Introduction

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The dopamine transporter (DAT) is a neuronal protein that clears dopamine from the synaptic space, controlling synaptic dopamine concentrations and regulating dopamine availability for pre- and postsynaptic receptors. DAT is a major target for psychostimulant drugs such as cocaine (Kuhar et al., 1991), and for neurotoxins such as 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (Kinemuchi et al., 1987). DAT and related neurotransmitter transporters are integral membrane proteins believed to consist of 12 transmembrane spanning domains (TMs), extracellularly oriented glycosylation sites, and intracellularly oriented N and C termini (Povlock and Amara, 1997). Although some of the structural aspects of these proteins predicted from primary sequence have been experimentally verified, many important details of structure, including relative proximity of transmembrane spanning helices and identification of substrate and antagonist active sites, remain to be elucidated.

Although the binding properties of cocaine and other structurally diverse dopamine uptake blockers have been extensively characterized (Mash and Staley, 1997), it is not clear how they bind to DAT or how this results in transport inhibition. Different ligands could bind to the same site, to different sites, or to overlapping but nonidentical sites. Some studies compatible with the latter have suggested that slight differences in protein-ligand interactions may lead to subtle differences in transporter function. For instance, cocaine and GBR analogs display dissimilarities in their in vitro binding characteristics (Madras et al., 1989; Pristupa et al., 1994) and produce different behavioral profiles in vivo (Rothman et al., 1992; Izenwasser et al., 1994). Specific residues and domains of DAT are also thought to contribute differentially to transport and ligand binding (Kitayama et al., 1992; Buck and Amara, 1994, 1995; Giros et al., 1994).

Using irreversible DAT antagonists we have directly identified distinct sites of protein-ligand interactions. [¹²⁵I]DEEP, a GBR-based photoaffinity ligand, and [¹²⁵I]GA-II-34, a benztropine derivative, become incorporated in TMs 1 to 2, whereas [¹²⁵I]RTI-82, a cocaine analog, becomes incorporated in a region of the protein containing TMs 4 to 7 (Vaughan 1995; Vaughan and Kuhar 1996; Vaughan et al., 1999). Thus, although the tropane ring is an essential component of the cocaine pharmacophore (Carroll et al., 1992), it is insufficient to produce identical targeting of [¹²⁵I]GA-II-34 and [¹²⁵I]RTI-82. However, we were unable to determine the basis for this differential ligand incorporation or the functional and structural relationship between the two protein domains.

The present study examines a newly developed ligand generated from GBR structures (Dutta et al., 1996, 1997, 1998a,b). Altering the GBR piperazine ring into a piperidine ring, in addition to other modifications (Fig. 1), produced high-affinity DAT ligands that showed striking increases in DAT/SERT selectivity (Dutta et al., 1997, 1998a,b). To investigate the molecular basis of binding of these compounds we developed a corresponding photoaffinity derivative, 4-[2-(diphenylmethoxy)ethyl]-1-[(4-azidophenyl)methyl]-piperidine ([¹²⁵I]AD-96-129) (Dutta et al., 2001b), and mapped its photoincorporation sites in comparison to the known labeling patterns of [¹²⁵I]DEEP, [¹²⁵I]RTI-82, and [¹²⁵I]GA-II-34. Although [¹²⁵I]AD-96-129 interacts with the same regions as the other photoaffinity labels, it does so with a two-site pattern of incorporation not previously observed for DAT. This strongly implicates the three-dimensional proximity of the labeled domains and provides one of the first indications of the three-dimensional nature of DAT antagonist binding sites.

View larger version (16K): [in this window] [in a new window]   Fig. 1.   DAT photoaffinity labels whose sites of incorporation have been identified. The structures show the positions of the azido (N3) moiety, through which the ligands become covalently attached to the protein.

