From the Howard Hughes Medical Institute, Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710
Received for publication, February 26, 2002, and in revised form, November 11, 2002
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ABSTRACT |
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The dopamine transporter (DAT) is a
presynaptic plasma membrane protein responsible for the termination of
dopaminergic neurotransmission in the central nervous system. While
most studies have focused on structure/function analysis, much less
information is available regarding the assembly and the trafficking of
this protein. To address this problem, we performed a mutational
analysis of the DAT protein, combined with biochemical,
immunological, and functional approaches. In mammalian cells
co-expressing differentially tagged DAT molecules, HA-tagged DAT
co-purified with 6His-tagged DAT demonstrating a physical interaction
between transporter proteins. Evidence for the functional
oligomerization of DAT was obtained using dominant-negative mutants of
DAT. Two loss-of-function mutant transporters (Y335A and D79G) that
were targeted to the cell surface inhibited wild-type DAT uptake
activity without affecting the membrane targeting of the wild-type
transporter. Moreover, non-functional amino and carboxyl
termini-truncated mutants of DAT inhibited wild-type DAT function by
interfering with the normal processing of the wild-type transporter to
the cell membrane. Mutations in the leucine repeat of the second
transmembrane domain of the transporter could eliminate the
dominant-negative effect of all these mutants. In addition, a
small fragment comprising the first two transmembrane domains of DAT
inhibited wild-type transporter function but not when the leucine
repeat motif was mutated. Taken together, our results suggest that the
assembly of DAT monomers plays a critical role in the expression and
function of the transporter.
The dopamine transporter
(DAT)1 belongs to a large
family of Na+/Cl Hydrophobicity analysis of their deduced amino acid sequence reveals
that Na+/Cl Despite the increasing numbers of studies reporting DAT function and
regulation, much less information is available regarding the mechanisms
involved in the cellular processing of this transporter. Recent
biochemical evidence suggests that neurotransmitter transporters exist
as oligomeric complexes in cells, but the relevance of this process to
transporter function as well as the molecular determinants involved in
transporter assembly are unclear. Oligomers of SERT have been detected
by co-immunoprecipitation of differentially tagged monomers (13).
Fluorescence resonance energy transfer analysis has also
provided evidence for the oligomerization of GABA transporters (14). In
addition, electrophysiological and freeze fracture electron microscopic
studies revealed a pentameric structure for the unrelated neuronal
glutamate transporter EAAT3 in the plasma membrane of
Xenopus oocytes (15). More recently, Hastrup et
al. (16) in an elegant series of experiments, demonstrated that
the human DAT could be cross-linked as a homodimer at the plasma
membrane of HEK-293 cells and provided evidence for a role of TM6 as an
oligomerization domain (16). The only exception to the transporter
oligomerization rule so far appears to be the glycine transporter
(GlyT), another member of the
Na+/Cl Given the importance of DAT in normal and abnormal brain function, it
becomes important to understand how these proteins are regulated at the
cellular level. In this report, we used mutational analysis combined
with biochemical, immunological, and functional approaches to examine
the assembly and trafficking properties of the human DAT expressed in
mammalian cells.
Materials--
[3H]DA (31.6 Ci/mmol) was supplied
by PerkinElmer Life Sciences, Taq polymerase was from Fisher
Scientific, restriction enzymes were from Takara Biomedicals, and DNA
purification kits were from Qiagen. The rat anti-DAT and rabbit
anti-DAT antibodies were from Chemicon, anti-HA antibody was from
Roche, and the anti-His antibody was from Sigma. Secondary antibodies
conjugated with HRP, FITC, or Texas Red were from Jackson
Immunoresearch. Sulfo-NHS-SS-biotin and ultralink avidin beads were
from Pierce.
DNA Constructs and Mutagenesis--
The full-length cDNA
encoding the human DAT was subcloned into the mammalian expression
vector pcDNA3.1 (Invitrogen). PCR-based mutagenesis (36 cycles at
94 °C for 30 s, 55 °C for 30 s, 72 °C for 3 min) was
used to incorporate the His6 (HHHHHH) or the HA (YPYDVPDYA)
epitopes into the amino terminus of DAT. Mutations in the coding region
of DAT including Y335A, D79G, and the substitution of leucine residues
from TM2 or TM9 to alanine residues were performed by site-directed
mutagenesis combined with overlapping PCR. Amino-terminal deletions of
DAT were generated by introducing initiator methionine residues at
amino acid positions 11, 20, 48, and 60. Carboxyl-terminal transporter
truncations were created by introducing stop codons in the coding
region of DAT at amino acid positions 611, 601, 591, 582, 293, and 141. Asparagine residues from N-linked glycosylation consensus
sequences were replaced by glutamine residues by site-directed mutagenesis. After PCR mutagenesis, restriction fragments containing mutated sequences were digested with appropriate restriction enzymes, subcloned into pcDNA3.1 and verified by automated sequencing.
Cell Culture and Transfections--
HEK-293 cells were grown to
60-80% confluency in 100-mm tissue culture dishes and transiently
transfected using the Ca2PO4 precipitation
method with 5 µg of total DNA. Cells were incubated with the
Ca2PO4-DNA mixture at 37 °C for 16 h,
followed by 48 h recovery in minimal essential medium supplemented
with 10% fetal bovine serum, 50 units/ml of penicillin, and 50 units/ml of gentamycin. Subsequent experiments were performed 48-72 h
after transfections.
