From the Department of Pharmacology and Neuroscience Program, University of Colorado Health Sciences Center, Denver, Colorado 80262
Received for publication, October 18, 2002 , and in revised form, May 5, 2003.
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Newly synthesized DAT acquires glycosylation in the endoplasmic reticulum (ER) and Golgi complex and is then trafficked to the plasma membrane. Typically, a large pool of mature DAT molecules are found at the cell surface of dopaminergic neurons and when DAT is heterologously expressed in tissue culture cells. However, DAT localization can be altered rapidly. Acute exposure of cells to either phorbol esters or substrates reduces the number of plasma membrane DATs and thus DAT function, and this reduction is due to acceleration of DAT endocytosis through a dynamin-dependent mechanism (36). Recently, it has been shown that DAT can interact with the anchoring protein PICK 1 and the adaptor protein Hic-5 (7, 8). However, in general, the molecular mechanisms controlling DAT trafficking are not yet well understood.
The DAT molecule is predicted to have 12 membrane-spanning sequences with
both amino and carboxyl termini oriented intracellularly. The specific
function of DAT transmembrane (TM) motifs and termini are not well
established. TM domains may play a role in intra- and intermolecular
interactions. Many membrane receptors and other integral membrane proteins
require dimerization or higher oligomerization for their activity. Several
lines of evidence have suggested that monoamine transporters are also dimers
or oligomers. Results with dominant negative forms of the serotonin
transporter (SERT) and norepinephrine transporter were consistent with this
idea (9,
10). Oligomerization of SERT
was demonstrated directly by co-immunoprecipitation
(11). The results of
fluorescence resonance energy transfer (FRET) studies further confirmed that
SERT and a -aminobutyric acid transporter (GAT-1) are homo-oligomers
(12,
13). Early radiation
inactivation studies also suggested that DAT exists as a dimer or oligomer
(14,
15). Recently, the potential
for DAT to exist as dimer or higher-order oligomer in the plasma membrane has
been demonstrated using chemical cross-linking
(16). More recently, Torres
and co-workers (17) reported
detection of DAT oligomerization by co-immunoprecipitation. In general, the
mechanisms and functional roles of oligomerization of monoamine transporters
remain to be defined. However, it has been proposed that SERT oligomerization
may be important for its transport activity
(9). Oligomerization may be
also required for proper trafficking of the newly synthesized transporter to
the plasma membrane (13,
17).
Hence, we generated fluorescent human DAT fusion proteins, which allow visualization of DAT interactions during trafficking in living cells, and then used these proteins in conjunction with high resolution FRET microscopy to examine DAT oligomerization in various cellular compartments. The effective energy transfer between cyan (CFP) and yellow fluorescent protein (YFP) occurs when two proteins are 15 nM apart. This close proximity typically requires direct interaction of proteins tagged with YFP and CFP. Measurements of sensitized FRET signals (fluorescence of the acceptor due to energy transfer from excited donor) on a pixel-by-pixel basis are designed to visualize protein-protein interactions in individual compartments of single living cells. Using this approach, we found that DAT is oligomerized in all cellular compartments in which it is localized, including post-synthetic compartments, plasma membrane, and endosomes, where DAT is accumulated when endocytosis is stimulated by phorbol esters or the DAT substrate amphetamine.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
PlasmidsTo generate the fusion proteins of human DAT
(18) with YFP (YFP-DAT) or CFP
(CFP-DAT), a DNA fragment encoding the full-length DAT was obtained by PCR
using Pfu polymerase (Stratagene Cloning Systems, La Jolla, CA), with
a KpnI restriction site introduced into the 5'-end and a
SmaI site into the 3'-end, and cloned into the pEYFP-C1 vector
or pECFP-C1 vector (Clontech, Palo Alto, CA), respectively. To generate
YFP-DAT'C615, a stop codon was created at the position corresponding to
amino acid residue 616 in the full-length DAT/pEYFP-C1 using a QuikChange
site-directed mutagenesis kit according to the manufacture's protocol
(Stratagene). To generate YFP-N-DAT lacking amino-terminal cytoplasmic
portion, a DNA fragment encoding the DAT sequence corresponding to amino acid
residues 66620 was obtained by PCR using Pfu polymerase with a
KpnI restriction site introduced into the 5'-end and a
SmaI site into the 3'-end, and cloned into the pEYFP-C1
vector.
Human Rab5a and Rab11a cDNAs were obtained from Guthrie cDNA Resource Center (Guthrie Research Institute, Sayre, PA). To generate YFP fusion proteins, full-length Rab5a and Rab11a were amplified by PCR and ligated into pEYFP-C1 by using the BglII and XhoI restriction sites. All constructs were verified by dideoxynucleotide sequencing. The construct EGFR-CFP was previously described (19). YFP-Hrs (hepatocyte-growth factor receptor substrate) was provided by Dr. H. Stenmark (Radium Institute, Oslo, Norway).
