 30 min, 37°C. Control and treated striatal homogenates were then washed twice by centrifugation (40,000× g for 10 min, 4°C) using 0.32 M sucrose/10 mM Na2HPO 4 buffer, pH 7.4, 4°C, before finally resuspending control and treated pellets to a tissue concentration of 10 m g / m i original wet weight. [3H]WIN 35,428 binding assay. [3H]WlN 35,428 binding was performed as previously described 5. Aliquots (100 /.tl) of control or treated striatal homogenates were added to tubes containing [3H]WlN 35,428 (4 or 40 nM) and incubation buffer (0.32 M sucrose/10 mM Na2HPO4, pH 7.4, 4°C) to give a total volume of 0.5 ml. Binding was allowed to proceed for 120 min at 4°C. To some tubes 30/xM (-)cocaine was added to determine non-specific binding. Incubation was terminated by filtration through Whatmann G F / B filters previously soaked in 0.05% PEI (polyethylenimine) followed by 3 × 5 /xl washes with the above incubation buffer. Radioactivity was determined using a Beckman LS 3801 scintillation counter (45-50% efficiency).
Materials [125I]DEEP (spec. act. 1400-1800 Ci/mmol) was prepared as previously described (Wilson et al.36). [3H]WIN 35,428 (spec. act 82.7 Ci/mmol) was obtained from New England Nuclear (Boston, MA). Mazindol was obtained from Sandoz (NJ). Neuraminidase and alpha-mannosidase were obtained from Sigma (St. Louis, MO) and N-glycanase was from Genzyme (Boston, MA). Rainbow molecular weight markers were from Amersham (Arlington Hgts, IL).
RESULTS
We have previously demonstrated that specific [~25I]DEEP binding in rat striatum has the pharmacological properties of binding to membrane preparations of the dopamine transporter 12. In Fig. 1, specific [~2SI]DEEP binding in the rat nucleus accumbens was also observed to have the pharmacological properties of the dopamine transporter. Mazindol, GBR 12909, (-)cocaine and nomifensine (at 10 /xM concentra-
268 tions) blocked [125I]DEEP binding to a protein with an apparent molecular weight of about 76,000 Da. Desipramine (norepinephrine uptake inhibitor) and citalopram (serotonin uptake inhibitor) did not significantly inhibit [125I]DEEP incorporation to this protein. Furthermore, the (+)isomer of cocaine (10 /xM), did not inhibit DEEP binding to this 76,000 Da protein, thus demonstrating stereoselectivity. Consequently, no pharmacological differences were observed between the dopamine transporters from the striatum and nucleus accumbens. It can be seen in Fig. 1 that [t25I]DEEP labels multiple proteins in rat striatum and nucleus accumbens, although only the band at 70,000-80,000 Da displays the appropriate pharmacology. Because these non-specifically labeled bands could interfere with subsequent analytical techniques transporter samples were subjected to electroelution after SDS-PAGE. When re-electrophoresed on SDS gels electroeluted transporters from each brain region co-migrated with the same apparent M r as transporters from SDS-solubilized membranes i.e. electroelution had no apparent effect on the molecular weight of transporters from either brain region (not shown). Electroeluted samples were thus free of radioactive contaminants and could be analyzed with a greater degree of confidence. In order to determine if differential glycosylation of dopamine transporters was the source of the molecular
Fig. 1. Pharmacological characterization of [lzSI]DEEP binding to membranes from the nucleus accumbens. Experiments were carried out as described in Materials and Methods. Mazindol, GBR 12909, nomifensine and ( - )cocaine, which block dopamine transport, inhibited [125I]DEEP binding to a protein with an apparent molecular weight of 76,000 Da, while citalopram, desipramine and (+)cocaine, which do not inhibit dopamine transport, did not prevent [125I]DEEP attachment to this protein.
Mr l 200
97 68
45
30
STR
NA
STR
NA
Fig. 2. Effect of neuraminidase (NEURAMIN) treatment on the [lasI]DEEP-labeled dopamine transporter from rat striatum (STR) and nucleus accumbens (NA). Treatment with neuraminidase reduced the apparent molecular weight of the transporter from both brain regions but did not eliminate the difference in molecular weight between the two brain regions thus suggesting that the transporter from the two regions have the similar sialic acid contents. Experiments were repeated four times with the same results.