Transport and Binding Measurements--
The conditions for
dopamine uptake in HEK-293 cells have been adopted from Giros et
al. (18). Briefly, 48-72 h after transfections, medium was
removed, and uptake was measured following incubation of cells for 5 min with 250 µl of uptake buffer (in mM: 5 Tris base, 7.5 HEPES, 120 NaCl, 5.4 KCl, 1.2 CaCl2, 1.2 MgSO4,
1 ascorbic acid, and 5 glucose, pH 7.4) containing 20 nM
[3H]DA (31.6 Ci/mmol) and increasing concentrations of
cold DA ranging from 100 nM to 30 µM. After
rinsing with 1 ml of NaCl-free uptake buffer, cells were solubilized in
0.5 ml of 1% SDS, and the radioactivity incorporated into the cells
was measured by liquid scintillation counting. Nonspecific uptake was
determined in the presence of 2 µM mazindol or 10 µM cocaine. The protein concentration was measured using
the BCA protein assay kit (Pierce). Data are presented as the mean ± S.E. For assessment of whole cell DAT levels, transiently transfected HEK-293 cells were grown in 150-mm dishes. The medium was
removed, and cells were washed twice with PBS. Cells were lysed at
4 °C for 20 min with 2 mM HEPES and 1 mM
EDTA buffer and then scraped and centrifuged at 31,000 × g for 20 min. The resultant pellet was homogenized in
binding buffer (0.25 M sucrose and 10 mM
Na2HPO4, pH 7.4) using a polytron homogenizer.
For saturation experiments, ~100 µg of protein aliquots were
incubated for 1 h at room temperature with 4 nM
[3H]CFT (83.9 Ci/mmol) and increasing
concentrations of cocaine ranging from 1 nM to 100 µM. The reaction was terminated by filtering the samples
through Whatman GF/C glass fiber filters with a Brandel cell harvester.
Nonspecific binding was determined in the presence of 2 µM mazindol. The protein concentration was measured using the BCA protein assay kit (Pierce). Data are presented as means ± S.E.
Immunocytochemistry and Confocal Microscopy--
For
immunostaining experiments, transiently transfected HEK-293 cells grown
on glass coverslips were placed in 6-well dishes at a density of 5 × 105 cells/well, followed by fixation in 4%
paraformaldehyde. After three washes with PBS, cells were permeabilized
in PBS containing 0.1% Triton X-100 for 10 min and incubated in
blocking solution (1% bovine serum albumin, 5% goat serum in PBS) for
1 h. Cells were incubated with rat anti-DAT (1:1000) or a rabbit
anti-DAT (1:1000) antibodies for 1 h at room temperature followed
by incubation with Texas red-conjugated anti-rat or FITC-conjugated
anti-rabbit secondary antibodies. Cells were then washed three times in
PBS, and the coverslips were mounted on glass slides using Vectashield (Vector Laboratories). Immunofluorescent images were generated using a
Zeiss laser scanning confocal microscope at 585 nm for Texas Red and
488 nm for FITC.
Cell Surface Biotinylation--
Transiently transfected
monolayers of HEK-293 cells were washed three times with PBS and then
incubated with gentle agitation for 30 min at 4 °C with 1 ml of 1 mg/ml sulfo-NHS-SS-biotin prepared in 150 mM NaCl, 2 mM CaCl2, 10 mM triethanolamine, pH
7.8. The reaction was quenched by incubating the cells for an
additional 10 min with 50 mM glycine in PBS. Cells were
then washed three times in PBS and incubated in radioimmune
precipitation assay buffer (RIPA) (10 mM Tris, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% Triton
X-100, and 1% sodium deoxycholate, pH 7.4) at 4 °C for 1 h.
Each sample was divided into two aliquots. One aliquot was used for
isolation of biotinylated proteins with ultralink-immobilized neutravidin beads. The second aliquot was used to determine total DAT
levels. Samples were analyzed by Western blotting with the rat anti-DAT
antibody and an HRP-conjugated secondary antibody.
His6-DAT Purification and Western
Blotting--
HEK-293 cells expressing His6-DAT with or
without HA-DAT were lysed in PBS containing 1% digitonin for 1 h
at 4 °C. The lysates were run through a nickel column (Qiagen),
fractionated on 10% acrylamide gels, and transferred to nitrocellulose
membranes. Western blotting was performed using anti-HA or anti-His
antibodies, secondary antibodies conjugated with HRP, and
immunoreactive bands were detected with the ECL system (Amersham Biosciences).
Physical Association between Differentially Tagged DAT
Proteins--
As a first step to understand the cellular regulation of
the human DAT, we investigated whether DAT proteins could form
oligomeric complexes in intact cells. DAT proteins were differentially
tagged with either the His6 (His6-DAT) or the
HA (HA-DAT) epitopes and assayed for protein-protein interaction by a
nickel-based purification assay in transfected HEK-293 cells. To ensure
that these epitope-tagged transporter molecules retained their
structural and functional integrity, uptake assays were first performed
in HEK-293 cells transfected with the individual constructs. As seen in
Fig. 1A the tagged
transporters were functional and displayed kinetic and pharmacological
properties similar to the wild-type transporter. Purification of
His6-DAT on nickel columns under non-denaturing conditions
allowed the detection of HA-DAT when both proteins are expressed
simultaneously in the same cell, demonstrating that DAT proteins form
tight complexes in living cells (Fig. 1B, third lane). No interaction was detected when lysates from cells
expressing individual constructs were mixed prior to purification on
nickel columns (Fig. 1B, second lane) indicating
that the interaction occurs only when both tagged proteins are
co-expressed in the same cell and is not promoted during protein
solubilization.
Non-functional DAT Mutants Inhibit Wild-type DAT Function through
Protein-Protein Interactions--
A physical interaction between DAT
proteins strongly suggest the formation of an oligomeric complex. To
provide direct functional evidence for DAT oligomerization, we reasoned
that mutant transporter molecules devoid of uptake activity might still
associate with the wild-type DAT and confer a dominant-negative effect
on wild-type transporter function when co-expressed in cells. In
searching for critical amino acids involved in transporter function and expression, we identified several residues that when mutated produced transporter proteins that exhibited little or no uptake activity. One
of these residues, a tyrosine at amino acid position 335 located on the
third intracellular loop between TMs 6 and 7, is conserved in all
members of the Na+/Cl
To test the possibility that this non-functional mutant might exhibit a
dominant-negative effect, mutant and wild-type transporters were
co-expressed in cells, and uptake activity was assayed. Co-expression of DATY335A with wild-type DAT results in a significant reduction of
[3H]DA uptake as compared with that observed in cells
expressing wild-type DAT alone (Fig.