	Experimental Procedures

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Photoaffinity Labeling. [¹²⁵I]DEEP, [¹²⁵I]RTI 82, and [¹²⁵I]AD-96-129 (specific activity 1600-2000 Ci/mmol) were synthesized as described previously (Lever et al., 1993; Dutta et al., 2001b) and used to label rat striatal membranes (Vaughan and Kuhar, 1996). Striatal tissue was homogenized with a Polytron homogenizer in sucrose-phosphate buffer (10 mM sodium phosphate plus 0.32 M sucrose, pH 7.4), and homogenates were centrifuged at 12,000g for 12 min. The resulting membranes were resuspended at 15 mg/ml original wet weight in the same buffer, and radioligand was added to a final concentration of 5 nM. After a 60-min incubation on ice, ligand was covalently incorporated into DAT by irradiating the sample with ultraviolet light for 45 s, and membranes were washed with buffer. The photolabeled membranes were either solubilized with SDS-PAGE sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, and 5 mM dithiothreitol) followed by electrophoresis, autoradiography, and electroelution of DAT (Vaughan, 1995), or were suspended in 50 mM Tris-HCl, pH 8.0, for in situ proteolysis and immunoprecipitation (Vaughan and Kuhar, 1996). Rats were housed and maintained in accordance with guidelines established by the University of North Dakota Animal Care and Use Committee and the National Institutes of Health.

Proteolysis. Gel purification and electroelution of photolabeled DAT were performed for experiments in which it was desired to remove other radiolabeled proteins present in striatal membranes. Proteolysis of these samples allows visualization of all DAT photolabeled fragments, regardless of their ability to be immunoprecipitated. For these experiments (Fig. 2), 25 µl of electroeluted sample was incubated for 1 h at 22°C with 25 µl of trypsin prepared in 50 mM Tris-HCl, pH 8.0. Reactions were stopped by adding 1 mg/ml trypsin inhibitor and 100 µl of SDS-PAGE sample buffer, followed by electrophoresis and autoradiography on 13% SDS-PAGE gels. For in situ proteolysis of membrane suspensions (Figs. 3-5 and 7), 25 µl of photolabeled membranes was incubated for 10 min at 22°C with 25 µl of trypsin in 50 mM Tris-HCl, pH 8.0. Reactions were stopped by adding 1 mg/ml trypsin inhibitor and centrifugation at 11,000g for 9 min. Supernatants were removed, and membranes were solubilized with 0.5% SDS followed by immunoprecipitation. Fragments generated from native and denatured protein correspond to comparable domains based on their similar immunoprecipitation properties (Vaughan, 1995; Vaughan and Kuhar, 1996). All experiments were repeated two to five times with similar results.

View larger version (89K): [in this window] [in a new window]   Fig. 2.   Peptide maps of DATs labeled with [125I]AD-96-129, [125I]DEEP, and [125I]RTI 82. Gel-purified photoaffinity-labeled DATs were treated with the indicated concentrations of trypsin, followed by electrophoresis on 13% SDS polyacrylamide gels and autoradiography. Molecular mass standards for all gels are shown in kilodaltons.

Immunoprecipitation, Electrophoresis, and Autoradiography. Epitope-specific immunoprecipitation of DAT and DAT fragments was performed as previously described using antiserum 16 generated against amino acids 42 to 59 or antiserum 5 generated against amino acids 225 to 238 (Vaughan, 1995; Vaughan and Kuhar, 1996). For peptide competition experiments, diluted antisera were preincubated with 50 µg/ml peptide 16 or peptide 5 before addition of DAT samples. Precipitated samples were electrophoresed on 13% or 9 to 16% SDS-polyacrylamide gels, and subjected to autoradiography using Kodak BioMax MS film for 1 to 4 days or PhosphorImager analysis and quantitation using a Molecular Dynamics PhosphorImager and ImageQuant software. The migration of DAT and DAT fragments relative to molecular mass standards varies slightly depending on the gel system used (Vaughan, 1995) and, in this study, are referred to by the previously ascribed values, which reflect our most accurate estimates of their masses (Vaughan, 1995; Vaughan and Kuhar, 1996). Photolabeling, proteolysis, and immunoprecipitation of DATs labeled with each ligand were done exactly in parallel. Autoradiographic exposures were adjusted to equalize band intensities between photolabeled samples, which differed due to differences in ligand affinities and specific activities.