weight heterogeneity three deglycosylases with differing specificities were used. Figure 2 shows the effect of neuraminidase, which cleaves terminal sialic acid residues 35. In control samples (no enzyme added) the molecular weight difference between the striatal transporter and the nucleus accumbens transporter is observed (Fig. 2, lanes 1 and 2). Treatment with neuraminidase reduced the apparent molecular weight of the dopamine transporter from both brain regions by approximately 8 kDa (Fig. 2, lanes 3 and 4). However, the apparent molecular weight of the dopamine transporter from the nucleus accumbens after neuraminidase treatment was still greater than that from the striatum. Thus, it appears that the dopamine transporter from the nucleus accumbens and striatum have simliar sialic acid content. In order to determine if the neuraminidase reaction had gone to completion further experiments were performed in which samples were given additional neuraminidase (2 U / m l ) after the first incubation. This additional treatment had no further effect on the apparent molecular weight change of the transporter from either region (data not shown); thus deglycosylation by neuraminidase had gone to completion in the first reaction. Previous studies have demonstrated that the dopamine transporter from rat striatum is devoid of mannose sugar residues 23'32. The possibility of mannose as a component of the carbohydrate structure of the dopamine transporter from the nucleus accumbens
269 could explain the apparent molecular weight difference. However as shown in Fig. 3, treatment with alpha-manosidase (12 U / m l ) did not have any effect on the apparent molecular weight of the dopamine transporter from either brain region, indicating that mannose is not present to a significant degree in the transporter from the nucleus accumbens or striatum as previously observed 23'32. T r e a t m e n t of the [125I]DEEP-labeled dopamine transporter from both brain regions with N-glycanase (which cleaves the whole carbohydrate structure from the polypeptide at N groups on asparagine residues 8'28) substantially reduced the apparent molecular weight of the transporter from both brain regions to about the same value of 56 kDa (Fig. 4). Thus, the difference in molecular weight between the two brain regions was apparently eliminated by N-glycanase treatment. This result was observed in each of four separate experiments. When samples were given an additional amount of N-glycanase at the end of the incubation period there was no further reduction in molecular weight and the results were the same as described above (data not shown). Effect o f neuraminidase treatment on [ 3 H ] W I N 35,428 binding in rat striatum To determine the effect of partial deglycosylation on [3H]WlN 35,428 binding to the dopamine transporter rat striatal homogenates were p r e p a r e d and treated with neuraminidase (2 U / m l ) as described in Materials and Methods. [3H]WlN 35,428 binding to predomi-
MI
TABLE I Effect of neuraminidase (2 U / ml) on [3H]WIN 35,428 binding to rat striatal membranes Rat striatal membranes (2 mg wet weight) in 0.1 /zl of 0.1 M sodium acetate buffer, pH 7.4, and 0.1 mM PMSF were incubated at 37°C in the presence or absence of neuraminidase (2 U/ml) for 30 min. Membranes were collected and resuspended in 0.32 M sucrose/10 mM Na2HPO4 buffer, pH 7.4, at 10 mg/ml. [3H]WIN 35,428 (4 and 40 nM) binding was conducted on ice for 2 h in triplicate in the absence and presence of 30/xM ( - )cocaine to determine non-specific binding. Values are mean + S.E.M.
Control Neuraminidase
[ 3H]WIN 35, 428 specifically bound (pmol / m g protein) 4 nM 40 nM 0.28 + 0.02 1.29 + 0.09 0.26 + 0.02 1.22 + 0.05
nantly high or low affinity binding sites on the dopamine transporter 5'25 was performed by incubating control and treated tissues with [3H]WlN 35,428 at 4 or 40 nM concentrations respectively. Table I shows that at both concentrations specific binding (as expressed in p m o l / m g protein) was not affected by neuraminidase treatment compared to controls. To ensure that neuraminidase treatment under the above conditions was complete, control and treated striatal homogenates were photo-labeled with [125I]DEEP and subjected to S D S - P A G E electrophoresis and autoradiography. Autoradiograms (not shown) revealed that neuraminidase treatment under the present conditions reduced the apparent molecular weight of the striatal dopamine transporter to the same apparent molecular size as previously observed 23. Thus it would appear that at least sialic acids are not required for [3H]WlN 35,428 to bind to the dopamine transporter. DISCUSSION
STR N A STR N A
Fig. 3. Effect of alpha-mannosidase treatment on [l~i]DEEP.labeled dopamine transporter. Alpha-mannosidase had no effect on the molecular weight of the transporter from either brain region. Experiments were carried out four times and yielded the same results.