3A). In contrast, no decrease
in uptake activity of the wild-type DAT was observed when co-expressed
either with an empty vector or with the unrelated
In the second mutant examined, an aspartate residue at position 79 in
the first TM of DAT was replaced with glycine (D79G). This residue is
conserved only in monoamine transporters, whereas in the rest of the
members of the Na+/Cl Role of the Intracellular Carboxyl-terminal Domain in DAT
Assembly--
Next, we sought to determine which domains are involved
in transporter oligomerization. We first turned our attention to the intracellular carboxyl-terminal of DAT. This domain has been shown to
interact with intracellular proteins such as PICK1 and synuclein (21,
22) and thus represent a candidate domain for monomer-monomer interaction. We made a series of deletions in the carboxyl-terminus of
DAT at amino acid positions 611, 601, 591, and 582 (designated S582*
through Q611*, Fig. 5A) by
introducing stop codons at the respective positions. All
carboxyl-terminal-truncated proteins expressed well in HEK-293 cells
and gave proteins of the appropriate molecular size (data not shown).
Uptake experiments from HEK-293 cells expressing each of these mutants
revealed that progressive deletions of the carboxyl terminus of DAT
(Q611*, R601*, and L591*) produced a progressive decrease in transport
activity (Fig. 5B). L591* exhibited less than 1% of
wild-type uptake function. A further deletion in the tail of DAT
(S582*) completely abolished transporter function (Fig. 5B),
suggesting that S582* lacks transport activity and/or is not expressed
at the plasma membrane. Confocal microscopy analysis of transfected
cells stained with the anti-DAT antibody demonstrate that the lack of
function of S582* is due to the impaired targeting of this mutant to
the plasma membrane (Fig. 5C).
To examine the possibility that the S582* mutant might inhibit the
function of the wild-type DAT, we expressed both constructs simultaneously in HEK-293 cells and measured uptake activity. No
detectable uptake activity was observed in cells expressing the S582*
mutant (Fig. 6A). However, the
uptake activity of DAT is decreased in the presence of the S582* mutant
as compared with that of DAT alone. Under conditions in which the whole
cell levels of wild-type DAT remains constant, there is a dramatic
decrease in the amount of cell surface wild-type DAT when co-expressed with S582* (Fig. 6B, inset). Thus, the decreased
uptake of the wild-type DAT when co-expressed with the deletion mutant
was apparently due to a physical trapping of the wild-type and mutant
DAT proteins in the cytoplasm. We further explored this possibility by
examining the subcellular localization of wild-type and mutant
transporters when expressed individually or in combination in HEK-293
cells. The wild-type transporter was tagged with GFP at the amino
terminus whereas the deletion mutant was tagged with the HA epitope
also at the amino terminus. This strategy allows for the differential visualization of wild-type and mutant proteins when co-expressed in the
same cell. When expressed alone, the HA-tagged truncated transporter
showed an intracellular staining pattern contrasting the clear plasma
membrane distribution observed in cells expressing the wild-type
GFP-tagged transporter (Fig. 6C, upper panels). When wild-type and mutant transporters were co-expressed, the distribution of the wild-type transporter changed dramatically. Immunofluorescent experiments demonstrate that wild-type DAT
colocalizes with the mutant transporter intracellularly (Fig.
6C, middle panels). As a control, we
co-transfected the GFP-tagged Functional Expression of DAT Amino-terminal-truncated
Mutants--
Amino-terminal deletions of DAT were generated to examine
the role of this domain in the functional expression of the
transporter. We created DAT deletion mutants lacking the first 10, 20, 48, and 60 amino acids (designated
Next, we examined the ability of the amino-terminal deletion A Leucine-repeat Motif in TM2 Is Required for DAT Oligomerization
and Trafficking--
We next investigated the role of two leucine
zipper-like motifs present within the second and the ninth TMs of DAT.
This class of motif, which consists of four leucine residues
periodically spaced by six amino acids and arranged in an
Having demonstrated that the substitution of the leucine repeat from
TM2 results in a non-functional transporter, we next explored whether
this mutant might still interact with the wild-type transporter. To
examine this possibility, we co-transfected wild-type DAT with TM24LA
and assayed uptake activity. As seen in Fig. 8D, [3H]DA uptake was similar in cells expressing DAT and
TM24LA compared with cells expressing DAT alone. In addition, we failed
to detect a protein-protein interaction between the HA-tagged TM24LA
and the His-tagged DAT (data not shown). Thus, these results suggest that the inability of TM24LA to inhibit the function of the wild-type DAT is due to the lack of interaction between this mutant transporter and the wild-type DAT. To support this possibility, we performed immunofluorescent analysis in cells co-expressing the GFP-tagged full-length DAT and the HA-tagged TM24LA mutant transporter. As shown
in Fig. 8E, the HA-tagged TM24LA mutant is not expressed at
the cell membrane as compared with the full-length GFP-tagged DAT. In
addition, the trafficking of the GFP-tagged DAT to the cell surface was
not altered in the presence of the TM24LA mutant suggesting that the
TM24LA transporter mutant is unable to associate with the wild-type
DAT. Furthermore, the dominant negative effect exhibited by the Y335A,
D79G, S582*, and Role of N-linked Glycosylation in Transporter Oligomerization and
Trafficking--
As shown above, elimination of the leucine repeat
from TM2 in DAT results in a non-functional transporter devoid of
dominant-negative effect. However, the observation that the size of
this mutant protein was much smaller than that of the wild type and
similar to the expected size for the non-glycosylated form, raised the possibility that lack of glycosylation might explain the lack of
function in the mutant transporter. To examine this possibility, we
investigated the role of N-linked glycosylation in the
functional expression of DAT. The human DAT contains three putative
N-linked glycosylation sites in the second extracellular
loop at positions 181, 188, and 205. We generated a series of mutants
by replacing each of the asparagines to alanine residues by
site-directed mutagenesis. These glycosylation mutants were transiently
transfected in HEK-293 cells and analyzed for transporter activity,
protein size, and subcellular distribution. Individual substitution of
asparagine residues from N-glycosylation consensus sequences
resulted in a reduction in the size of the protein (Fig.
