The C-terminal Tail of the Metabotropic Glutamate
Receptor Subtype 7 Is Necessary but Not Sufficient for Cell Surface
Delivery and Polarized Targeting in Neurons and Epithelia*
J. Brian
McCarthy
§,
Seung T.
Lim¶,
N. Barry
Elkind
,
James S.
Trimmer¶,
Robert M.
Duvoisin**,
Enrique
Rodriguez-Boulan**, and
Michael J.
Caplan
From the Departments of
Cellular and Molecular
Physiology and
Cell Biology, Yale University School of Medicine,
New Haven, Connecticut 06520, the ¶ Department of Biochemistry and
Cell Biology and Institute for Cell and Developmental Biology, State
University of New York at Stony Brook, Stony Brook, New York
11794-5215, and the ** Dyson Vision Research Institute, Departments of
Ophthalmology and Cell Biology, Weill Medical College, Cornell
University, New York, New York 10021
Received for publication, September 11, 2000, and in revised form, November 21, 2000
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ABSTRACT |
Complex neuronal functions rely upon the
precise sorting, targeting, and restriction of receptors to specific
synaptic microdomains. Little is known, however, of the molecular
signals responsible for mediating these selective distributions. Here
we report that metabotropic glutamate receptor subtype 7a
(mGluR7a) is polarized at the basolateral surface when expressed
in Madin-Darby canine kidney (MDCK) epithelial cells but is not
polarized when expressed in cultured hippocampal neurons.
Truncation of the mGluR7 cytoplasmic tail produces a protein
that is restricted to a perinuclear intracellular compartment in both
neurons and MDCK cells, where this protein colocalizes with a
trans-Golgi network antigen. The mGluR7 cytoplasmic domain appended to
the transmembrane portion of the vesicular stomatitis virus G protein
and the ectodomain of human placental alkaline phosphatase is
distributed over the entire cell surface in cultured neurons. When
expressed in MDCK cells, this construct remains in an intracellular
compartment distinct from endosomes or lysosomes. Thus, the cytoplasmic
tail domain of mGluR7 is necessary but not sufficient for polarized
targeting in MDCK monolayers, whereas in neurons the cytoplasmic tail
is sufficient for cell surface expression but not polarization.
Additional mechanisms are likely required to mediate mGluR7 neuronal
polarization and synaptic clustering.
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INTRODUCTION |
Specialized domains at the neuronal cell surface orchestrate
complex synaptic functions. Accurate sorting, trafficking, and restriction of proteins to these microdomains ensures the precise distribution of synaptic proteins for proper neuronal function. Proteins destined for secretion or incorporation into the plasma membrane transit to the cell surface through a series of subcellular compartments. Synthesis occurs in the soma at the rough endoplasmic reticulum (RER),1 and
postsynthetic processing continues in the Golgi complex, with sorting
of proteins into specific transport vesicles occurring in the TGN (1,
2). Information contained within targeted proteins function as sorting
signals to specify their ultimate destination within the cell (3, 4).
Although progress is being made in the categorization of sorting
signals, their diversity and complexity, as well as the underlying
mechanisms through which cells carry out polarized targeting, remain
unresolved (1).
Neurotransmitter receptors are among the complex array of proteins at
synaptic microdomains that participate in the generation and
propagation of neuronal activity. Glutamate serves as the principle
excitatory amino acid neurotransmitter at central nervous system
synapses. Activity at glutaminergic synapses is mediated by both
ionotropic and metabotropic glutamate receptors (5). Metabotropic
glutamate receptors couple with heterotrimeric G-proteins to regulate
cell excitability and synaptic transmission (6, 7) and have been
implicated in synaptic plasticity (8). Currently eight distinct mGluR
subtypes have been recognized (6). These have been observed to exhibit
subtype- and region-specific differences in distribution along the
neuronal cell surface in vivo. The mGluR7 subtype, in
particular, highlights the complexity of protein targeting. mGluR7
receptors have been reported to be targeted to postsynaptic sites in
the olfactory bulb (9) and locus coeruleus (10, 11), to axons in the
hippocampus (12, 13), to both pre- and postsynaptic sites in the retina
(14), and to both axons and dendrites of cultured hippocampal neurons (15). In the hippocampal formation additional complexity is evident,
because the distribution of mGluR7 is apparently dependent upon the
nature of the postsynaptic target neuron. Within individual axons,
mGluR7 is found to be present only in distinct subpopulations of
presynaptic terminals (16). The subtype- and region-specific placement
of mGluR7 at distinct membrane domains requires the existence of
mechanisms whereby sorting information is interpreted, so that
receptors can be distributed precisely. mGluR7 is a member of the
larger seven transmembrane or G-protein-coupled receptor family of
receptors. Little is known about the cellular mechanisms that govern
the intracellular trafficking of the individual members of this
critically important protein family.