Cell Culture and TransfectionsPorcine aortic endothelial (PAE) cells were grown in F12 medium containing 10% fetal bovine serum, antibiotics, and glutamine. HEK293T cells (further referred to as HEK293) were grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, antibiotics, and glutamine. Cells were grown to 5080% confluency and transfected with appropriate plasmids using Effectine (Qiagen, Hilden, Germany). In transient transfection experiments, the cells were split 1 day after transfection onto glass coverslips and used for experiments on the second or third day at about 50% confluency. For biochemical immunoprecipitation and biotinylation experiments the cells were plated into 100- or 35-mm dishes, respectively, and used for experiments on the second day at about 90% confluency. Entactin-Collagen IV-Laminin coating (Upstate Biotechnology, Lake Placid, NY) was applied to 35-mm dishes to increase HEK293 cell adherence. The immortalized dopaminergic cell line 1RB3AN27 was kindly provided by Dr. K. Prasad (University of Colorado Health Sciences Center) (20). These cells were grown in RPMI medium supplemented with 10% fetal bovine serum and antibiotics.
For selection of stable clones, PAE cells transfected with CFP-DAT,
YFP-DAT, DAT, or YFP-N-DAT were grown with G418 (400 µg/ml) to
obtain a pool of constitutively expressing cells. The single cell clone
selection was then carried out by the limited dilution method in 96-well
dishes. PAE cells stably expressing DAT were transfected with a mixture of
YFP-
N-DAT and pSUMS-Hygro constructs (10:1) and grown with G418 and
hygromycin (0.2 µg/ml) to obtain clones stably expressing both DAT and
YFP-
N-DAT.
Uptake AssaysPAE cells expressing hDAT, CFP-DAT, or YFP-DAT were grown in 12-well plates for 23 days. The cells were rinsed and then assayed in Krebs-Ringer HEPES buffer (KRH; 120 mM NaCl, 4.7 mM KCl, 2.2. mM CaCl2, 1.2 mM Mg SO4, 1.2 mM KH2PO4, 10 mM glucose, 10 mM HEPES, pH 7.4) supplemented with 10 µM pargyline, 10 µM ascorbic acid, and 10 µM catechol. Assays (1 ml) included 50 nM [3H]DA plus increasing amounts of unlabeled DA (final concentrations 50 nM to 50 µM). Nonspecific [3H]DA accumulation was determined in the presence of 1 mM cocaine and averaged 2.0 ± 0.2% of total. After 10 min of incubation at 37 °C, uptake was terminated by quickly washing the cells three times with 1 ml of ice-cold KRH. Cells were then solubilized in 0.5 ml of 3% trichloroacetic acid for 30 min with gently shaking. Accumulated [3H]DA was determined by liquid scintillation counting. Data were analyzed using GraphPad Prism (San Diego, CA).
ImmunoprecipitationPAE cells stably expressing DAT and/or
YFP-N-DAT grown on 100-mm dishes to about 90% confluency were washed
three times with Ca2+,Mg2+-free
phosphate-buffered saline (CMF-PBS). The cells were then solubilized by
scraping with a rubber policeman in lysis buffer (50 mM NaCl, 2
mM MgCl2, 20 mM HEPES, pH 7.2, 10% glycerol,
10 mM dithiothreitol (DTT), 1 mM EGTA, 1 mM
EDTA, 10 mM sodium fluoride, 1 mM phenylmethylsulfonyl
fluoride, 10 µg/ml leupeptin, 544 µM iodoacetamide, 10
µg/ml aprotinin, 1% Triton X-100, 1% sodium deoxycholate) and by further
incubating for 10 min at 4 °C. The lysates were then cleared by
centrifugation for 10 min at 16,000 x g and incubated with
polyclonal antibodies to GFP (18 µg per dish) overnight at 4 °C. Under
these conditions, at least 95% of YFP-
N-DAT was immunoprecipitated. The
precipitates were washed twice with lysis buffer supplemented with 100
mM NaCl, washed once without NaCl, and then denatured by heating in
sample buffer for 5 min at 95 °C. The immunoprecipitates and the aliquots
of cell lysates were resolved on 7.5% SDS-PAGE, and the proteins were
transferred to the membrane. Western blotting was performed with monoclonal
antibodies to GFP or DAT followed by species-specific secondary antibodies
conjugated with horseradish peroxidase. The enhanced chemiluminescence kit was
purchased from Pierce (Rockford, IL).
Surface BiotinylationPAE or HEK293T cells were grown in 35-mm dishes and used at 100% confluency. Cells were washed twice with cold phosphate-buffered saline containing 0.1 mM CaCl2 and 1 mM MgCl2 (PBS) and incubated for 20 min on ice with 1 mg/ml sulfosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate (EZ-LinkTM sulfo-NHS-S-S-biotin, Pierce) in PBS, followed by a second incubation with fresh NHS-S-S-biotin. After biotinylation, the cells were washed twice with cold PBS, incubated 20 min on ice with 0.1 M glycine in PBS, washed with PBS again, and solubilized in lysis buffer (see above) supplemented with 10 mM Tris-HCl (pH 6.8) in which DTT was omitted. Cell lysates were precleared by centrifugation at 16,000 x g; the biotinylated proteins were precipitated with NeutrAvidinTM beads (Pierce), washed five times with lysis buffer, incubated in sample buffer supplemented with 100 mM DTT for 10 min to cleave disulfide bonds, and denatured by heating the beads in sample buffer at 95 °C for 5 min. Supernatants from the NeutrAvidin precipitation and the aliquots of cell lysates were further subjected to immunoprecipitation with rat anti-DAT or polyclonal anti-GFP. NeutrAvidin beads and immunoprecipitates were analyzed by electrophoresis and Western blotting.
