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The Journal of Neuroscience, December 15, 2002, 22(24):10643-10652
A Novel Sorting Motif in the Glutamate Transporter Excitatory
Amino Acid Transporter 3 Directs Its Targeting in Madin-Darby Canine
Kidney Cells and Hippocampal Neurons
Chialin
Cheng1,
Greta
Glover2,
Gary
Banker2, and
Susan G.
Amara1
1 Howard Hughes Medical Institute and Vollum Institute
and 2 Center for Research on Occupational and Environmental
Toxicology, Oregon Health and Science University, Portland, Oregon
97239
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ABSTRACT |
The glutamate transporter excitatory amino acid transporter 3 (EAAT3) is polarized to the apical surface in epithelial cells and
localized to the dendritic compartment in hippocampal neurons, where it
is clustered adjacent to postsynaptic sites. In this study, we analyzed
the sequences in EAAT3 that are responsible for its polarized
localization in Madin-Darby canine kidney (MDCK) cells and neurons.
Confocal microscopy and cell surface biotinylation assays demonstrated
that deletion of the EAAT3 C terminus or replacement of the C terminus
of EAAT3 with the analogous region in EAAT1 eliminated apical
localization in MDCK cells. The C terminus of EAAT3 was sufficient to
redirect the basolateral-preferring EAAT1 and the nonpolarized EAAT2 to
the apical surface. Using alanine substitution mutants, we identified a
short peptide motif in the cytoplasmic C-terminal region of EAAT3 that
directs its apical localization in MDCK cells. Mutation of this
sequence also impairs dendritic targeting of EAAT3 in hippocampal
neurons but does not interfere with the clustering of EAAT3 on
dendritic spines and filopodia. These data provide the first evidence
that an identical cytoplasmic motif can direct apical targeting in
epithelia and somatodendritic targeting in neurons. Moreover, our
results demonstrate that the two fundamental features of the
localization of EAAT3 in neurons, its restriction to the
somatodendritic domain and its clustering near postsynaptic sites, are
mediated by distinct molecular mechanisms.
Key words:
dendritic sorting signal; apical sorting signal; glutamate transporter; polarized trafficking; EAAT3; excitatory amino
acid carrier
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INTRODUCTION |
The actions of glutamate, the major
excitatory neurotransmitter in the mammalian CNS, are terminated by
sodium-dependent glutamate transporters located on the plasma membrane
of neurons and glia. Glutamate transporters constitute a distinct gene
family of which five human glutamate transporters [excitatory amino
acid transporters 1-5 (EAAT1-5)] have been identified (Arriza et
al., 1994 , 1997; Fairman et al., 1995). The rodent homologues of EAAT1
(glutamate/aspartate transporter) and EAAT2 (glutamate
transporter-1) are primarily expressed in glia (Rothstein et
al., 1994; Lehre et al., 1995), whereas EAAT3 (EAAC1) and EAAT4 are
expressed predominantly in neurons (Furuta et al., 1997; Dehnes et al.,
1998). EAAT3 is widely expressed in the CNS as well as in epithelial
cells in the kidney and gut. Surprisingly, the carrier is not found on
the axons or presynaptic terminals, which release glutamate. Instead,
EAAT3 and EAAT4 are targeted to the somatodendritic domain and
concentrated near postsynaptic sites (Coco et al., 1997; Furuta et al.,
1997; Conti et al., 1998; Dehnes et al., 1998; He et al., 2000) where they are thought to regulate synaptic signaling by limiting the diffusion of glutamate to extrasynaptic receptors (Brasnjo and Otis,
2001).
The Madin-Darby canine kidney (MDCK) cell line has been used
extensively as a simple system to study the sorting of polarized membrane proteins (Scannevin et al., 1996; Kryl et al., 1999; Poyatos
et al., 2000; Martinez-Maza et al., 2001; McCarthy et al., 2001). In
MDCK cells, apical and basolateral proteins are segregated from each
other as they exit the trans-Golgi network and then subsequently
delivered to the appropriate surface. In the case of basolateral
proteins, sorting depends on short motifs present in the cytoplasmic
tails of the proteins. Apical sorting is less well understood. Some
proposed apical sorting mechanisms include
glycosylphosphatidylinositol linkage, raft association, and
glycosylation (Winckler and Mellman, 1999). More recently, evidence has
emerged that cytoplasmic regions of proteins may also contain
information important for apical sorting. For instance, a 39-amino acid
sequence in the cytoplasmic C terminus of rhodopsin directs its apical
localization in MDCK cells (Chuang and Sung, 1998). Similarly, a
32-amino acid sequence at the C terminus of GABA transporter 3 (GAT-3)
confers apical localization (Muth et al., 1998). This region contains a
motif that may bind to postsynaptic density-95/Drosophila
disc large protein/zona occludens protein 1 domains (Muth et
al., 1998), but it is not known whether this mediates its targeting.
Discrete, well defined sorting motifs have yet to be defined in these,
or other, apically targeted proteins.