Because of the technical difficulties inherent in studying sorting and
trafficking in neurons, cultured cell lines have been utilized as a
means both to study sorting in a polarized cell system and to allow
biochemical investigations of the underlying cellular mechanisms. Among
these culture models, epithelial cells have been especially useful in
studies of neuronal membrane protein targeting (17). This utility may
be in part based upon the developmental origin of neurons from
neuroepithelial stem cells (18). In some but not all instances,
Madin-Darby canine kidney epithelial cells (MDCK) and neurons seem to
employ similar signals and mechanisms to generate polarized protein
distributions (as discussed in Refs. 4, 15, and 19). Many neuronal
proteins found in axons can sort to the apical surface of epithelia,
whereas most dendritic proteins can sort to the basolateral surface. We
report here an analysis of the subcellular targeting of the mGluR7
receptor. By coordinated investigation in both epithelial cell lines
and in cultured hippocampal neurons, we are able to examine whether the
requirements for the polarized sorting of the protein are the same in
both cell types. We report that mGluR7 achieves a polarized
steady-state distribution at the basolateral membrane when expressed in
epithelial monolayers. The cytoplasmic tail domain of mGluR7 is
necessary but not sufficient for this polarized targeting in MDCK
cells. In neurons however, exogenously expressed mGluR7 does not
achieve a polarized distribution. The cytoplasmic tail is not
sufficient for polarization, but without it the protein does not
achieve a cell surface distribution.
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EXPERIMENTAL PROCEDURES |
Construction of Fusion Proteins--
Cloned mGluR7a cDNA in
the pBluescript plasmid was a kind gift from S. Nakanishi (20). An
XbaI site was introduced at the Bluescript polylinker
NotI site by insertion of an oligonucleotide linker. This
XbaI site was used along with the pBluescript polylinker ClaI restriction site and the AatII restriction site within
mGluR7a for subcloning and fusion protein construction. Fusion PCR was used to construct the GFP·mGluR7 fusion protein. PCR was used to generate three products from which the final fusion products were
made by use of the same primers. The first PCR product was generated
from the mGluR7 wt cDNA using a 5'-primer complimentary to and
including the pBluescript polylinker ClaI restriction site and a 3'-primer that included both the last five codons before the
intended site of GFP incorporation (10 amino acids following the signal
sequence) and the first seven codons of the GFP sequence. The GFP
sequence was generated in a similar manner from pEGFP-C1 (CLONTECH, Palo Alto Ca.) using primers that
included both the mGluR7 sequence before the site of intended insertion
and the sequence on either end of the GFP encoding sequence. The
downstream mGluR7 product was generated with a 5'-primer including both
the final five codons of GFP and the initial six codons following the
insertion site and a 3'-primer past the unique AatII
restriction site in mGluR7. The first two PCR products were combined
and allowed to prime off each other, outside primers were added, and
the fusion product was generated. This initial fusion product was then
used in a similar manner with the third PCR product from the initial set to produce the complete fusion PCR product. The resulting fragment
was digested at the unique ClaI and AatII
restriction sites for replacement into the original cDNA plasmid of
mGluR7. The GFP·mGluR7-tail deletion construct was generated by PCR
with a 5'-primer at the interface of the GFP and mGluR7 fusion and a
3'-primer selective to a sequence following the last transmembrane domain. The primer was designed to incorporate a stop codon 11 amino
acids following the final transmembrane domain and an XbaI restriction site. The resulting fragment was digested at the unique XbaI and AatII restriction sites for replacement
into the cDNA plasmid of GFP·mGluR7. The PLAP·mGluR7-tail
fusion construct was generated using a PLAP·VSVG plasmid (21). A PCR
fragment containing the cytoplasmic tail of mGluR7 was generated using
a 5'-primer containing following the VSVG transmembrane domain up to
and including a HindIII restriction site, and a 3'-primer to
incorporate an XbaI restriction site following the stop
codon. The resulting fragment was digested at the XbaI and
HindIII restriction sites for replacement into the cDNA
plasmid of PLAP·VSVG. All constructs were sequenced through the
respective ligation points. The constructs were then subcloned into the
mammalian expression vector pCB6 (kindly provided by M. Roth,
University of Texas, Southwestern) between the ClaI and
XbaI sites prior to transfection.