Immunofluorescence StainingPAE cells expressing CFP-DAT were grown on glass coverslips and treated with Me2SO, PMA, or amphetamine at 37 °C. The cells were then washed with CMF-PBS and fixed with freshly prepared 4% paraformaldehyde (Electron Microscopy Sciences, Ft. Washington, PA) for 15 min at room temperature and mildly permeabilized using a 3-min incubation in CMF-PBS containing 0.1% Triton X-100 and 0.5% bovine serum albumin at room temperature. Cells were then incubated in CMF-PBS containing 0.5% bovine serum albumin at room temperature for 1 h with a monoclonal antibody to EEA.1 and then incubated for 30 min with a secondary donkey anti-mouse IgG labeled with Cy3 (Jackson Laboratories, West-Glove, PA). Both primary and secondary antibody solutions were precleared by centrifugation at 100,000 x g for 10 min. After staining, the coverslips were mounted in Fluoromount-G (Southern Biotech Inc., Birmingham, AL) containing 1 mg/ml para-phenylenediamine. To obtain high resolution three-dimensional images of cells, the fluorescence imaging workstation consisted of a Nikon inverted microscope equipped with a 100x oil immersion objective lens, cooled charge-coupled device Sensi-Cam QE 16 MHz (Cooke, Germany), z-step motor, dual filter wheels, and a xenon 175-watt light source, all controlled by SlideBook 3.0 software (Intelligent Imaging Innovation, Denver, CO). Typically, 2540 serial two-dimensional images were recorded at 100- to 200-nm intervals. A Z-stack of images obtained was deconvoluted using a modification of the constrained iteration method. Final arrangement of all images was performed using Adobe Photoshop.
Live Cell Microscopy and FRET ImagingPAE or HEK293 cells transiently or stably co-expressing various YFP/CFP-tagged proteins were grown on glass coverslips. The coverslips were mounted in a microscope chamber and placed on a microscope stage. To visualize CFP and YFP, consecutive images were acquired through corresponding filter channels.
The method of sensitized FRET measurement has been described previously (19, 21). We previously validated this technique by measuring FRET intensities in various control interaction pairs and by using the donor fluorescence recovery method (21). Briefly, images were acquired sequentially through YFP, CFP, and FRET filter channels. Filter sets used were: YFP (excitation, 500/20 nm; emission, 535/30 nm), CFP (excitation, 436/10 nm; emission, 470/30 nm), and FRET (excitation, 436/10 nm; emission, 535/30 nm). An 86004BS dichroic mirror (Chroma, Inc.) was utilized. Binning 2 x 2 modes and 100- to 250-ms integration times were used. The background images were subtracted from the raw images prior to carrying out FRET calculations. Corrected FRET (FRETC) was calculated on a pixel-by-pixel basis for the entire image using Equation 1: FRETC = FRET (0.50 x CFP) (0.02 x YFP), where FRET, CFP, and YFP correspond to background-subtracted images of cells co-expressing CFP and YFP acquired through the FRET, CFP, and YFP channels, respectively. 0.50 and 0.02 are the fractions of bleed-through of CFP and YFP fluorescence, respectively, through the FRET filter channel. The negative FRETC values obtained in some control experiments are due to slight overestimation of the bleed-through coefficients.
FRETC values were calculated from the mean fluorescence intensities for each selected sub-region of the image containing individual ruffles, phylopodia and lamellipodia (plasma membrane), diffuse fluorescence of the plasma membrane, endosomes, and tubular-vesicular ER structures according to Equation 1. Normalized sensitized FRET (FRETN) values for individual cellular compartments were calculated according to Equation 2: FRETN = FRETC/YFP x CFP, where FRETC, CFP, and YFP are the mean intensities of FRETC, CFP, and YFP fluorescence in the selected sub-region. In these calculations, the fluorescence intensity of CFP was underestimated because of donor fluorescence quenching due to FRET. The decrease in donor fluorescence was calculated from FRETC values using the conversion coefficient G (3.2 in all experiments) as described (22) and was found to be about 510% of CFP fluorescence intensities. Therefore, the underestimation did not significantly affect FRETN values. Because FRETN displays a non-linear dependence when donor or acceptor are in a significant molar excess over each other, FRETN values were calculated in the sub-regions of the cell in which the amount of the donor did not exceed the amount of the acceptor more than 2-fold and vice versa.