9B), suggesting that the three
consensus sequences are glycosylated. Analysis of transporter activity
in cells transfected with the single mutants revealed uptake activity
similar to that observed for the wild-type transporter (Fig.
9A), indicating that single glycosylation sites are not essential for transporter function. Cell surface biotinylation experiments and immunostaining demonstrated that these mutant transporters are properly targeted to the plasma membrane (Fig. 9,
B and C). We next examined whether removal of all
glycosylation sites resulted in any alteration in transporter
expression and function. As shown in Fig. 9B, the triple
glycosylation mutant (TGM) migrated as a protein of ~70
kDa, consistent with the contribution of the three glycosylation sites
to the size of the transporter. Uptake assays revealed an approximate
50% decrease in Vmax with no alterations in
Km (1.7 µM in cells expressing the wild-type DAT versus 1.9 µM in cells
expressing the TGM transporter). Subcellular localization of the
non-glycosylated transporter using immunofluorescence microscopy and
cell surface biotinylation showed that the reduction in transport
function is associated with an increase in the intracellular retention
of the mutated transporters (Fig. 9, A and C).
Thus, removal of all three glycosylation sites partially impairs the
trafficking of DAT to the plasma membrane. However, the
non-glycosylated transporter is still capable of forming functional
transporter proteins at the plasma membrane.
Transporter Fragments Containing TM2 Inhibit Wild-type Transporter
Activity--
Although the results obtained with the TM24LA mutant
suggest that TM2 is an interacting domain within DAT, they do not
exclude the possibility that the lack of interaction observed might be due to the improper folding of the mutant protein. To provide direct
evidence for the contribution of TM2 in transporter assembly, we
generated two transporter fragments containing the leucine repeat,
TM1-2, and TM1-5 (Fig.
10A). TM1-2 contains a stop
codon at the end of the first intracellular loop at amino acid position 141, whereas TM1-5 contains a stop codon in the extracellular loop of
DAT between TMs 5 and 6 at amino acid position 293. As predicted, these
transporter fragments are not functional when expressed in HEK-293
cells (Fig. 10B). However, when co-expressed with the
wild-type transporter, each of these fragments, showed dominant-negative effect (Fig. 10B). Confocal microscopy
images of cells transfected with the full-length DAT and either TM1-2 or TM1-5 mutant proteins revealed an increase in the intracellular distribution of the full-length DAT (data not shown). To further support the involvement of the leucine-repeat motif from TM2 in DAT
assembly, we generated two additional fragments. TM1-EL1 contains a
stop codon at the beginning of TM2 at amino acid position 94, whereas
TM1-2(4LA) contains the first two TMs of DAT except that the
leucine-repeat motif from TM2 has been mutated to alanine residues as
described in Fig. 8. These fragments failed to inhibit uptake activity
when co-expressed with the wild-type transporter in HEK-293 cells (Fig.
10B). Taken together, these results are consistent with a
role of the leucine-repeat motif of TM2 in the assembly of DAT.
In this study, we have examined several aspects of the cell
biology of the human dopamine transporter expressed in HEK-293 cells.
The main conclusions from the experiments presented here are that DAT
exists as an oligomeric complex in intact cells and that the assembly
process is required for the proper trafficking of the transporter
complex to the plasma membrane. Several independent lines of evidence
support these conclusions. First, we have provided biochemical evidence
for the physical association between differentially epitope-tagged
transporter proteins, indicating that the transporter exist as an
oligomeric complex in intact cells. Second, we have used non-functional
mutants of DAT that are expressed at the plasma membrane and exhibit
dominant-negative effect when co-expressed with the wild-type
transporter. This effect is not the result of reduced levels of the
wild-type transporter at the plasma membrane but is due to specific
protein-protein interactions between wild-type and mutant transporter.
Third, trafficking-defective amino and carboxyl-truncated transporter
mutants that do not reach the plasma membrane reduce the function of
the wild-type transporter by a mechanism involving a decrease in the
levels of cell surface DAT. Immunofluorescence analysis demonstrated
that the truncated transporters accumulate in the cytoplasm where they
retain the wild-type transporter and greatly reduce its ability to
reach the cell surface. We conclude that dominant-negative mutants
reduce the activity of the transporter by forming oligomeric complexes
at the cell membrane (Y335A and D79G) or by interfering with the normal
processing and trafficking of the wild-type DAT ( The present findings also highlight the importance of the
leucine-repeat motif from TM2 as an important domain potentially contributing to transporter assembly. Substitutions of this repeat by
mutagenesis results in a mutant transporter devoid of uptake activity
due to the inability of this protein to be delivered to the plasma
membrane. This mutant protein does not exhibit dominant-negative effect
upon co-expression with the wild-type transporter suggesting that the
leucine repeat participate in monomer-monomer interaction. However, an
alternative explanation of our results is that the leucine-repeat
mutant presents a defect in folding steps necessary for assembly and
trafficking. The latter alternative does not appear to be consistent
with our additional findings. A small fragment of DAT containing only
the first two TMs is also capable of inhibiting wild-type DAT function,
an effect that is lost upon mutation of the leucine residues in the
motif. Thus, it appears that the leucine motif is important for the
initial steps in the assembly of DAT. Leucine-repeat motifs have been
implicated in the assembly process of several membrane proteins. In
several cases, as in the human immunodeficiency virus type 1 transmembrane glycoprotein (24, 25), the murine coronavirus spike
protein (26), and the Arabidopsis thaliana somatic
embryogenesis receptor kinase 1 protein (27), these motifs are not part
of the transmembrane domains. However, there are examples such as the
cardiac ion channel phospholamban (28, 29) and the erythropoietin
receptor (30), in which leucine repeats are part of transmembrane
domains that mediate oligomerization. Interestingly, a similar leucine
repeat located in TM9 does not appear to be required for transporter oligomerization. Substitutions of the leucine repeat in TM9 did not
impair the trafficking or the function of the transporter. These
results suggest that specificity in the location of the leucine repeat
is important in determining assembly.