Cell Culture and Transfection--
COS-1 cells were grown in
Dulbecco's modified Eagle's medium (DMEM) supplemented with 10%
fetal bovine serum, 2 mM L-glutamine, 50 units/ml penicillin, and 50 µg/ml streptomycin. Type-II MDCK cells
were grown in minimal essential media (Life Technologies, Inc.)
supplemented with 10% fetal bovine serum, 2 mM
L-glutamine, 50 units/ml penicillin, and 50 µg/ml
streptomycin. Cells were grown in a 37 °C incubator with 5%
CO2 and passaged twice weekly through exposure to 0.05%
trypsin, 0.5 mM EDTA (Life Technologies, Inc.).
COS-1 cells were transiently transfected using DEAE-dextran (22). Cells
(2 × 105 per 35-mm dish) were plated on glass
coverslips the night before transfection. Cells were washed in
Tris-buffered saline and exposed to 5 µg of DNA, and 0.5 mg/ml
DEAE-dextran (Amersham Pharmacia Biotech) was added for 40 min at
37 °C. The solution was discarded. and the cells were incubated in 1 ml of DMEM containing 10% fetal bovine serum and 0.1 mM
chloroquine for 3 h at 37 °C. The cells were washed in
Tris-buffered saline and incubated in complete DMEM for 48-72 h prior
to immunofluorescent immunohistochemistry. Subconfluent MDCK cells were
prepared as previously described (22) and transfected by the PerFect
lipid method (Invitrogen, San Diego, Ca.), with fusion constructs that
had been subcloned into the pCB6 vector. This vector carries resistance
to the antibiotic G418 (Life Technologies, Inc.). After selection in
1.8 mg/ml G418, clones were screened for expression by
immunofluorescence, Western blotting, or phosphatase assay (BluePhos
Microwell, Kirkegaard & Perry, Gaithersburg, MD).
Primary hippocampal cultures were prepared as described (19). Cultured
neurons were transfected at stage 5, a stage when synapses have been
formed and endogenous synaptic receptors have polarized and localized
to the synapses. Cultured hippocampal neurons were transfected at 7 days in vitro (DIV) by the LipofectAMINE 2000 method (Life
Technologies). In brief, 1 µg of DNA was diluted into 50 µl of
serum-free medium and incubated at room temperature for 5 min. Then, 3 µl of LipofectAMINE 2000 reagent was diluted into 50 µl of
serum-free medium in a second tube. The diluted DNA and the diluted
LipofectAMINE 2000 reagent were combined, mixed, and incubated for 15 min at room temperature. Neurons were transferred to a new 6-well plate
that contained 1 ml of the neuronal maintenance medium from the
hippocampal culture. The DNA-LipofectAMINE 2000 reagent complexes were
mixed into the medium bathing the cells and incubated at 37 °C with
5% CO2 for 2 h. The coverslips were then placed back
into the original 6-well plate and incubated for 2 days.
Immunofluorescent Microscopy--
Immunofluorescence was
performed as described (4) on stably expressing cell lines. Briefly,
cells were grown to confluence on Transwell porous cell culture inserts
(Corning Costar Corp., Cambridge, MA) or, for screening clones, on
8-well Lab-Tek slides (Nalge Nunc International, Naperville, IL) and
fixed in
20 °C methanol for 7 min at room temperature. Cells were
then permeabilized in a phosphate-buffered saline-based wash buffer
containing 0.3% Triton X-100 and 0.1% bovine serum albumin for 15 min. Nonspecific binding of antibody was blocked by incubating the
cells in goat serum dilution buffer (16% filtered goat serum, 0.3%
Triton X-100, 20 mM NaPi, pH 7.4, 0.9% NaCl)
at room temperature for 1 h. Chimeras were detected either by
native GFP fluorescence, anti-mGluR7 wt (kindly provided by J. Conn,
Ref. 12), anti-GFP (CLONTECH Laboratories, Inc.),
or anti-PLAP (Fitzgerald Inc.) (1:50). Secondary goat anti-mouse or
anti-rabbit antibodies (1:200) were conjugated to either rhodamine or
fluorescein (Sigma). All antibody incubations took place in goat serum
dilution buffer for 1 h at room temperature. Between primary and secondary antibody incubations, the cells were subjected to
three 5-min washes in the phosphate-buffered saline-based wash buffer.