Materials. Tosyl-phenylalanyl-chloromethyl ketone-treated trypsin and trypsin inhibitor were from Worthington Biochemical Corporation (Lakewood, NJ), protein A Sepharose CL4B and High and Low Range Rainbow Molecular Weight Markers were from Amersham Pharmacia Biotech (Piscataway, NJ), and other reagents were from Fischer Scientific (Pittsburgh, PA) or Sigma Chemical (St. Louis, MO).

	Results

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Peptide Maps of DATs Labeled with [¹²⁵I]AD-96-129, [¹²⁵I]DEEP, and [¹²⁵I]RTI 82. The overall peptide mapping pattern of DATs labeled with [¹²⁵I]AD-96-129 was first examined in comparison to the well characterized patterns generated from DATs labeled with [¹²⁵I]DEEP and [¹²⁵I]RTI 82 (Fig. 2). This experiment was performed using preparations of gel-purified DAT in which DAT is the only radiolabeled protein in the sample, thus all fragments shown derive from DAT and can be visualized regardless of their ability to be immunoprecipitated. The mapping pattern of DATs labeled with [¹²⁵I]DEEP was consistent with previous studies showing major fragments of about 45 and 14 kDa that originate from N-terminal regions of the protein, and a lightly labeled fragment at about 32 kDa that originates from C-terminal regions. Proteolysis of [¹²⁵I]RTI 82-labeled DATs also produced previously described fragments of about 32 and 16 kDa that originate from the C-terminal and central regions of the protein and a larger fragment at ~50 kDa distinct from the [¹²⁵I]DEEP-labeled 45-kDa fragment (see below). The relationship of these fragments to the primary sequence is described more thoroughly below and is summarized in Fig. 6. The peptide map of [¹²⁵I]AD-96-129-labeled DATs contained major photolabeled fragments evident at 45 and 32 kDa, as well as smaller fragments in the 14- to 16-kDa range. Since the different fragments generated from DATs labeled with [¹²⁵I]DEEP or [¹²⁵I]RTI 82 result from incorporation of the ligands in different regions of the DAT primary sequence, the pattern of fragments obtained from [¹²⁵I]AD-96-129 indicated the possibility that the ligand was incorporated into different protein domains. The substantial levels of radioactivity present in the fragments indicate that they represent major sites of photolabeling, but do not exclude the possibility that potential fragments derived from labeling of distinct sites might be lost due to small size.

Epitope-Specific Immunoprecipitation of Photolabeled Fragments. To identify the incorporation site(s) of ¹²⁵I-AD-96-129 in the DAT primary sequence, we treated labeled membrane suspensions with trypsin, removed the protease by washing the membranes, and immunoprecipitated the solubilized membranes with DAT antiserum 16, which recognizes amino acids 42 to 59 in the N-terminal tail just before TM1, or antiserum 5, which recognizes amino acids 225 to 238 in EL2 just before TM4 (Fig. 3). Proteolysis and immunoprecipitation were performed in parallel with samples labeled with [¹²⁵I]DEEP and [¹²⁵I]RTI 82 to serve as controls and points of reference. For both the [¹²⁵I]AD-96-129- and [¹²⁵I]DEEP-labeled samples (Fig. 3, left), serum 16 precipitated full-length DATs that migrate at about 80 kDa and tryptic fragments of approximately 45 and 14 kDa (Fig. 3, left arrows). Precipitation of the same [¹²⁵I]AD-96-129-labeled sample with antiserum 5 (Fig. 3, right) resulted in the extraction of full-length protein and 32- and 16-kDa fragments that comigrated with [¹²⁵I]RTI 82-labeled fragments (Fig. 3, right arrows), and a slight amount of the 50-kDa fragment. The 50-kDa [¹²⁵I]RTI 82-labeled fragment is not the same as the 45-kDa fragment labeled with [¹²⁵I]DEEP because it does not immunoprecipitate with serum 16 (data not shown). Some nonspecific [¹²⁵I]AD-96-129-labeled bands (e.g., at 32 kDa) were present at times but do not seem to be derived from DAT because they were present in nonprotease-treated samples, and may result from residual carryover of a prominent 32-kDa protein present in the starting sample (Fig. 5). The fragments identified in these experiments are retained in membranes after proteolytic treatment, indicating that they contain transmembrane spanning structure and that the binding of these ligands is closely associated with TM helices.