Many proteins that are dendritically localized in neurons are
concentrated on the basolateral surface of MDCK cells, and in some
cases, the same motifs mediate sorting in both cell types. The
endogenous localization of EAAT3 represents an exception to this idea:
EAAT3 is present at the apical membrane in kidney cells and on the soma
and dendrites of neurons (Coco et al., 1997; Shayakul et al., 1997). If
the sorting of EAAT3 depends on its association with lipid rafts or its
glycosylation, as observed for some apical proteins, this would be the
first instance in which such signals mediated dendritic targeting in
neurons. It would be equally unusual to find a cytoplasmic, dendritic
targeting motif that mediated apical sorting in MDCK cells.
Alternatively, it may be that different motifs mediate sorting in the
two cell types. To elucidate the motifs involved in the sorting of
EAAT3 and to resolve this paradox, we have investigated the sorting of
EAAT3 in MDCK cells and in hippocampal neurons. By expressing a series
of mutated proteins, we have identified a novel 11-amino acid motif in
the cytoplasmic tail of EAAT3 that mediates its apical sorting in MDCK
cells and its somatodendritic localization in hippocampal neurons.
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MATERIALS AND METHODS |
Cell culture
MDCK cells (American Type Culture Collection, Manassas, VA) were
maintained in DMEM (Invitrogen, Carlsbad, CA) supplemented with 10%
fetal bovine serum, 89 U/ml penicillin, and 89 µg/ml streptomycin.
Media for stable MDCK cell lines were further supplemented with 500 µg/ml geneticin (G418) (Invitrogen). The cells were plated at
high density onto 12 or 24 mm Transwell-COL filters (Costar, Cambridge,
MA) and grown to confluency before use.
Primary hippocampal cultures were prepared from embryonic day 18 rats
as described previously (Banker and Goslin, 1998). Neurons were plated
at 300,000 cells per 6 cm dish.
Mutagenesis
Various PCR-based mutagenesis protocols were used to create the
mutant constructs. All the mutant constructs were sequenced to confirm
that no unintended mutations were introduced. All constructs were
subcloned into the pEGFPC vector (Clontech, Palo Alto, CA).
To construct the EAAT1/EAAT3 C-terminal chimeras (E1CT3 and E3CT1),
silent mutations at
E501L502 and
L470 of EAAT1 and EAAT3 cDNAs,
respectively, were introduced by using the three-primer PCR mutagenesis
technique (Seal and Amara, 1998) to create the SacI
restriction site. The C termini of EAAT1 and EAAT3 were exchanged at
the SacI restriction site to produce the resultant E1CT3 and
E3CT1 constructs. EAAT3 C-terminal deletion constructs were made with
the last amino acid of the mutant being the following:
E469,
P484, S497,
and V510 for E3 470-524, E3485-524,
E3498-524, and E3511-524, respectively. The constructs
containing C-terminal regions of EAAT3 fused to EAAT2 were created by
attaching EAAT3 C-terminal cDNA fragments to full-length EAAT2 cDNA
separated by a linker (containing the sequence GCC GGA TCT GCC). The
amino acid residues of EAAT3 contained in the EAAT3 C-terminal
fragments are denoted in parentheses. For instance, E2-3(469-524)
contains the amino acids
E469-F524 of
EAAT3 fused to full-length EAAT2. The QuikChange site-directed mutagenesis kit from Stratagene (La Jolla, CA) was used to create the
triple or double alanine substitution mutants. The template was green
fluorescent protein (GFP)-E2-3(498-524), and primers were created
with the least nucleotide changes to the alanine codon.
Transfection
Stable transfection into MDCK cells. MDCK cells (at
50-80% confluency) in each well of a six-well plate were transfected
with a mixture containing 2 µg of DNA and 40 µg of LipofectAMINE
(Invitrogen) in serum-free DMEM. After 5 hr, 10% fetal bovine serum,
89 U/ml penicillin, and 89 µg/ml streptomycin were added to the
mixture. Twenty-four hours after the start of transfection, the
DNA-LipofectAMINE-DMEM mixture was replaced with fresh DMEM containing
serum, penicillin, streptomycin, and 500 µg/ml G418 (G418 media).
Forty-eight to 72 hr after the start of transfection, three wells
containing the greatest number of fluorescent cells were replated at
low density and placed in G418 media for 2-3 weeks to select for
stably transfected cell lines. G418-resistant cell lines were screened for detectable GFP fluorescence, and cell lines with higher GFP fluorescence levels were chosen for use in this study. For each construct, results were verified with at least two independently derived stable cell lines.
Transient transfection into MDCK cells. For each construct
to be transfected, 1.2 µg of DNA was combined with 2.4 µg of
LipofectAMINE 2000 (Invitrogen) in serum-free DMEM and incubated at
room temperature for 30 min. The mixture was then combined with MDCK
cells in media and plated at high density onto a 12 mm Transwell-COL
filter. The bottom of the transwell filter was filled with media.
Twenty-four hours after the start of transfection, the wells of
transfected cells were replaced with fresh media. The cells were
allowed to grow to confluency for a further 48-72 hr before use.