FRETC images are presented in pseudocolor mode. FRETC intensity is displayed stretched between the low and high renormalization values, according to a temperature-based lookup table with blue (cold) indicating low values and red (hot) indicating hot values. To eliminate the distracting data from regions outside of cells, the YFP channel was used as a saturation channel, and the FRETC images are displayed as YFP intensity-modulated images. In these images, data with YFP values greater than the high threshold of the saturation channel are displayed at full saturation, whereas data values below the low threshold are displayed with no saturation (i.e. black). All calculations were performed using the Channel Math and FRET modules of the SlideBook software.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
The presence of functional transporters in PAE cells stably expressing YFP/CFP-DAT was confirmed by measurements of [3H]DA uptake. All cell lines exhibited specific [3H]DA uptake (Fig. 1B), defined as the difference between [3H]DA uptake in the absence and presence of 1 mM cocaine. Kinetic analysis confirmed that each cell line contained high affinity, cocaine-sensitive [3H]DA uptake with Km values in the range of 216 µM. CFP-DAT was expressed in PAE cells at higher levels than YFP or untagged DAT and thus tended to yield higher Km values. Importantly, however, Km values measured in PAE cells expressing YFP-DAT, CFP-DAT, or untagged DAT were not consistently different from each other (Fig. 1B) and were similar to values measured in our previous work with DAT expressed in Xenopus oocytes (24).
The plasma membrane expression of DAT fusion proteins in PAE cells was
confirmed by surface biotinylation assays using a membrane-impermeable
derivative of biotin. Biotinylated untagged DAT and YFP-DAT migrated on
SDS-PAGE as smeared bands of 7375 kDa and 103105 kDa (the
difference corresponds to
26 kDa of YFP), respectively
(Fig. 1C). Minor
biotinylated bands of
180190 kDa for DAT and
250 kDa for
YFP-DAT were also detected. These forms may represent SDS-resistant dimers of
DAT and YFP-DAT, correspondingly. Several species of DAT and YFP-DAT were also
detected in the supernatants from the NeutrAvidin affinity column. The
non-biotinylated forms, which migrated similar to the biotinylated forms, are
likely to correspond to surface-exposed DAT (especially, in the case of
YFP-DAT) that were not biotinylated due to the limited efficiency of
biotinylation or internalized, endosomal DAT. Other forms with molecular
weights different from the surface forms probably represent monomeric and
dimeric species of DAT and YFP-DAT located intracellularly. A 75- to 80-kDa
band of YFP-DAT (IC-1) recognized by antibodies to DAT
(Fig. 1C) and GFP
(data not shown) represents non-glycosylated YFP-DAT. The corresponding form
of DAT (50 kDa) was poorly detected because of the overlap with IgG, but was
clearly detectable in cell lysates (data not shown). A 180- to 190-kDa form
(IC-2) may correspond to SDS-resistant dimers of IC-1
(Fig. 1C).
That YFP- and CFP-DAT were efficiently transported along the biosynthetic pathway to the cell surface was also demonstrated by fluorescence microscopy. Fig. 2 shows that CFP-DAT and YFP-DAT co-expressed in PAE cells were mainly localized in the plasma membrane, often concentrating in ruffle- and phylopodia-like structures (Fig. 2). In addition, a pool of fluorescent DAT could be also seen in the perinuclear and Golgi area of many cells. These transporters probably represent newly synthesized YFP/CFP-DAT located in the ER and Golgi complex.
|
FRET between CFP-DAT and YFP-DATDA uptake and cell surface
biotinylation experiments validated the use of YFP-DAT and CFP-DAT expressed
in PAE cells to examine the oligomeric state of these proteins in various
cellular compartments. In previous studies we described a three-filter method
of FRET microscopic analysis in living cells
(19). This method allows rapid
measurement of sensitized FRET signals with high spatial resolution.
Fig. 2A shows that
positive CFP YFP FRETC signals were detected in the diffuse
fluorescence areas and phylopodia/ruffle structures of the plasma membrane
when YFP-DAT and CFP-DAT were co-expressed in PAE cells. These data suggest
that the amino termini of the two proteins are in close proximity to each
other and that DAT forms dimers or higher oligomers at the cell surface.
As a control for FRET analysis of the CFP/YFP-DAT pair, we measured FRET signals in cells co-expressing YFP-DAT with an unrelated integral membrane protein, the receptor for epidermal growth factor (EGFR) tagged with CFP (EGFR-CFP). Co-expressed YFP-DAT and EGFR-CFP were highly co-localized in the plasma membrane (Fig. 2B). However, no FRETC signals were detected in these cells, suggesting that the proteins did not form complexes.