View larger version (109K): [in this window] [in a new window]   Fig. 3.   Epitope-specific immunoprecipitation of [125I]AD-96-129 proteolytic fragments. Rat striatal membranes labeled with [125I]AD-96-129, [125I]DEEP, or [125I]RTI 82 were treated with the indicated concentrations of trypsin, followed by immunoprecipitation with antiserum 16 (left) or antiserum 5 (right). Samples were electrophoresed on 9 to 16% SDS-polyacrylamide gels followed by autoradiography. Arrows on the left indicate positions of 45- and 14-kDa fragments labeled with [125I]AD-96-129 or [125I]DEEP; arrows on the right indicate positions of 32- and 16-kDa fragments labeled with [125I]AD-96-129 or [125I]RTI 82.

View larger version (83K): [in this window] [in a new window]   Fig. 4.   Specificity of photoaffinity-labeled fragment immunoprecipitation. Rat striatal membranes labeled with [125I]AD-96-129, [125I]DEEP, or [125I]RTI 82 were treated with 10 µg/ml trypsin followed by immunoprecipitation with serum 16 (left) or serum 5 (right). During precipitation samples received either no addition, 50 µg/ml peptide 16, or 50 µg/ml peptide 5, as indicated. Arrows on the left indicate position of 45- and 14-kDa fragments labeled with [125I]AD-96-129 or [125I]DEEP, arrows on the right indicate positions of 32-, 16-, and 10-kDa fragments labeled with [125I]AD-96-129 or [125I]RTI 82. Note the slightly different migration patterns of the standards for the two panels.

View larger version (114K): [in this window] [in a new window]   Fig. 5.   Pharmacological specificity of [125I]AD-96-129 labeling of DAT and DAT tryptic fragments. Striatal membranes were labeled with [125I]AD-96-129 in the absence or presence of 10 µM () cocaine as indicated. A, labeled membranes were electrophoresed directly on an 8% gel followed by electrophoresis and autoradiography. The arrow shows the position of full-length DAT at 80 kDa. B, [125I]AD-96-129-labeled membranes were treated with 50 µg/ml trypsin followed by immunoprecipitation with serum 16 or serum 5, electrophoresis on a 9 to 16% gel, and autoradiography. The arrows on the left show the positions of the intact DAT and the 45-kDa fragment precipitated with serum 16, the arrows on the right show the position of intact DAT and the 32-kDa fragment precipitated with serum 5.

View larger version (11K): [in this window] [in a new window]   Fig. 6.   Summary of DAT photoaffinity ligand binding sites. Schematic diagram of rat dopamine transporter primary amino acid sequence, with transmembrane domains indicated by filled rectangles; antibody 16 and 5 epitopes shown by open, numbered rectangles; consensus N-glycosylation sites indicated by open circles; and lysine and arginine residues (potential trypsin proteolysis sites) shown by tic marks. The arrow indicates the position of the initial tryptic proteolysis site at R218. The bars above the protein sequence represent the major photoaffinity-labeled fragments identified in these studies, with the mass of each fragment indicated to the left or right. The ligands that become incorporated in the fragments are indicated above the bars.

Protease Sensitivity. In previous studies we observed that DATs labeled with [¹²⁵I]DEEP or [¹²⁵I]RTI 82 displayed a pronounced difference in protease sensitivity, with [¹²⁵I]DEEP-labeled DATs being readily proteolyzed, whereas those labeled with [¹²⁵I]RTI 82 were markedly protease resistant (Vaughan, 1995; Vaughan and Kuhar, 1996). Because the initial tryptic proteolysis site that cuts the protein into the 45- and 32-kDa fragments is on the C-terminal side of EL2 near TM4, we speculated that [¹²⁵I]RTI 82 incorporation in the TM4 to 6 region may be occurring close to the protease site, obscuring its availability for proteolysis and resulting in protease resistance, whereas the incorporation of [¹²⁵I]DEEP in TMs 1 to 2, which may be more distant from the protease site, does not affect its availability and leaves the protein susceptible to proteolysis. We noted during characterization of [¹²⁵I]AD-96-129 binding that DATs labeled with this compound displayed a protease sensitivity intermediate to those of DATs labeled with [¹²⁵I]DEEP or [¹²⁵I]RTI 82. In Fig. 4, for example, after treatment with 10 µg/ml trypsin, the amount of full-length (unproteolyzed) DAT remaining relative to that of the 45- or 32-kDa fragments is very low for DATs labeled with [¹²⁵I]DEEP, is high for DATs labeled with [¹²⁵I]RTI 82, and is intermediate for DATs labeled with [¹²⁵I]AD-96-129. A comparable trend is evident in Fig. 3.