Transient transfection of dissociated hippocampal neuronal
cultures. Constructs were introduced into cultures at 8-10 d
in vitro using Effectene-mediated transfection (Qiagen,
Valencia, CA). Briefly, for each construct, 1 µg of DNA was mixed
with 8 µl of enhancer and then 10 µl of Effectene according to the
manufacturer's instructions. After addition of the DNA-Effectene
complexes to the media, the cells were incubated for 2 or 9 d at
37°C. The cells were then fixed in 4% paraformaldehyde and 4%
sucrose and permeabilized in 0.25% Triton X-100, and coverslips were
mounted on slides using elvanol. GFP fluorescence was used to measure cell surface distribution. Expression of the constructs for a minimum
of 2 d allowed for accumulation at the cell surface and minimized
the contribution of intracellular fluorescence. Under these conditions,
the GFP signal appears enriched at the surface when examined at high
magnification. Moreover, expression of the EAAT constructs had reached
a steady state at these times.
Immunohistochemistry and confocal microscopic imaging
All steps were performed at room temperature. The cells were
initially rinsed twice with PBS (containing 100 µM
CaCl2 and 1 mM
MgCl2) before they were fixed with 4%
paraformaldehyde (with 50 mM HEPES in PBS, pH 7.4) for 20 min. Then the cells were rinsed twice with PBS and incubated with a
blocking and permeabilization solution containing 5% horse serum, 1%
BSA, and 0.2% Triton X-100 in PBS for 30 min. Subsequently, staining
for E-cadherin was performed with the monoclonal antibody
anti-uvomorulin clone DECMA-1 (Sigma, St. Louis, MO). The cells were
incubated in the primary antibody for 2-3 hr. The cells were washed
three times for 3 min with PBS and probed with rhodamine red-conjugated
secondary antibody (Jackson ImmunoResearch, West Grove, PA). The cells
were washed three times for 3 min with PBS before the filter containing
the cells was excised from its support and mounted onto a slide with
ProLong antifade reagent (Molecular Probes, Eugene, OR). Images were
constructed by measuring the fluorescence signal using confocal
microscopy (MRC 1024 system; Bio-Rad, Hercules, CA) with excitation
lines of 488 and 568 nm for the GFP and rhodamine signals,
respectively. The z-series was collected by initially
focusing on the middle section of the cells (position 0) and scanning 8 µm above to 8 µm below this plane at 0.1 µm intervals.
Cell surface biotinylation assay
Cell surface expression of the EAAT constructs was assayed with
modifications of the method described previously (Daniels and Amara,
1998). All steps before the Western blot analysis were performed at
4°C unless otherwise specified. Briefly, the cells were washed once
quickly with room temperature PBS and then three times for 10 min with
cold PBS. The cells were then incubated with 2 mg/ml sulfosuccinimidyl
2-(biotinamido)ethyl-1,3-dithiopropionate (sulfo-NHS-SS-biotin) (Pierce, Rockford, IL) in biotinylation buffer (in mM: 2 CaCl2, 150 NaCl, and
10 triethanolamine, pH 7.5) for 40 min placed either on the top or
bottom of the transwell filter to assay for apical or basolateral
expression of the transporters, respectively. The side not incubated
with sulfo-NHS-SS biotin was incubated with biotinylation buffer only.
The reaction was quenched by incubation with 100 mM glycine
in PBS for 20 min. The cells were washed with PBS and then lysed with
lysis buffer [1% Triton X-100, 150 mM NaCl, 5 mM EDTA, and 50 mM Tris, pH 7.5, containing a
protease inhibitor mixture (1×; Roche Molecular Biochemicals, Indianapolis, IN)], and the cell lysate was collected after
centrifugation at 14,000 × g for 10 min. The cell
lysate was incubated with Ultralink immobilized NeutrAvidin beads
(Pierce) for 2-3 hr. The beads were separated from the supernatant by
centrifugation at 5000 × g for 15 min. The beads
contained cell surface proteins, whereas the supernatant contained
cytosolic proteins. The beads were washed three times with lysis
buffer, twice with high-salt wash buffer (0.1% Triton X-100, 500 mM NaCl, 5 mM EDTA, and 50 mM Tris, pH 7.5), and once with no-salt wash
buffer (50 mM Tris, pH 7.5). Subsequently, the
proteins on the beads were released by incubation with SDS loading
buffer containing 200 mM DTT. The proteins were separated on 8% polyacrylamide gels and then transferred onto Immobilon-P blots (Millipore, Bedford, MA). The blots were probed for
the expression of EAATs with either polyclonal antibodies to the C
terminus of the EAATs or a monoclonal antibody to GFP (JL-8; Clontech)
and visualized with horseradish peroxidase-conjugated secondary
antibody and chemiluminescent reagent (PerkinElmer, Beverly, MA). Blots
were exposed to film to obtain nonsaturated exposures, which could be
used for densitometric quantitation of chemiluminescent signals. Films
were scanned using a Duoscan F40 (Agfa, Ridgefield Park, NJ), and band
intensities in each lane (same area size in each lane) were
background-subtracted and quantitated by using the software program
TINA (Fuji Medical Systems, Stamford, CT). Statistical significance was
determined with the Newman-Keuls multiple-comparisons test for
independent groups using WINKS evaluation software (Texasoft, Cedar
Hill, TX).