Because the efficiency of energy transfer depends on the amount of donor and acceptor capable of interaction, FRETC values were divided by the product of YFP and CFP intensities on a pixel-by-pixel basis to obtain normalized FRETC (FRETN) values for various regions of the cell (see "Experimental Procedures"). FRETN values correlate with the equilibrium binding constant for the measured interaction and may, therefore, serve as a measure of the relative affinity of interaction (22). FRETN values in cells expressing CFP-DAT/YFP-DAT were similar in the diffuse fluorescence areas and ruffle/phylopodia structures of the plasma membrane (Table I). These values were substantially higher than in control cells expressing two co-localized but non-interacting proteins, EGFR-CFP and YFP-DAT (Table I).
|
DAT has been shown to be capable of rapid endocytosis when heterologously expressed (3, 5, 6, 25). To test whether DAT remains oligomerized during endocytic trafficking, the endocytosis of CFP- and YFP-DAT in PAE cells was induced by PMA. Exposure to PMA at 37 °C for 530 min (1 µM) caused accumulation of YFP- and CFP-DAT in vesicular structures in the perinuclear area in most of the cells, and this occurred concomitantly with a decrease in the plasma membrane fluorescence (Fig. 2). Down-regulation of DAT has also shown to be triggered by amphetamine in HEK293 cells, although the endosomal localization of DAT in amphetamine-treated cells was not confirmed (6). Using fluorescent DAT proteins we show here that YFP- and CFP-DAT accumulated in vesicular organelles after 2-h treatment of PAE cells at 37 °C with 4 µM amphetamine (Fig. 2A). In contrast to PMA-treated cells, visible accumulation of DAT in vesicular organelles was observed in a limited fraction of the amphetamine-treated cell population, and the extent of endosomal accumulation of DATs was smaller than in cells treated with PMA. Importantly, positive FRETC signals were detected in vesicular organelles containing YFP- and CFP-DAT in cells treated with either PMA or amphetamine (Fig. 2A). FRETN values varied significantly in different cells but were consistently higher in endosomes (see Table I).
Phorbol esters are known to cause transient down-regulation and endosomal accumulation of EGFR (26). Interestingly, PMA treatment of PAE cells co-expressing YFP-DAT and EGFR-CFP resulted in translocation of both cargoes into the same vesicular organelles (Fig. 2B). As in the case of untreated cells, no FRETC signals between DAT-CFP and EGFR-YFP were detected in PMA-treated cells, suggesting that the proteins did not form complexes (Table I).
Having shown FRET between co-expressed CFP- and YFP-DAT in endothelial cells, we examined whether similar oligomerization can be observed in dopaminergic cells. To this end, we used an immortalized dopaminergic cell line 1RB3AN27 (20). These cells begin to express tyrosine hydroxylase and differentiate under conditions of growth in the presence of dibutyryl cAMP (20). Oligomerization of CFP/YFP-DAT was also observed in 1RB3AN27 cells expressing CFP-DAT and YFP-DAT (Fig. 3). CFP- and YFP-DAT displayed localization similar to that observed in PAE cells. FRETC signals were readily detectable at the cell surface in control cells, in endosomes in cells treated with PMA (Fig. 3 and Table I), and in the ER (data not shown).
|
Accumulation of CFP-DAT in Early Endosomes and Late Recycling Compartment in Cells Treated with PMA and AmphetamineTo confirm that vesicular structures containing DAT in cells treated with PMA or amphetamine represent endosomal compartments, the localization of DAT was compared with that of endosomal markers. PAE cells stably expressing CFP-DAT (PAE/CFP-DAT) were transiently transfected with YFP-fused Rab5a (YFP-Rab5), a resident protein of early endosomes (27) or with YFP fusion of Rab11a (YFP-Rab11), a marker of early and late recycling endosomes (27, 28). As shown in Fig. 4A, a number of DAT-CFP containing structures also contained YFP-Rab5 in cells treated with PMA or amphetamine, whereas there was very minimal, if any, localization of CFP-DAT in Rab5-positive endosomes in untreated cells. The pattern of distribution of two proteins within the endosomes was not, however, identical. High resolution images of living cells showed that CFP-DAT was typically distributed in the entire endosomal membrane and, possibly, inside the endosome. In contrast, YFP-Rab5 was often concentrated in one or several poles of the endosome, in so-called "Rab5 domains" (29).
|
DAT was also well co-localized with another endosomal protein, Hrs (Fig. 4B). This protein is involved in targeting of ubiquitylated cargo to the internal membranes of multivesicular bodies and lysosomal degradation pathway (30). Hrs overexpression results in the blockade of endosomal trafficking, recruitment of clathrin to endosomes, and enlargement of endosomes (31). Interestingly, however, DAT was mainly excluded from the Hrs-containing microdomains/coats within individual endosomes (Fig. 4B), suggesting that the endosomal sorting of DAT is not mediated by the Hrs pathway.