View larger version (34K): [in this window] [in a new window]   Fig. 7.   Differential protease sensitivity of photoaffinity labeled DATs. A, rat striatal membranes labeled with [125I]AD-96-129, [125I]DEEP, or [125I]RTI 82 were treated with the indicated concentrations of trypsin followed by immunoprecipitation with serum 16, electrophoresis, and autoradiography. B, densitometric quantitation of full-length DAT from each of the photolabeled samples, expressed as amount of photoaffinity-labeled protein remaining relative to untreated control samples. Values shown are the average of two experiments, error bars indicate standard deviation.

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

Potential Proximity of Labeled Domains. This study documents the incorporation of a novel dopamine transporter photoaffinity ligand, [¹²⁵I]AD-96-129, into two distinct regions of the DAT primary sequence. These regions encompass TMs 1 to 2 near the N terminus of the protein and TMs 4 to 6 in the central third of the protein, both of which are major sites of incorporation for three other DAT photoaffinity ligands, [¹²⁵I]DEEP, [¹²⁵I]RTI 82, and [¹²⁵I]GA II 34. Previous studies have shown that [¹²⁵I]DEEP and [¹²⁵I]GA II 34 become incorporated primarily in the TM1 to 2 site, whereas [¹²⁵I]RTI 82 and a very slight amount of [¹²⁵I]DEEP become incorporated in the TM 4 to 6 region. In contrast to the specific or near-specific incorporation of these photolabels into one or the other region, [¹²⁵I]AD-96-129 incorporation occurred at comparable levels in both sites at a concentration (5 nM) well below its IC₅₀ value of 154 nM for inhibiting binding of the cocaine analog [³H]WIN 35,428 (Dutta et al., 2001b). These results strongly suggest that these regions are in spatial proximity and argue against the possibility that one of the domains represents a spatially distinct low-affinity site. Dual incorporation of photoaffinity labels in distinct regions of the primary sequence on VMAT (Sievert and Ruoho, 1997) and the multidrug resistance transporter, P-glycoprotein (Bruggemann et al., 1992; Greenberger 1993; Dey et al., 1997) has also been taken as evidence for the three-dimensional proximity of the labeled domains. Interestingly, one of the labeling sites on VMAT is near TM1, indicating a potential similarity to the DAT TM 1 to 2 site. Dual incorporation of [¹²⁵I]AD-96-129 may also represent ligand interaction with different transport cycle states of DAT. A similar conclusion has recently been obtained for the two photolabel sites on P-glycoprotein, but the labeled domains are nevertheless thought to be in proximity (Dey et al., 1997).

View larger version (25K): [in this window] [in a new window]   Fig. 8.   Model of photoaffinity label binding to DAT. Schematic cross-sectional view of a section of DAT shown from the extracellular side of the membrane. Transmembrane spanning helices are indicated as numbered circles and are primarily arranged in the clockwise order suggested to provide favorable helix packing (Edvardsen and Dahl, 1994); interhelical connecting loops are not shown. Shading indicates the photoaffinity-labeled domains identified in these studies. A-C, hypothetical orientation of DEEP, AD-96-129, and RTI 82 for N3 group incorporation in the TM 1 to 2 domain (A), the TM 4 to 6 domain (C), or either domain (B). DEEP and AD-96-129 are modeled as having substantial similarity in their binding site orientations. The positioning of other aspects of the ligands with relative to the protein is not known and as shown is arbitrary. Incorporation of ligands was modeled to occur in TMs 1 and 4 of the possible helices in each shaded domain (see text for details).