Imaging and quantitation of GFP fluorescence in
hippocampal neurons
Neurons were chosen for analysis only if they were sufficiently
separated from other transfected cells to ensure that all labeled
processes arose from the cell pictured. Images were acquired on a Leica
(Nussloch, Germany) DMIRBE microscope linked to a Princeton Instruments
Micromax chilled CCD camera controlled by Metamorph imaging software
(Universal Imaging, Downingtown, PA). For cells at 11 d in
vitro, low-magnification images were acquired using a 16×, 0.5 numerical aperture (NA) Plan Fluotar objective, and high-magnification
images for quantification were acquired using a 63×, 1.32 NA Plan Apo
objective. Cells at 19 d in vitro were imaged on the
same microscope at 63× by acquiring a through-focal series of images
at 0.2 µm intervals (~30 planes). Stacks of these images were
subject to constrained-iterative deconvolution (15 iterations) using
Deltavision SoftWoRx 2.5 software (Applied Precision, Issaquah, WA) based on the point spread function determined for our
microscope. High-magnification images of 19 d in vitro
cells are displayed as single planes of the resulting deconvolved stacks.
Images for quantification were corrected for background fluorescence
and uneven field illumination. The average fluorescence intensity in
axons and dendrites was obtained by drawing a series of ~10
one-pixel-wide lines down the center of processes on corrected high-magnification digital images. For each cell, the fluorescence for
each line in the axons and dendrites was averaged to obtain the final
values for axon and dendrite intensity. These averages were used to
calculate the axon/dendrite ratio. Theoretically, one would expect a
uniformly distributed protein to have an axon/dendrite ratio of 1. In
practice, the axon/dendrite ratio for nonpolarized proteins averages
between 0.5 and 1. This is probably because of a greater contribution
of out-of-focus fluorescence in the thicker dendrites compared with the
relatively thin axons. Statistical significance of differences between
groups was determined by performing the Newman-Keuls
multiple-comparisons test for independent groups using WINKS evaluation software.
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RESULTS |
Localization of wild-type EAATs in MDCK cells
We stably expressed EAAT1-5 tagged on the N terminus with GFP in
polarized MDCK cells and examined their localization using confocal
microscopy and cell surface biotinylation. EAAT3 was extremely
polarized to the apical surface, whereas EAAT1 and EAAT2 were
expressed at both the apical and
basolateral surfaces (Figs. 1,
2B) with EAAT1 more
basolateral-preferring than EAAT2 (22 ± 8% apical versus 54 ± 1% apical; Table 1). It is important
to note that our stably transfected, GFP-tagged EAAT3 retained the normal apical localization of endogenous EAAT3 in kidney epithelial cells (Shayakul et al., 1997). EAAT4 and EAAT5 were expressed at lower
levels than the other EAATs, but they appeared to be present at both
cell surfaces (data not shown). Therefore, EAAT3 is the only glutamate
transporter that is completely polarized in MDCK cells.
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Figure 1.
Localization of wild-type EAAT1-3 in MDCK cells.
MDCK cells were stably transfected with EAAT1-3 that was tagged with
GFP at the N terminus and grown on transwell filters. A,
Representative Western blots from a cell surface biotinylation assay.
This assay assessed the stable expression level of each EAAT at each
cell surface by the application of biotin at the apical or basolateral
surfaces. There is no signal when biotin was not included in the assay.
The control blots show that
Na+/K+-ATPase is detected at the
basolateral but not the apical cell surface fraction, and actin is only
detected in the intracellular fraction. B,
Representative confocal images of EAAT1-3 in a vertical section
(z-series) illustrating the localization of GFP-EAAT
(green) and a basolateral marker, E-cadherin
(red). The top panel of each construct is
a composite image illustrating both GFP and basolateral marker signals,
and the bottom panel contains only the GFP signal. In
the vertical section, a fluorescence signal at the apical surface
appears as a horizontal line at the top
of the cell, whereas a signal at the basolateral surface appears as
vertical lines at the sides of the cell.
Occasionally, there is fluorescence attributable to the underlying
filter, which appears as a horizontal line at the
bases of all the cells. This fluorescence signal is not
scored as basolateral localization. All EAATs are expressed at both
surfaces, except EAAT3, which is restricted to the apical surface. Each
blot and image is representative of at
least two additional experiments. Scale bar, 20 µm.
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Figure 2.
Schematic representation and localization of the
EAAT3 mutants. A, Topology of EAAT1 (Seal et al., 2000)
and regions that are exchanged with EAAT3. The C terminus
(orange), the large second extracellular loop
(purple), and the N-terminal region
(blue) are shown. Numbers denote
transmembrane domains. OUT, Extracellular; IN,
intracellular. B, Schematic diagram of the EAAT1/3
C-terminal chimeras, EAAT3 deletions, and EAAT3 C terminus attached to
the end of full-length EAAT2. Regions containing sequences from EAAT1,
EAAT2, and EAAT3 are denoted by yellow,
red, and green, respectively. The
localization of the stably transfected mutants was determined by both
confocal microscopic imaging and a cell surface biotinylation assay.
The presence or absence of a signal at a domain is denoted by + or  ,
respectively.