YFP-Rab11 was mainly localized in tubular-vesicular and vesicular compartments in the Golgi area and cell periphery (Fig. 5). As in the case of Rab5, a pool of CFP-DAT was co-localized with YFP-Rab11 in endosomes upon treatment with PMA or amphetamine. A small amount of CFP-DAT was seen co-localized with Rab11 in untreated cells, presumably as the result of constitutive internalization and recycling of CFP-DAT. These data demonstrate in living cells that DAT is internalized in response to PMA and amphetamine treatment into various types endosomal compartments that are involved in major sorting and recycling pathways.
|
Confocal microscopy was used to confirm the accumulation of DAT in early endosomes using an endogenous marker of these compartments. To this end, CFP-DAT localization was compared with localization of the early endosomal marker EEA.1 in fixed cells using image reconstitution by deconvolution of three-dimensional images (Fig. 6). The optical sectioning revealed significant co-localization of CFP-DAT and EEA.1 in endosomes (Fig. 5). Together, Figs. 4, 5, 6 demonstrate the PMA- or amphetamine-induced endocytosis of tagged DAT into early-recycling endosomal compartments, in which, according to FRET analysis, DAT molecules are oligomerized (Figs. 23).
|
Co-immunoprecipitation of Wild-type Untagged and Mutant YFP-tagged
DATFRET microscopic analysis provided strong evidence for the
constitutive oligomerization of DAT in living PAE cells. To confirm the
oligomeric status of DAT using a different assay, co-immunoprecipitation
experiments were performed. To this end, wild-type DAT or CFP-DAT was
constitutively or transiently co-expressed with YFP-N-DAT, a mutant in
which the entire amino-terminal portion (65 amino acid residues) was deleted
and replaced by YFP. PAE cells constitutively expressing DAT or
YFP-
N-DAT alone were used as controls. YFP-
N-DAT was efficiently
transported to the surface in PAE cells under all expression conditions based
on surface biotinylation (Fig.
7A) and fluorescence imaging experiments
(Fig. 7B). A similar,
but untagged, DAT mutant was retained in the ER
(17). It is possible that the
attachment of YFP to the truncated DAT in our experiments had a stabilizing
effect on the transporter molecule, thus allowing proper folding of
YFP-
N-DAT in the ER and effective ER export. Transient co-expression of
YFP-
N-DAT with CFP-DAT in PAE cells resulted in co-localization of the
two proteins in the plasma membrane and positive FRETC signals
(Fig. 7B), similar to
what was observed with wild-type CFP- and YFP-DAT proteins
(Fig. 2). Despite expression at
the cell surface, however, no [3H]DA uptake by YFP-
N-DAT was
detected (data not shown). Interestingly, a DAT mutant, in which the 59 amino
terminal residues of DAT were deleted, is delivered to the plasma membrane and
displayed normal dopamine transport in Madin-Darby canine kidney cells
(32). This may suggest a
critical role of residues 5965 in DAT transport activity.
Alternatively, the discrepancies between trafficking and transport activities
of amino-terminally truncated DAT mutants observed by Gu and co-workers
(32), Torres and co-workers
(17), and in our experiments
may be due to different cellular backgrounds.
|
In co-immunoprecipitation experiments, YFP-N-DAT was precipitated
from the lysates of PAE cells stably co-expressing DAT and YFP-
N-DAT
using a GFP antibody. The immunoprecipitates were probed with rat anti-DAT
antibody that recognizes the full-length DAT but not the YFP-
N-DAT
mutant. The wild-type DAT migrating on SDS-PAGE at the position corresponding
to monomeric plasma membrane DAT (7375 kDa, see also
Fig. 1) was detected in GFP
immunoprecipitates, indicative of YFP-
N-DAT/DAT association
(Fig. 7C). Other forms
of DAT were not detected in significant amounts in immunoprecipitates and cell
lysates in these experiments. Based on densitometric analysis of the intensity
of chemiluminescence signals from DAT immunoreactivity in anti-GFP
immunoprecipitates and lysates,
20% of total cellular DAT was
co-immunoprecipitated with YFP-
N-DAT. Together with FRET analysis,
these data strongly support the existence of DAT in an oligomeric state.
Oligomerization of YFP-DAT and CFP-DAT in the Biosynthetic CompartmentsTo test whether newly synthesized DAT exists as an oligomer during its anterograde transport through the ER and Golgi, a mutant of DAT with impaired transport to the plasma membrane was constructed. In this mutant the last five amino acid residues of the carboxyl terminus that include a PDZ (PSD/Discs large/zona occludens-1) domain-binding sequence important for plasma membrane transport (7) were deleted (YFP-DAT-C'615). When transiently expressed in PAE or other cells for 12 days, this mutant was primarily localized in the tubular-vesicular network seen throughout the entire cell, often in the nuclear envelope membrane and concentrated in the Golgi area (Fig. 8). This pattern of localization is typical of the distribution of newly synthesized proteins in the ER, intermediate compartment, and Golgi complex (further referred as "ER localization"). A small pool of YFP-DAT-C'615 could be detected at the cell surface after 3 days of expression, in contrast to DAT mutants lacking 35 or 42 amino acid residues of the carboxyl terminus, which were not transported to the plasma membrane under any conditions (data not shown).
|
When YFP-DAT-C'615 was co-expressed with CFP-DAT, both proteins were co-localized in the ER in some cells. In other cells CFP-DAT displayed strong plasma membrane fluorescence, whereas YFP-DAT-C'615 was located in the ER. Analysis of the relative expression of the two transporters in individual cells revealed that in cells with a relatively high CFP/YFP ratio (for example, 3:1 in Fig. 8A), most of the CFP-DAT reached the cell surface. In contrast, in cells expressing relatively higher levels of YFP-DAT-C'615 (for example, 1:1 CFP/YFP ratio in Fig. 8A), both wild-type and mutant DAT were retained in the ER. In the latter case, positive FRETC signals were detected, suggesting CFP-DAT/YFP-DAT-C'615 oligomerization in the ER (Fig. 8A and Table I).