Molecular Determinants of Ligands. The incorporation profiles of the four irreversible ligands we have examined may begin to elucidate the structural determinants that contribute to and affect their binding. All of the compounds that label the TM 1 to 2 domain, [¹²⁵I]DEEP, [¹²⁵I]AD-96-129, and [¹²⁵I]GA II 34, contain a diphenyl moiety, indicating the potential for these ring structures to orient the ligands similarly within the binding pocket, possibly via - interactions with aromatic groups on the protein. Molecular differences that may produce greater association of [¹²⁵I]AD-96-129 than [¹²⁵I]DEEP with the TM 4 to 6 domain include the ¹²⁵I-AD-96-129 piperidine ring. The N-atom in this group has a different pK_a than the piperazine N-atoms in [¹²⁵I]DEEP, which may result in different hydrogen or ionic bonding interactions with the protein. Another difference between [¹²⁵I]DEEP and [¹²⁵I]AD-96-129 is the length of the aromatic-alkyl chain connecting to the N-atom of the piperidine or piperazine ring, which may give rise to differential steric properties or ligand flexibilities. It is intriguing that dual incorporation, albeit to different extents, is seen for the GBR-like compounds [¹²⁵I]DEEP and [¹²⁵I]AD-96-129, whereas no evidence for multisite incorporation has been found for the tropane ring-containing compounds [¹²⁵I]RTI 82 and [¹²⁵I]GA II 34. It may be that conformational flexibility of GBR-like compounds allows ligand bending or movement within the binding pocket that promotes multisite incorporation. Cocaine and WIN compounds display substantial structural constraints imposed by the tropane ring (Zhu et al., 1999), which may minimize ligand movement and lead to the more discrete incorporation observed for [¹²⁵I]RTI 82 and [¹²⁵I]GA II 34. Future studies involving closely related structural variants of these ligands will help to further elucidate these issues.

Protease Sensitivity Effects of Ligands. Our finding that DATs labeled with different ligands display widely different protease sensitivities may be an indication of the existence of different structural states of ligand-complexed protein. These protease sensitivity studies were performed with native preparations of DAT, compatible with the results reflecting authentic variations in protein structure. Two possible scenarios may explain the finding that protease resistance is correlated with the extent of ligand incorporation in the TM 4 to 6 region. In one scenario, ligands that become incorporated in TMs 4 to 6 sterically cover the EL2 protease site and interfere with protease access, whereas ligands incorporated in TMs 1 to 2 are not near the site and leave the protein susceptible to proteolysis. This would imply a region of ligand-protein proximity along the C-terminal side of EL2 for some but not all ligands, and if ligand binding occurs within a membrane-embedded pocket as is conventionally thought, would also imply that this region of EL2 would be situated close to the lipid bilayer. An alternative scenario is that ligand incorporation in TMs 4 to 6 induces a conformational change in the protein that results in the EL2 protease site becoming inaccessible to enzyme, or conversely that ligand incorporation in TM 1 to 2 promotes a conformation in which the protease site becomes more accessible. In either case, site-specific ligand incorporation produces different conformational changes, indicating that different blockers induce different conformational states of the protein. These results may suggest a relationship between conformational movements in EL2 and transport inhibition, and suggest that differences in the response of the protein to different blockers may be related to the subtle differences in DAT response observed for cocaine and GBR analogs. Stabilization of EL2 may also underlie the zinc-induced inhibition of dopamine transport (Loland et al., 1999).

Summary. This study provides the first direct evidence that distinct domains of DAT can interact with a single ligand, thus implicating the proximity of the irreversibly labeled domains to other regions that may contain additional binding determinants. This advances our understanding of the structure of ligand binding sites, which are likely to consist of residues that are far apart in primary sequence but close together three dimensionally. Although a computer model based on primary sequence has been developed for DAT (Edvardsen and Dahl, 1994), a three-dimensional structure based on NMR or crystallization is currently not available. The results shown here therefore contribute to a framework for understanding the structural basis of neurotransmitter transporter function.