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Table 1.
Quantitative analysis of the cell surface expression of
stably transfected EAAT constructs in MDCK cells
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The C-terminal motif directs apical localization of EAAT3 in
MDCK cells
To identify the region of EAAT3 important for its apical
localization, we made chimeras between EAAT1 and EAAT3, exchanging the
three large less conserved regions: the N terminus, the large second
extracellular loop (which contains consensus N-linked glycosylation sites), and the C terminus (Fig. 2A). Replacing the C
terminus of EAAT3 with the C terminus of EAAT1 (E3CT1) abolished apical targeting (Figs. 2B, 3; Table 1). Conversely,
replacing the C terminus of EAAT1 with the analogous C-terminal region
of EAAT3 (E1CT3) conferred specific apical localization, changing from 22 ± 8% apical localization of wild-type EAAT1 to 97 ± 1%
apical localization of E1CT3 (Table 1). Replacing the large second
extracellular loop or the N-terminal region of EAAT3 with the
corresponding regions of EAAT1 did not affect the polarization of EAAT3
(although the chimera including the N-terminal region of EAAT1 showed
little cell surface expression). Replacing these domains of EAAT1 with the corresponding regions of EAAT3 did not cause EAAT1 to become apically localized. These substitutions indicated that the cytoplasmic C terminus of EAAT3 contains information important for its apical localization.
To further test the role of the EAAT3 C terminus, we constructed
addition and deletion mutants. We first tested whether this small
region of EAAT3 could redirect a nonpolarized protein to the apical
domain. The addition of the C terminus of EAAT3 to full-length EAAT2
[E2-3(469-524)] changed its localization from nonpolarized to
apically polarized as judged by cell surface biotinylation assays (Fig.
3A, Table 1) and confocal
microscopy (Fig. 3B). The C terminus of EAAT3 similarly
redirected the taurine transporter to the apical surface (data not
shown). Conversely, deleting the C terminus of EAAT3 (E3470-524)
resulted in a nonpolarized localization (54 ± 3% apical); this
mutant could be detected at both the basolateral and apical surfaces
(Figs. 2B, 3). Together, the results from stably
transfected chimeric, addition, and deletion mutants in MDCK cells
demonstrate that the C terminus of EAAT3 is necessary for its apical
sorting and sufficient to confer apical localization on a nonpolarized
protein.
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Figure 3.
The cytoplasmic C terminus of EAAT3 is important
in its apical localization. Representative Western blot analysis
(A) and confocal vertical
(z-series) images (B) of MDCK
cells stably transfected with EAAT1-3 C-terminal chimeras or
truncation mutant constructs are shown. The presence of the C terminus
of EAAT3 in the mutant proteins confers stable expression predominantly
at the apical surface, whereas its absence results in a more
nonpolarized localization. Results are representative of at least two
additional experiments. Scale bar, 20 µm.
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Next, we sought to narrow down the region within the C terminus of
EAAT3 that is important for its apical localization. First, we made
successively smaller truncations of the C terminus of EAAT3. All the
truncated EAAT3 mutants have reduced apical localization (Fig.
2B, Table 1). Subsequent analyses (see next
paragraph) showed that all of the truncations eliminated all or part of
the sequence required for apical sorting. We also used a complementary approach of adding successively smaller regions of the EAAT3 C terminus
to the end of full-length EAAT2. This strategy revealed the importance
of the region between the last 27 and 14 amino acids of EAAT3. The
mutant E2-3(498-524) showed an apical localization, whereas
E2-3(511-524) had a significantly less polarized distribution (Figs.
2B, 4; Table 1).
Similar results were found when the same regions of the C terminus of
EAAT3 were fused to the full-length taurine transporter (data not
shown). These results show that amino acids between
D498 and D511
of the C terminus of EAAT3 contain an apical localization signal.
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Figure 4.
A 14-amino acid sequence in the C
terminus of EAAT3 contains the signal for apical localization.
Representative Western blots (A) and confocal
images (B) of MDCK cells stably transfected with
constructs containing full-length EAAT2 fused to various regions of the
EAAT3 C terminus are shown. The mutant proteins containing at least the
most distal 27-amino acid sequence of the EAAT3 C terminus attached to
EAAT2 [E2-3(498-524)] shows an apical localization,
whereas the mutant with a smaller distal region (14-amino acid
sequence) of the C terminus of EAAT3 [E2-3(511-524)]
has a more nonpolarized localization. These results suggest that
the region in EAAT3 between 27 and 14 amino acids from the C-terminal
end contains the sorting motif. Results are representative of at least
two additional experiments. Scale bar, 20 µm.