To control for the specificity of FRET signals in the ER, YFP-DAT-C'615 was co-expressed with EGFR-CFP for 1618 h. At this time after transfection, a large pool of EGFR-CFP was moving through the biosynthetic pathway and was highly co-localized with YFP-DAT-C'615 (Fig. 8B). However, no positive FRET signal was detected (Fig. 8B). The FRETN values of ER-localized YFP/CFP-DAT in PAE cells were on average lower than these values in other cellular compartments, although they were significantly higher than FRETN of the negative control, EGFR-CFP/YFP-DAT-C'615 (Table I). The data presented in Fig. 8A suggest that the formation of heterooligomers between the wild-type DAT and the ER export-defective DAT mutant may interfere with the normal delivery of wild-type DAT to the cell surface.
To further substantiate this conclusion, the effect of overexpression of DAT-C'615 on plasma membrane expression of wild-type DAT was examined using a surface biotinylation assay. DAT expression levels were relatively low in PAE cells, particularly, when the two DAT fusion proteins were co-expressed. Therefore, for these biochemical experiments we used HEK293 cells, which allow high efficiency of DAT expression in a large fraction of the cell population. These cells have previously been well characterized as a DAT expression system (6). Transiently co-expressed CFP-DAT and YFP-DAT were located at the plasma membrane in many HEK293 cells (Fig. 9A), although in cells with very high levels of expression, the two proteins were often retained in the ER. As observed in PAE cells, CFP-DAT was retained together with the traffic-defective mutant YFP-DAT-C'615 in the ER of HEK293T cells when the mutant was expressed at near equimolar or higher levels compared with CFP-DAT (Fig. 9A).
|
Co-expression of YFP-DAT-C'615 with untagged DAT significantly and concentration-dependently reduced the amount of DAT detectable by surface biotinylation (Fig. 9B). Only small amounts of a 105-kDa form of YFP-DAT-C'615 (corresponding to the monomeric plasma membrane form of YFP-DAT, see Fig. 1) was detected by blotting with anti-GFP in lysates (Fig. 9B, right panel) and the biotinylated fraction (data not shown). The major forms of the mutant (7580 kDa and 180190 kDa bands) were not accessible to surface biotinylation and were, therefore, located intracellularly (Fig. 9B). These data are consistent with the imaging analysis demonstrating an impaired transport of YFP-DAT-C'615 to the cell surface. The dominant-negative effect of the ER export-deficient DAT mutant on surface expression of DAT further suggested that oligomerization of the newly synthesized DAT occurs in the ER and that proper DAT oligomerization is necessary for the efficient transport of DAT from the ER to the plasma membrane.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Phorbol ester-induced endocytosis of DAT has been demonstrated previously in several heterologous-expression systems (3, 5). Accordingly, exposure to 1 µM PMA caused a dramatic accumulation of fluorescent DAT in endosomes of PAE cells, as well as in an immortalized dopaminergic cell line 1RB3AN27. These endosomes contained Rab5, EEA.1, and Hrs and can be classified as early and intermediate compartments. To a lesser extent, DAT was localized in the late recycling compartment identified by Rab11. Interestingly, however, DAT was mainly excluded from Rab5 and Hrs microdomains in endosomes. This observation suggests that DAT is not specifically recruited to the Hrs-dependent degradation pathway used mainly by ubiquitylated cargo (30).
Moreover, using PAE/DAT cells we were able to demonstrate endocytosis of DAT induced by 4 µM amphetamine into the same early/recycling endosomal compartments. This observation provides direct support for the hypothesis that DAT down-regulation observed upon similar exposure to amphetamine in HEK293 cells is the result of DAT internalization to endosomes (6). In all of these studies, including our own, relatively long exposure times were used. However, evidence from experiments following in vivo exposure to methamphetamine and DA supports the relevance of substrate-induced down-regulation in brain (4, 33, 34). Furthermore, protein kinase C inhibitors block substrate-induced DAT down-regulation in in vitro models, suggesting that protein kinase C-induced endocytosis is involved in the regulation mediated by substrates (4, 35, 36).
The capability to visualize trafficking of functional YFP/CFP-DAT in PAE cells prompted us to use FRET to analyze DAT oligomerization. Biochemical methods of detection of dimerization, such as immunoprecipitation and cross-linking, require cell disruption and cannot guarantee that dimers are not formed during cell lysis. In contrast, the FRET technique allows detection of dimerization in intact living cells. Modification of the three-filter method to measure sensitized FRET was previously employed in our studies of proteins involved in trafficking and signaling of EGFR (19). The high resolution of this technique allows analysis of FRET efficiencies in individual organelles in living cells. The three-filter method also allows normalization of FRET signals to the amount of donor and acceptor on a pixel-by-pixel basis. The resulting FRETN values obtained for each individual pixel or group of pixels corresponding to a cellular organelle can be then compared.