Results of this study confirm earlier observations that the transporter from the striatum is a glycoprotein with an N-linked carbohydrate enriched in sialic acid a n d / o r N-acetylglucosamine but not in mannose 1,a3,3z. Additionally, this study shows for the first time that the transporter from the nucleus accumbens is also a glycoprotein with similar properties. Studies by Missale et al. a6 have reported that dopamine uptake in striatum is different from that in the nucleus accumbens. However, in the present study no pharmacological differences were observed between the dopamine transporters from striatum and nucleus accumbens, at least in so far as [125I]DEEP binding shows. This latter result is in agreement with several other studies which show that the dopamine transporters in the striatum and nucleus accumbens have
270 similar [3H]cocaine binding characteristics and [3H] dopamine uptake properties 6'1~. A striking finding was that the difference in apparent molecular weight of the transporter in the two brain regions was eliminated by treatment with Nglycanase but not with neuraminidase or alpha-mannosidase. This suggests that the carbohydrate structure is the major source of the apparent size heterogeneity and that the sialic acid component of the transporters in both regions are similar. The exact nature of the difference of the carbohydrate moieties is yet to be determined. Furthermore, it is therefore possible that the two proteins are the same gene product. Recent studies indicate that a cDNA for a single protein from rat midbrain libraries can confer dopamine uptake, as well as cocaine binding, in transfected cells 7'19'33. This protein has several potential glycosylation sites on a proposed extracellular loop between transmembrane regions three and four. The results shown here and elsewhere 12'23'32, that glycosylation of dopamine transporters occurs in brain, are consistent with results of these recent cloning experiments. Furthermore, data in this communication indicate that glycosylation patterns for the dopamine transporter are different in different brain regions. Data from the cloning experiments suggest a M r of about 69,000 for the deglycosylated transporter, which is higher than is suggested here (Fig. 4, approximately 56,000). This discrepancy may be at least partly acMr
STR
N A STR
NA
Fig. 4. Effect of N-glycanase on [125I]DEEP-labeled dopamine transporter from striatum (Str) and nucleus accumbens (Na). Treatment with N-glycanase reduced the apparent molecular weight of the transporter from both brain regions (compared to control samples incubated in the absence of N-glycanase) and eliminated the difference in apparent molecular weight. Experiments were carried out four times with essentially the same results.
counted for by the behavior of this protein in SDSP A G E gels. The higher the acrylamide content of the gels, the higher the apparent molecular weight of the transporter (R. Vaughan, unpublished observations with glycosylated protein). In this study, an acrylamide content of 10% was selected because it reliably resolved the apparent M r difference between the two brain regions. There are many known cases of different glycosylation patterns exhibited by identical gene products 29. In particular, Stiles et alY, have reported /32-adrenoce ptors in hamster lung and rat erythrocytes are either high mannose- or complex-type glycoproteins. Furthermore, the CRF receptor in rat brain and anterior pituitary have apparent molecular weights of 58 and 75 kDa, respectively, and this difference is due to different glycosylation patterns 1°'11. Similar observations have also been reported for the DI and D 2 dopamine receptors~7, TM. A variety of functions have been described for the carbohydrate portion of glycoproteins 27. Several studies show glycosylation to be essential for protein translocation. The non-glycosylated form of the G-protein in vesicular stomatitis virus is expressed in tunicamycin-treated cells but is not transported to the cell surface compared to the glycosylated form 9. Glycosylation may also be involved in the functionality of cell surface receptors. In tunicamycin-treated A431 ceils the non-glycosylated /32-adrenoceptor exhibits similar binding affinity and number of sites compared to untreated cells. However, there is a loss in adenylate cyclase activity, which is due to a coupling defect at the receptor level, since other components of the signal transmission chain remained functional 4. In contrast, the non-glycosylated /3t-adrenoceptor from turkey erythrocytes exhibits enhanced adenylate cyclase activity compared to glycosylated /3~-adrenoceptor 3. In the present study removal of sialic acids from striatal dopamine transporters by neuraminidase treatment did not affect [3H]WIN 35,428 binding to the dopamine transporter at either 4 or 40 nM, respectively. This would suggest that sialic acids are not involved in cocaine binding to the dopamine transporter. Zaleska and Erecinska 3v have reported neuraminidase treatment of rat brain synaptosomes to reduce dopamine uptake (Vmax) without affecting the affinity (K m) of dopamine for the transporter. Since affinity was not affected this would support the present observation that sialic acids are not involved in the binding of dopamine to the transporter. However, since dopamine uptake (Vmax) was reduced by removal of sialic acid, it is possible that sialic acids are involved in the chain of events that occurs following binding of dopamine to
271 the transporter. Indeed, in several transport systems it has been suggested that terminal sialic acids may either form part of the binding site for Na ÷ or they are involved in the active conformation of the carrier protein that binds Na + 38. In summary, the present study demonstrates that the apparent heterogeniety of the dopamine transporter in rat striatum and nucleus accumbens is due to differences in glycosylation patterns and that the polypeptide portion of the dopamine transporter in both regions have similar apparent molecular weights.
17
18 19 20 21
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