While our results provide evidence for some involvement of TM2 in the
assembly of DAT, these findings do not rule out the possibility that
other domains might also participate in the oligomerization of this
transporter complex. Indeed, during the course of our study, Hastrup
et al. (16) demonstrated that DAT could be cross-linked as a
homodimer at the plasma membrane of HEK-293 cells (16). These complexes
were revealed via the selective chemical cross-linking of a cysteine
residue on the extracellular side of TM6. The TM6 in this family of
transporters contains a glycophorin A motif that has been shown to
mediate dimerization of membrane proteins (31). Interestingly,
mutations of the conserved glycine residues of this motif in DAT led to
loss of function and lack of cell surface targeting (16), similar to
the DAT TM2 leucine-repeat motif mutant reported here. Hastrup's
findings and those reported here are not mutually exclusive as many
membrane-bound proteins are known to oligomerize through interactions
mediated by multiple domains. The best characterized example is the
muscle nicotinic acetylcholine receptor (nAChR), which contains four
different subunits that combine to form pentameric hetero-oligomers.
nAChR complexes are assembled using a stepwise pathway (32-34).
Critical motifs involved in initial subunit assembly events reside in
several domains of each channel subunit. It remains to be determined
whether a multi-step process involving different interacting domains
also occurs in the assembly of DAT. Alternatively, it is possible that TM2 might be involved in early steps during the assembly process of
DAT, whereas residues in TM6 might be important in maintaining the
oligomeric structure of DAT at the plasma membrane as demonstrated by
Hastrup et al. (16).
Interestingly, the mutant lacking the leucine repeat from TM2 was
not glycosylated. A conceivable explanation is that assembly precedes
glycosylation during the trafficking of the transporter and thus, lack
of assembly in the leucine-repeat mutant results in lack of
glycosylation. Alternatively, it is also plausible that the impaired
delivery of this mutant to the plasma membrane results as a consequence
of lack of glycosylation. Our results using glycosylation mutants
demonstrate that glycosylation is not essential, under our experimental
conditions, for the functional expression of the transporter. In the
case of SERT, Tate and Blakely (35) demonstrated that lack of
glycosylation did not affect the affinity of serotonin for the
transporter. The carbohydrate moieties of glycoproteins in general are
believed to be important for a variety of functions including normal
protein folding, protection from proteolytic degradation, intracellular
trafficking, and/or cell surface targeting (36). For instance, the loss
of N-linked glycosylation impaired the trafficking of the
AT1A receptor to the plasma membrane (37). On the other hand, Bisello
et al. (38) showed that N-linked glycosylation is
not essential for the expression of the parathyroid hormone-related
protein receptor on the plasma membrane (38). Thus, the loss of
N-linked glycosylation appears to have a spectrum of effects
on membrane-bound proteins, and no general predictions can be made
based exclusively on the occurrence of glycosylation for any given
protein. Our results demonstrate that glycosylation is not essential
for DAT functional expression and suggest that oligomerization takes
place during the early stages of transporter processing and appears to
be required for the integration of the complex into intracellular
membranes for subsequent glycosylation and trafficking.
Interestingly, our data indicate that the carboxyl terminus of DAT
plays a critical role in the targeting of the protein to the plasma
membrane. Progressive truncation of the transporter causes progressive
reduction of function as a result of the impairment of these mutants to
reach the plasma membrane. It is tempting to speculate that the tail of
DAT contains sequences that mediate interactions with intracellular
proteins and that those interactions are important for the proper
trafficking of the transporter to the plasma membrane. In fact,
DAT-interacting proteins that regulate transporter trafficking through
interactions mediated by the carboxyl terminus have been described (21,
22).
Since the molecular identification of plasma membrane
neurotransmitter transporters, it was originally proposed that these proteins function as monomeric units. However, several studies in
recent years have now begun to establish that some transporters function as oligomeric complexes. The neuronal glutamate transporter was shown to exist as multimers in brain tissue (39). Recently, elegant
experiments using freeze-fracture electron microscopy combined with
functional analysis demonstrated that the human glutamate transporter
EAAT3 exists as a pentameric structure (15). There is also compelling
evidence, both biochemical and functional, indicating that the
serotonin and GABA transporters form oligomeric complexes in intact
cells (13, 14).
Taken together, data from the present study and from Hastrup et
al. (16) indicate that the human DAT exist as an oligomeric complex in intact cells. The formation of the complex involves possibly
several domain interacting regions and appears to be essential for the
proper trafficking of the transporter complex to the cell membrane.
While our findings shed light into several aspects of the cell biology
of DAT, some important questions still remain to be addressed. What is
the stoichiometry of the oligomeric complex? At which step in the
synthesis and processing of DAT does oligomerization take place? What
are the domains involved in the formation of the substrate and/or the
ion pore of the transporter? Finally, by which mechanisms
i.e. inter- versus intramolecular interactions,
mutant plasma membrane transporters inhibit the function of DAT? While
future studies should be aimed at a more mechanistic understanding of
the functional consequences of transporter oligomerization, the ability
of mutant transporters to exhibit dominant-negative effect may have
important physiological implications.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-dependent
plasma membrane transporters that also includes the closely related
norepinephrine and serotonin transporters (NET and SERT, respectively),
and carriers for GABA, glycine, proline, taurine, and betaine. In the
central nervous system, DAT mediates the re-uptake of released dopamine
(DA) from the synaptic cleft back into the nerve terminal for
subsequent storage and release. Pharmacological and genetic studies
highlight the DAT-mediated re-uptake process as the main mechanism for
the termination of dopamine neurotransmission (1). In addition, DAT
represents the main target site for commonly abused drugs such as
cocaine and amphetamine as well as some therapeutic agents used in the management of affective disorders (2).