FRET analysis of interactions between YFP-DAT and CFP-DAT revealed positive FRETC and FRETN values in all cellular compartments, indicative of close proximity of two proteins and consistent with dimerization or higher oligomerization. The stringent quality control in the ER could be the reason for relatively homogeneous but low level expression of DAT in PAE cells and, therefore, relatively weak FRETC signals. It should be pointed out that even under conditions of an equimolar ratio of YFP- and CFP-DAT, the probability of heterodimerization of YFP- and CFP-DAT is only 50%, which reduces the maximal FRETC and FRETN values by 2-fold compared with the values that would be observed for the same affinity interaction of two different proteins. In general, FRETN values in Table I have relatively high standard deviations, as have been observed in all previous studies using this technique (22, 37). Furthermore, the biological significance of the differences among FRETN values obtained in different cellular compartments is uncertain. The ER in PAE cells tended to yield the lowest FRETN values, presumably due to low local concentrations of YFP- and CFP-DAT in this organelle in these cells. The highest FRETN values were obtained in endosomes, possibly because of the high local concentrations of DAT fusion proteins in these compartments. Overall, the results of the FRET analysis suggest that DAT is oligomerized constitutively and that it is unlikely that oligomerization is involved in the regulation of endocytic trafficking.
Dimerization or higher oligomerization may be, however, important for DAT trafficking from the ER to the plasma membrane. Figs. 8 and 9 show that overexpression of an ER export-defective mutant has a dominant-negative effect on the plasma membrane transport of wild-type DAT. The same effects were convincingly documented in a report by Torres and co-workers (17). These observations are consistent with the model whereby ER export-competent DAT molecules must oligomerize to traffic through the ER-Golgi system to the cell surface. The importance of oligomerization of GAT-1 for transport to the plasma membrane has been directly demonstrated by Scholze and co-workers (13).
The mechanisms responsible for DAT-DAT intermolecular interactions have recently begun to be elucidated. It is likely that TM domains are responsible for oligomerization. For example, in our experiments deletion of the amino terminus of DAT did not prevent DAT-DAT oligomerization. The predicted leucine zipper motif in TM2 appears to be involved in the intermolecular interactions (17), similar to what was demonstrated for GAT-1 oligomerization (13). TM6 has also been proposed to be one of the interaction interfaces (16). Studies of DAT molecular size using radiation inactivation predicted that DAT is a tetramer (14, 15). The tetrameric state of SERT was also predicted based on co-immunoprecipitation experiments (11). However, our FRET microscopic analysis does not provide a reliable way to distinguish tetramerization from dimerization.
Although live-cell FRET analysis requires expression of transporters in heterologous systems, DAT oligomerization has been demonstrated in an immortalized dopaminergic cell line (Fig. 3) and rat striatum by both cross-linking and radiation inactivation techniques (14, 16), suggesting that this property of the DAT molecule may be important for its function in dopaminergic neurons. The exact functional role of DAT oligomerization in the regulation of DA uptake remains to be elucidated. However, expression of SERT concatenations consisting of four SERT molecules resulted in chimeric proteins with increased affinities for serotonin uptake (9). Oligomerization of SERT has also been suggested to be important for efficient serotonin transport (11). Because TM domains are critical for both proper folding and oligomerization of DAT, mutations in TM domains can affect the transport capacity of DAT at the plasma membrane indirectly through the poor delivery of the newly synthesized transporter to the plasma membrane, as well as directly. Therefore, careful analysis of subcellular localization of TM mutants of DAT will be necessary to elucidate the role of oligomerization in the transport function of DAT.
![]() |
FOOTNOTES |
---|
To whom correspondence should be addressed: Dept. of Pharmacology, University
of Colorado Health Science Center, 4200 E. Ninth Ave., Denver, CO 80262. Tel.:
303-315-7252; Fax: 303-315-7097; E-mail:
alexander.sorkin{at}uchsc.edu./.
1 The abbreviations used are: DA, dopamine; DAT, dopamine transporter; GFP,
YFP, and CFP, green, yellow, and cyan fluorescent proteins; FRET, fluorescence
resonance energy transfer; FRETC, corrected FRET; PAE, porcine
aortic endothelial; ER, endoplasmic reticulum; PMA, phorbol 12-myristate
13-acetate; TM, predicted transmembrane domains; sulfo-NHS-S-S-biotin,
sulfosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate; SERT, serotonin
transporter; GAT-1, -aminobutyric acid transporter; EGFR, epidermal
growth factor receptor; CMF-PBS,
Ca2+,Mg2+-free phosphate-buffered
saline; DTT, dithiothreitol; FRETN, normalized sensitized FRET.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|