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We used two additional, complementary methods to pinpoint residues
within the 14 amino acids of the EAAT3 C terminus responsible for
apical targeting. For these experiments, constructs were expressed transiently, and localization was assessed by confocal microscopy. First, we examined constructs of EAAT2 fused to short segments from the
C terminus of EAAT3 (Fig. 5A;
see supplemental data on-line at www.jneurosci.org). Constructs
containing residues
D498-V510
were apically polarized, whereas constructs containing residues upstream of V504 were not. This indicates
that residues
N505-V510
(NGGFAV) are crucial for apical localization. Second, we made sequential triple or double alanine substitutions in the EAAT3 region
of the mutant consisting of EAAT2 attached to amino acids D498-F524 of
EAAT3 [E2-3(498-524)] (Fig. 5). The substitution with three alanine
residues between amino acids V504 and
A509 disrupted apical localization;
substitution of amino acids
K501-Y503
had a smaller effect (40% of transfected cells exhibited a polarized distribution). Alanine substitutions N-terminal to amino acid K501 or C-terminal to amino acid
A509 did not disrupt apical localization.
Next, we examined double alanine substitutions between amino acids
V504 and
D511. Substitution of the residues
V504N505 and
F508A509
resulted in loss of apical
polarity. Substitution of
residues V510D511
slightly disrupted the polarized distribution (70% of the transfected cells still showed apical localization). Replacing residues
G506G507 with
alanine did not alter apical localization of the mutant protein, but
this may be because alanine and glycine are similar in structure.
Substituting bulkier amino acid residues at positions 506 and 507 may
have an impact on the polarity of EAAT3. The substitution of
A509 with serine (the rat homolog of EAAT3
has a serine at this position) did not alter the apical localization of
the mutant construct (data not shown). Thus, the residues
V504N505 and
F508A509
within the motif are critical in apical sorting, whereas the neighboring residues may influence the specific localization motif. The
apical localization sequence motif is KSYVNGGFAVD (amino acids
K501-D511).
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Figure 5.
Delineation of the residues in the sorting motif
for apical localization of EAAT3. A, Representative
confocal images of MDCK cells transiently transfected with constructs
that contained full-length EAAT2 fused to either short regions of the
EAAT3 C terminus or the most distal 27-amino acid sequence of EAAT3
[E2-3(498-524)] with triple or double alanine
substitution mutations. Both E2-3(498-510) and
E2-3(485-497) contained a 12-amino acid sequence from
the C terminus of EAAT3, but only E2-3(498-510) was
apically localized. B, Sequences of the constructs
containing sequential triple or double alanine substitution of
E2-3(498-524) and localization of the mutants. Regions of EAAT2 and 3 are represented in red and green,
respectively, and the residues that are substituted with alanines are
denoted in black. Substitution of residues with alanine
of the following sequence disrupts the apical localization of the
parent construct: KSYVNGGFAVD (the critical
residues are gray). Results are representative of at least
three additional experiments in duplicate. Scale bar, 20 µm.
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The C-terminal sorting motif targets EAAT3 to the somatodendritic
region in neurons
Next, we tested whether the apical targeting motif in EAAT3 also
directs the localization of EAAT3 in neurons. We transfected both
wild-type and mutated EAATs tagged with GFP or yellow fluorescent protein (YFP) into polarized neurons in primary hippocampal cultures. We quantified the average fluorescence in axons and dendrites for each
cell and used the ratio of axon to dendrite fluorescence as a measure
of polarity. The wild-type EAAT3-YFP was extremely polarized to
dendrites (Figs. 6A,
7B), with an average
axon/dendrite ratio of 0.06 ± 0.02 (Table 2), a value comparable
with that of other dendritically polarized proteins such as transferrin and low-density lipoprotein receptors (G. Banker, unpublished observations). In older neurons (19 d in vitro), which are
highly innervated, EAAT3 was present in clusters on the dendritic
surface and on spines and filopodia (Fig. 7C). Some of these
clusters lie adjacent to synaptophysin-positive presynaptic
specializations (data not shown), consistent with the postsynaptic
localization of EAAT3 that has been reported previously. Thus, in all
respects, transfected GFP- or YFP-tagged EAAT3 showed the same
polarized distribution in neurons as for endogenous EAAT3 (Coco et al., 1997). When live cells were imaged, transport of EAAT3 carriers was
readily observed within the dendrites, but no carriers were visible in
the axon (data not shown). Thus in neurons, the polarization of EAAT3
in the dendritic membrane reflects its selective delivery to dendrites,
not its selective retention in the dendritic membrane.
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Figure 6.
Distribution of wild-type and mutant EAATs in
polarized hippocampal neurons. Hippocampal cultures were transiently
transfected with GFP- or YFP-tagged EAATs, and expression of the
constructs was assessed in 11-d-old hippocampal neurons. The EAAT3-YFP
signal (A) is completely restricted to dendrites,
whereas cotransfected soluble cyan fluorescent protein
(CFP; B) fills the entire cell, including
its axon. Mutant E3(504-509AAAAAA) with a six-alanine
replacement in the apical sorting domain is present in both axons and
dendrites (C), as is EAAT2
(D). Arrows indicate axons;
arrowheads indicate dendrites. Scale bar, 100 µm.
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View larger version (44K):
[in this window]
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|
Figure 7.