-dependent plasma
membrane neurotransmitter transporters are proteins containing twelve
transmembrane domains (TMs) with both the amino and the carboxyl
termini located on the intracellular side of the membrane. This
topological arrangement has been confirmed for several members of the
family, including DAT (3). Since the molecular cloning of this
transporter gene family, a great deal of information has been
accumulated concerning the relationship between the structure and
function of this class of proteins (4). Studies using mutagenesis and
heterologous expression systems have identified several amino acid
residues and domains involved in substrate and inhibitor binding.
Moreover, there is growing evidence suggesting that the subcellular
distribution of monoamine transporters is regulated by second messenger
systems (5). Activation of protein kinase C in cells expressing DAT,
SERT, or NET results in decreased transporter activity (6-8). This effect is believed to result from a rapid redistribution of transporter proteins from the cell surface to intracellular compartments (9-10). In the case of DAT, there is also evidence indicating that substrates and inhibitors appear to regulate the cellular distribution of this
transporter (11-12).
-dependent transporter
family. Biochemical analysis of the size of GlyT expressed in
Xenopus oocytes is not consistent with an oligomeric
structure for this transporter protein (17). This seemingly
contradictory finding suggest that oligomerization might not be a
common feature in transporter biology, or alternatively it is possible
that biochemical approaches used to study oligomerization might disrupt
the protein-protein interactions required to maintain oligomeric
complexes. In any case, these results point out to the need for
functional approaches to establish the oligomeric nature of a given
transporter complex and its functional relevance.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Physical association between differentially
tagged DAT proteins. A, uptake activity of wild-type,
His6-tagged, and HA-tagged DAT expressed in HEK-293 cells.
Uptake experiments were performed with 20 nM
[3H]DA in the absence or the presence of 10 µM cocaine (+C). B, HA-DAT
co-purifies with His6-DAT in HEK-293 cells. Protein lysates
from cells expressing His6-DAT and HA-DAT (lane
3) were subjected to Ni2+ chromatography and resolved
by 10% PAGE. For mixing experiments, lysates from cells transfected
with individual constructs (lane 2) were mixed before
Ni2+ chromatography. Proteins were separated on 10% gels
under non-reducing conditions and detected by Western blotting with the
anti-His6 or the anti-HA antibodies. The results are
representative of at least three independent experiments.
transporter family and
forms part of a putative consensus sequence for tyrosine-based
internalization motif (19). We replaced this residue to alanine by
site-directed mutagenesis and examined transporter activity in HEK-293
cells. When expressed in cells, the DATY335A mutant does not exhibit
detectable uptake activity (Fig.
2A), despite the fact that the
protein is properly targeted to the cell membrane as evidenced by cell
surface biotinylation experiments (Fig. 2B) and confocal
microscopy images of immunostained transfected cells (Fig.
2C). Recently, Loland et al. (20) have also shown that the Y335A mutation in DAT results in a transporter protein exhibiting less than 1% of uptake activity. In addition, these authors
provide evidence suggesting that this tyrosine is critical in mediating
conformational changes in DAT during activation-inactivation steps.
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Fig. 2.
The non-functional DATY335A mutant is
properly targeted to the plasma membrane. A, HEK-293
cells were transfected with wild-type DAT or DATY335A and assayed for
[3H]DA uptake as described under "Experimental
Procedures." B, Western blot analysis of whole cell
transporter proteins (left panel) or cell surface
transporters (right panel) from HEK-293 cells transfected
with wild-type DAT or DATY335A as revealed with the anti-DAT antibody.
C, confocal microscopy images from cells expressing the
wild-type DAT (left panel) or the DATY335A mutant
(right panel). Immunostaining was performed on
permeabilized cells with the anti-DAT antibody and a secondary antibody
conjugated with Texas-Red. The results are representative of at least
five independent experiments.
2-adrenegic receptor protein (Fig. 3C). The
reduction of wild-type DAT activity upon DATY335A co-expression was not
due to a decrease in the total expression levels of the wild-type DAT
or the amount of the wild-type transporter expressed at the cell
membrane as revealed by biotinylation of cell surface proteins using
the HA-tagged transporter (Fig. 3B). Kinetic analysis
revealed an approximate 60% decrease in Vmax
with a small alteration in Km (2.2 µM
in cells expressing wild-type DAT versus 3.6 µM in cells co-expressing wild-type DAT and Y335A). Thus,
these findings provide evidence for the formation of an oligomeric
complex between wild-type DAT and mutant Y335A transporter proteins at
the cell membrane.
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Fig. 3.
DATY335A exhibits dominant-negative effect
when co-expressed with wild-type DAT. A, uptake
experiments were performed from cells expressing HA-tagged wild-type
DAT alone (circles) or cells transfected with HA-tagged
wild-type DAT and DATY335A (in a 1:2 DNA ratio, squares).
B, biotinylation experiments showing cell surface HA-tagged
DAT when expressed alone (lanes 1, 3,
5) or with the untagged DATY335A mutant (lanes 2,
4, 6). Western blot analysis was performed with
the anti-HA antibody. C, uptake activity of wild-type DAT
when expressed alone or in combination with empty vector, the
2-adrenergic receptor or the DATY335A mutant. Results
are representative of three independent experiments.
-dependent
transporter family, this position is occupied by a glycine residue.
When expressed in HEK-293 cells, the D79G mutant did not exhibit
detectable uptake activity (Fig.