Higher-magnification images of wild-type and
mutant EAATs in polarized hippocampal neurons. EAAT3-YFP is enriched on
the surface of dendrites but absent from axons
(B). Coexpressed soluble cyan fluorescent protein
(CFP) fills the entire volume of the axon and dendrites
(A). The mutant EAAT3
[E3(504-509AAAAAA)] and EAAT2 are present in both
dendrites and axons (D, E). In older
neurons (transfected at 10 d in vitro and imaged
9 d later), both wild-type and mutant EAAT3 are frequently present
in clusters on spines and filopodia in the dendrites, and mutant EAAT3
is present in axons. Dendrites (arrowheads) are thicker,
taper as they extend away from the cell body, and have filopodia and
developing spines present all along them. Axons (arrows)
are thinner, maintain a relatively uniform diameter, and lack spines.
Scale bars: A, B, D, E, 10 µm; C, E, F,
2 µm.
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In contrast, two EAAT3 C-terminal truncation mutants lacking the
apical-targeting motif (E3470-524 and E3485-524) were
significantly less polarized (average axon/dendrite ratios, 0.28 ± 0.03 and 0.29 ± 0.05, respectively). Moreover, the
substitution of residues 504-509 within the sorting motif of EAAT3
with alanines [E3(504-509AAAAAA)] disrupted somatodendritic polarity
to the same extent as deleting the entire cytoplasmic tail (average
axon/dendrite ratio, 0.26 ± 0.02). In all cases, mutant EAAT3s
extended throughout the entire axonal arbor, a result not observed with
the wild-type protein. However, disruption of the sorting motif in
EAAT3 did not result in a complete loss of polarity (compare with
EAAT2, whose axon/dendrite ratio was 0.68). Therefore, other regions of
EAAT3 may also contain dendritic targeting information. Although such
mutations disrupted the polarization of EAAT3, they did not prevent its
clustering on dendritic spines (Fig. 7F). These
results suggest that the apical sorting signal is not required for the
synaptic clustering of EAAT3. Rather, these results show that the
apical sorting motif in EAAT3 is responsible for its dendritic
targeting in neurons.
We next investigated whether adding the EAAT3 sorting motif to a
uniformly distributed protein was sufficient to cause its redistribution to the dendrites. When EAAT2 was expressed in
hippocampal neurons, it was approximately equally distributed in axons
and dendrites, exhibiting an average axon/dendrite ratio of 0.68 ± 0.09 (Figs. 6D, 7D; Table 2), a value
comparable with that found for nonpolarized proteins such as
NgCAM-related cell adhesion molecule and CD8 (B. Sampo and G. Banker, unpublished observations). To test whether the EAAT3 sorting
motif could redirect EAAT2 to the dendrite, we fused the last 27 amino
acids of EAAT3 (containing the sorting motif) to full-length EAAT2
[E2-3(498-524)]. E2-3(498-524) was significantly more dendritic
than wild-type EAAT2 (axon/dendrite ratio, 0.31 ± 0.04) but was
less polarized than wild-type EAAT3. A similar mutant, which differed
only in that alanines were substituted for residues 504-506 within the
sorting motif [E2-3(498-524:504-506AAA)], was as nonpolarized as
wild-type EAAT2 (axon/dendrite ratio, 0.82 ± 0.10). This
indicates that the EAAT3 sorting motif has some ability to redirect a
uniform protein in neurons but is not sufficient to completely redirect
EAAT2, as it is in MDCK cells. It may be that EAAT2 itself contains its
own sorting information that influences its localization in neurons and
thus competes with the EAAT3 motif.
Together, our results clearly show that EAAT3 contains a sorting motif
in its cytoplasmic tail. This motif, KSYVNGGFAVD, directs the apical localization of EAAT3 in MDCK cells and is required
for its dendritic localization in hippocampal neurons.
| |
DISCUSSION |
In this study, we identified a novel sorting motif in the
cytoplasmic C-terminal region of EAAT3
(KSYVNGGFAVD) that directs its specific
localization to the apical domain of MDCK cells and the somatodendritic
region of hippocampal neurons. This is the first well defined motif
known to be required for apical sorting in MDCK cells and dendritic
sorting in hippocampal neurons and capable of redirecting the
localization of nonpolarized proteins to these domains. A database
search did not reveal any other known membrane proteins that contain
the motif VNGGFA, but other epithelial sorting motifs shared by
different proteins have proved difficult to identify solely on the
basis of the amino acid sequence.
The EAAT3 sorting motif is one of several different motifs found in the
cytoplasmic tails of neuronal proteins that target them to dendrites
(Jareb and Banker, 1998; Poyatos et al., 2000; Ruberti and Dotti,
2000). It is, however, one of the first instances of a defined
cytoplasmic motif that mediates apical sorting in MDCK cells.
Traditionally, it has been thought that basolateral and dendritic
sorting shared many common features, as did apical and axonal sorting
(Dotti and Simons, 1990). Over the past few years, it has become clear
that apical proteins are not always segregated to the axonal domain in
neurons. For example, other amino acid transporters, such as GAT-3 and
glycine transporter 2, are sorted to the apical domain in MDCK cells
but are uniformly distributed in neurons (Ahn et al., 1996; Poyatos et
al., 2000). Our results, however, represent the first instance in which
a well- defined dendritic targeting motif mediates apical targeting in epithelia.
The EAAT3 sorting motif likely directs dendritic and apical targeting
by interacting with other proteins involved in targeting. Proteins that
bind to the EAAT3 sorting motif have not yet been identified. Recently,
the protein glutamate transporter EAAC1-associated protein
(GTRAP)3-18 has been shown to interact with the C terminus of
the rat homolog of EAAT3 (Lin et al., 2001). However, increasing the
expression of GTRAP3-18 in cells decreases the affinity of the
transporter for its substrate but does not alter the cell surface
expression of the transporter. Thus, it is not likely that GTRAP3-18
is involved in the sorting of EAAT3 to the appropriate cell surface.
The mechanism by which the EAAT3 sorting motif may direct apical and
dendritic targeting could involve any of the steps in membrane protein
targeting. It is thought that sorting motifs, particularly those that
reside in the cytoplasmic domain, are recognized by cytoplasmic adaptor
proteins (APs). These adaptor proteins concentrate the membrane
proteins within discrete domains and link them with coat proteins. The
coat proteins then induce vesicle budding, hence leading to the sorting
of distinct membrane proteins into unique transport carriers (Gu et
al., 2001). For example, specific subunits of two adaptor complexes,
AP-1 and AP-4, have been shown to be necessary for the
basolateral sorting of some proteins containing C-terminal sorting
signals (Folsch et al., 1999; Simmen et al., 2002). It is possible that
neurons and epithelia express a common adaptor that recognizes the
EAAT3 motif and sorts EAAT3 protein into a specific population of
carrier vesicles but that the fate of these carriers differs in neurons and epithelia. Because there are profound differences in the
organization of microtubules in neurons and epithelia, it is possible
that differences in the microtubule-based transport of EAAT3 carriers contribute to their apparently divergent localization in neurons and
epithelia. In MDCK cells, microtubules are oriented with their minus
ends directed toward the apical surface and their plus ends directed
toward the basolateral domain (Bacallao et al., 1989). In neurons,
microtubules in the axons are oriented with the plus end directed away
from the cell body, whereas microtubules in the dendrites have a mixed
orientation (Baas et al., 1988). Thus a minus end-directed motor might
be expected to move carrier vesicles to the apical domain in MDCK cells
and to the dendritic domain in neurons, as proposed by Goldstein and
Yang (2000).
There is already evidence for the involvement of the minus end motor
dynein in the apical sorting of rhodopsin, an example of an apical
protein whose sorting is directed by a cytoplasmic motif (Chuang and
Sung, 1998; Tai et al., 2001). The 39-amino acid tail of rhodopsin
interacts with the dynein light chain TcTex-1 but not another light
chain dynein, RP3 (Tai et al., 1999). RP3 and TcTex-1 can
compete for binding to the dynein complex, and the overexpression of
RP3 in MDCK cells leads to a decrease in the expression of TcTex-1 as
well as the nonpolarized localization of rhodopsin (Tai et al., 2001).
Thus, the apical targeting of rhodopsin in MDCK cells is dependent on
TcTex-1-mediated dynein function. It would be of interest to determine
whether the sorting motif in EAAT3 interacts with components of dynein
or other minus end-directed motor proteins, such as kinesin family
member C2 (Goldstein and Yang, 2000; Yang et al., 2001).
The clustering of EAAT3-GFP on dendritic spines and filopodia also
deserves comment. In the brain, EAAT3 has a "perisynaptic" location, lying just beyond the active zone where postsynaptic glutamate receptors are most highly concentrated. This location suggests that neuronal glutamate transporters are poised to regulate the activity of extrasynaptic glutamate receptors by controlling the
diffusion of glutamate away from the synapse, as has been demonstrated
recently at parallel fiber synapses in the cerebellum (Brasnjo and
Otis, 2001). These results indicate that the correct localization of
glutamate transporters is critical to their role in regulating
neurotransmission. The EAAT3 clusters we observed, which might be
difficult to detect by immunostaining of intact tissue, bear a striking
resemblance to postsynaptic receptor clusters seen on cultured
hippocampal neurons (Allison et al., 2000; Naisbitt et al., 2000). The
clustering of EAAT3 is not affected by deletion or mutation of the
dendritic targeting signal, suggesting that it is mediated by a
different molecular mechanism. It would be of interest to know whether
interaction with membrane scaffolding proteins contributes to the
localization of postsynaptic transporters such as EAAT3.
| |
FOOTNOTES |
Received June 27, 2002; revised Sept. 23, 2002; accepted Sept. 30, 2002.
This work was supported by Howard Hughes Medical Institute and National
Institutes of Health Grants NS 33273 (S.G.A.), NS17112 (G.B.), and MH
66179 (G.B.). We thank Dr. D. M. Fass for critical review of this
manuscript and B. Smoody for preparing the neuronal cell cultures.
Correspondence should be addressed to Dr. Susan G. Amara, Howard Hughes
Medical Institute and Vollum Institute, Oregon Health and Science
University, L-474, 3181 Southwest Sam Jackson Park Road, Portland, OR
97239. E-mail: amaras{at}ohsu.edu.
| |
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C. Vanoni, S. Massari, M. Losa, P. Carrega, C. Perego, L. Conforti, and G. Pietrini
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TOP
ABSTRACT
INTRODUCTION