4A). Preliminary studies
suggest that Asp-79 in DAT is critical in determining substrate
selectivity.2 Cell surface
biotinylation and immunostaining revealed that the mutant protein was
efficiently delivered to the plasma membrane (Fig. 4A,
inset). Thus, as in the case of the Y335A mutant, D79G lacks
intrinsic transport activity. We then explored the possibility that the
D79G mutant might exhibit dominant-negative effect when co-expressed
with the wild-type transporter. As shown in Fig. 4B, at a
constant level of wild-type DAT, co-expression of D79G caused a
significant reduction of [3H]DA uptake. The
dominant-negative effect exhibited by the mutated transporter occurs
when both wild-type and mutated DAT are co-expressed at the cell
membrane. Thus, these results provide another example of a transporter
complex at the cell membrane formed by a functional and a
non-functional transporter.
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Fig. 4.
Dominant-negative effect of DATD79G on
wild-type DAT activity. A, uptake activity of wild-type
DAT and the DATD79G mutant expressed in HEK-293 cells. The
inset shows cell surface wild-type and mutant transporter
proteins detected by biotinylation experiments and Western blotting
using the anti-DAT antibody. B, transport activity of
wild-type DAT alone or when co-expressed with the DATD79G mutant in
HEK-293 cells. Results are representative of three independent
experiments.
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Fig. 5.
Truncations at the carboxyl terminus of DAT
impair the trafficking of the transporter to the plasma membrane.
A, schematic representation of wild-type (WT) and
carboxyl-terminal deletions Q611*, R601*, L591*, and S582* of DAT.
Shaded boxes represent TMs. B, transport activity
of wild-type and carboxyl-terminal deletions of DAT expressed in
HEK-293 cells. The mutant L591* exhibited less than 1% of wild-type
DAT activity whereas the S582* mutant did not show any detectable
transport activity. C, confocal microscopic images from
cells expressing wild-type and carboxyl-terminal deletion mutants of
DAT. Immunostaining was performed with the anti-DAT antibody. Note the
lack of cell surface expression of the S582* mutant. Results are
representative of five independent experiments.
1B-adrenergic receptor along with the
S582* mutant transporter. As shown in Fig. 6C (lower
panels) the membrane localization of the receptor is not altered
in the presence of the deletion mutant demonstrating that the mutant
transporter does not cause a general nonspecific effect on protein
processing. Taken together, our results indicate that the intracellular
tail of the transporter is essential for the trafficking of this
protein to the plasma membrane, and the inhibitory effect of the
truncated transporter on wild-type DAT function results from the
formation of oligomeric complexes unable to undergo normal processing
to the cell surface. Because the truncated mutant is still able to
associate with the wild-type transporter, these findings also suggest
that the intracellular tail of DAT does not appear to be essential for
DAT oligomerization.
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Fig. 6.
The S582* deletion mutant impairs the normal
processing of the wild-type DAT to the cell surface. A,
transport activity of wild-type and the deletion S582* mutant of DAT.
The inset shows the lack of cell surface expression of the
S582* mutant compared with the wild-type transporter as revealed by
biotinylation and Western blotting analysis using the anti-DAT
antibody. B, co-expression of S582* mutant with wild-type in
a 1:2 DNA ratio reduces the uptake activity of the wild-type
transporter. HEK-293 cells were transfected with either the wild-type
DAT alone or in combination with the S582* mutant. The inset
shows a reduction in the cell surface levels of DAT when co-expressed
with the S582* deletion mutant. C, confocal images of cells
expressing either the GFP-DAT or the HA-S582* transporters (upper
panels) or cells co-expressing GFP-DAT and HA- S582* mutant
(middle panels). Note that the GFP-tagged wild-type DAT
co-localizes with the HA-tagged S582* deletion mutant inside the cell
(lower panels) co-expression of the GFP-tagged
1B-adrenergic receptor and the HA-tagged DAT deletion mutant S582*.
Immunostaining was done with the HA antibody. The results are
representative of three independent experiments.
10,
20,
48, and
60,
respectively). In each case an initial methionine was engineered into
the truncated sequence to ensure correct translation. When expressed in
HEK-293 cells
10,
20, and
48 displayed transport activity
similar to the wild-type transporter. In all cases only
Vmax was reduced whereas Km
values were not significantly altered (Km values in
µM; wild-type DAT, 2.6 ± 0.7;
10, 2.8 ± 0.6;
20, 2.1 ± 0.5; and
48, 1.91 ± 0.6, n = 4). In contrast, cells expressing the
60
transporter mutant did not show any detectable uptake activity (Fig.
7A). Immunofluorescent
analysis with an anti-DAT antibody against the second extracellular
loop of DAT, revealed an intracellular distribution of the
60
deletion mutant in transfected HEK-293 cells when compared with cells
expressing the wild-type DAT (Fig. 7B). These results
demonstrate that the lack of transport displayed by the
60 mutant is
due to the improper processing and/or sorting of this protein leading
to the absence of transporter molecules on the plasma membrane.
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Fig. 7.
Role of the amino-terminal domain in
transporter assembly and trafficking. A, transport
activity of amino-terminal deletion mutants of DAT, 10,
20,
48, and
60 expressed in HEK-293 cells. B, confocal
microscopy analysis of cells transfected with wild-type (left
panel) or the amino-terminal deletion mutant
60 (right
panel). Immunostaining was done with an anti-DAT antibody that
recognizes an epitope located in the second extracellular loop of DAT.
C, the amino-terminal-truncated mutant
60 reduces the
transport activity of the wild-type DAT when co-expressed in a 1:2 DNA
ratio in HEK-293 cells. D, confocal microscopy images of
cells transfected with GFP-tagged DAT alone (left panel) or
in combination with the deletion mutant
60 (right panel).
The results are representative of three independent experiments.
60
mutant to co-associate with the full-length transporter by testing for
dominant-negative effect when co-expressed in HEK-293 cells. Similar to
the dominant-negative effect showed by the S582* mutant, the
60
mutant caused a significant reduction of wild-type transport activity
(Fig. 7C). Immunostaining of cells transfected with the
GFP-DAT in the presence of the
60 mutant revealed an increase in the
intracellular distribution of the full-length transporter when compared
with cells expressing the GFP-tagged DAT alone (Fig. 7D).
Hence, the dominant-negative effect of
60 on the wild-type
transporter results as a consequence of the association of mutant and
wild-type transporters inside the cells. These results also rule out
the involvement of the amino terminus of DAT as an essential domain in oligomerization.
helix-like structure, was originally described in DNA-binding proteins
and believed to mediate protein-protein interactions (23). Using
site-directed mutagenesis, we replaced the three leucine residues and
one methionine from TM2 at amino acid positions 99, 106, 113, and 120 by alanine residues (TM24LA). Cells expressing this mutant transporter
did not exhibit detectable uptake activity (Fig.
8A) despite the fact that the
protein was synthesized at similar levels compared with the wild-type
transporter (Fig. 8B). Interestingly, the size of this
mutated transporter was much smaller than the size of the wild-type
transporter. Cell surface biotinylation revealed that TM24LA was not
expressed at the plasma membrane (data not shown). The affinity of
cocaine for the transporter mutant was slightly decreased
(IC50 = 333.2 nM in cells expressing DAT
versus 832.5 nM in cells expressing TM24LA, Fig.
8C) suggesting that the mutant transporter is still capable
of forming functional binding sites. In contrast, replacement of the
four leucine residues in TM9 at amino acid positions 440, 447, 454, and
461 by alanine residues (TM94LA) resulted in a functional transporter
with similar kinetic and pharmacological properties compared with the
wild-type transporter (Fig. 8A). Despite the similarities
between these two domains, our results are consistent with a critical
role for the leucine repeat from TM2, but not for that from TM9, in the
functional expression of the transporter.
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Fig. 8.
Integrity of a leucine repeat in TM2 is
important for DAT assembly and trafficking. A,
transport activity of wild-type DAT, TM2 leucine-repeat mutant
(TM24LA), or TM9 leucine-repeat mutant (TM94LA)
in the absence or the presence of 10 µM cocaine
(TM4LA + C). Results are representative of three independent
experiments. B, Western blot analysis of whole cell
transporter proteins from HEK-293 cells transfected with wild-type DAT
or TM2L4A as revealed with the anti-DAT antibody. Arrows
represent the glycosylated and non-glycosylated forms of the
transporter. C, [3H]CFT binding experiments
performed in cells expressing wild-type DAT or TM24LA. The total
binding of the TM24LA mutant was ~25% of the wild-type transporter.
D, the TM2 leucine-repeat mutant does not exhibit
dominant-negative effect when co-expressed with the wild-type
transporter. E, confocal microscopy images shows lack of
co-localization between the GFP-tagged full-length DAT and the
HA-tagged TM2L4A leucine-repeat mutant.
60 mutants upon co-expression with the wild-type
transporter could be eliminated when the leucine repeat was mutated in
these mutants (Table I).
Dominant-negative effect of non-functional mutants on wild-type DAT
activity
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Fig. 9.
Role of N-linked
glycosylation in the functional expression of DAT. A,
transport activity of wild-type DAT, single N-linked
glycosylation mutants N181Q, N188Q, and N205Q, and the triple
glycosylation mutant (TGM). Results are representative of
three independent experiments. B, biotinylation of cell
surface wild-type DAT and N-linked glycosylation mutants
revealed with the anti-DAT antibody. Arrows represent the
sizes of the fully, partially, and non-glycosylated proteins.
C, confocal microscopy images from HEK-293 cells transfected
with wild-type DAT or N-linked glycosylation mutants.
Immunostaining was performed with the anti-DAT antibody.
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Fig. 10.
Fragments of DAT containing TM2 exhibit
dominant-negative effect on wild-type transporter activity.
A, schematic representation of wild-type and fragments of
DAT. TM1-2 represents a truncated fragment of DAT containing the first
two TMs, TM1-5 represent a fragment of DAT containing the first five
TMs, TM1EL1 represents a fragment containing the first TM and the first
extracellular loop, and TM1-2(4LA) represents a fragment containing
the first two TMs in which the leucine repeat from TM2 has been mutated
to alanine residues as described in the legend to Fig. 8. B,
transport activity of wild-type DAT, fragments TM1-2, TM1-5, or
wild-type DAT co-expressed with either TM1-2, TM1-5, TM1EL1, or
TM1-2 (4LA) at a 1:2 DNA ratio. Results are representative of three
independent experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
60 and S582*).
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ACKNOWLEDGEMENTS |
---|
We thank members of the Caron laboratory for helpful discussions. We would also like to thank Dr. Susan Amara (Vollum Institute, Portland, OR) for providing the GFP-tagged DAT cDNA.
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FOOTNOTES |
---|
* This study was supported by National Institutes of Health Grants NS-19576 (to M. G. C.) and DA-14150 (to G. E. T.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Investigator of the Howard Hughes Medical Institute. To whom
correspondence should be addressed: Dept. of Cell Biology, Duke University Medical Center, Rm. 489, CARL Bldg., Research Dr., Durham,
NC 27710. Tel.: 919-684-5433; Fax: 919-681-8641; E-mail: m.caron@cellbio.duke.edu.
Published, JBC Papers in Press, November 11, 2002, DOI 10.1074/jbc.M201926200
2 G. Torres, unpublished results.
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ABBREVIATIONS |
---|
The abbreviations used are:
DAT, dopamine
transporter;
DA, dopamine;
NET, norepinephrine transporter;
SERT, serotonin transporter;
GlyT, glycine transporter;
TMs, transmembrane
domains;
HEK, human embryonic kidney;
CFT, 2-carbomethoxy-3
-(4-fluorophenyl)tropane;
GFP, green
fluorescent protein;
HA, hemagglutinin;
PBS, phosphate-buffered
saline;
HRP, horseradish peroxidase;
FITC, fluorescein isothiocyanate;
GABA,
-amino butyric acid.
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REFERENCES |
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