After incubation with the secondary antibody, cells were washed in
phosphate-buffered saline three times for 5 min each and finally in 10 mM NaPi for 10 min before being mounted on
coverslips with Vectashield (Vector Laboratories, Burlingame, CA).
Confocal sections were taken using a Zeiss LSM 410 laser scanning
confocal microscope. Images are the product of 8-fold line averaging.
Xz cross sections were generated with a 0.2 micron motor step. Contrast and brightness were set so that all pixels were in the linear range.
Immunofluorescence staining of neurons was performed as described (19).
Immunofluorescent images were captured with IP Labs on a Zeiss Axiophot
epiflurescence photomicroscope. Fluorescent dextran uptake was
performed as described (23), cells were incubated for various times
with DMEM+ containing 1 mg/ml rhodamine-dextran (lysine fixable)
(Molecular Probes) Mr = 10,000 in both upper and
lower Transwell filter chambers.
Cell Surface Biotinylation--
Cells were grown on 24-mm
Transwell inserts (0.4-µm pore, Costar) for 1 week, and the medium
was replaced with complete MEM (without Geneticin) before the
experiment. Steady-state biotinylation of either apical or basolateral
proteins was carried out separately at pH 9.0 as described (22). Cells
were incubated with N-hydroxysulfosuccinimide (NHS)-biotin for 2× 20 min. Following biotinylation, filters were excised from the cups with a razor blade, and the attached monolayers were lysed in 1% Triton X-100 in 150 mM NaCl, 5 mM EDTA, 50 mM Tris, pH 7.5. The biotinylated
proteins (either apical or basolateral cell surface proteins) were
recovered from cell lysates by incubation with 100 µl of packed
immobilized streptavidin-agarose beads (Pierce). Bound proteins were
eluted from the beads in Laemmli sample buffer, analyzed by
SDS-polyacrylamide gel electrophoresis and Western blotting as
described previously (22). The blot was incubated with anti-GFP or
anti-PLAP (1:200) primary antibodies followed by horse radish
peroxidase-conjugated secondary antibodies (Sigma) and developed with
enhanced chemiluminescence reagent (Amersham Pharmacia Biotech).
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RESULTS |
Metabotropic Glutamate Receptor Constructs: Substitution, Deletion,
and GFP Fusion Protein Constructs--
mGluR7 is a 7-transmembrane
domain receptor with a large extracellular domain and a 60-amino acid
cytoplasmic tail. The construct designated R7 wt (Fig.
1, mGluR7 wt) was investigated
by use of a specific antiserum (11) directed against the C-terminal
cytoplasmic tail of mGluR7. For subsequent investigations we chose to
epitope-tag the receptor by creating GFP·mGluR7 fusion proteins. The
GFP·R7 fusion protein construct has GFP incorporated in frame at a
position 10 amino acids distal to the signal sequence (Fig. 1,
GFP-mGluR7). For investigations of the role of the
cytoplasmic tail in receptor localization, the cytoplasmic tail was
deleted from the GFP·R7 fusion protein at a position 11 amino acids
distal to the predicted end of the final transmembrane domain creating
the GFP·R7-tail-minus construct (Fig. 1, GFP-mGluR7 tail
minus). To investigate whether the cytoplasmic tail of mGluR7 may
function autonomously to direct localization, a fusion protein
PLAP·R7-tail (Fig. 1, PLAP-mGluR7 tail) was generated with
the mGluR7 cytoplasmic tail incorporated inframe with a construct
containing the VSVG transmembrane domain and placental alkaline
phosphatase as the extracellular domain.

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Fig. 1.
Metabotropic glutamate receptor
constructs. The predicted membrane topologies and relevant
sequence domains are illustrated for each mGluR7 cDNA construct.
mGluR7 wt contains the entire wild-type receptor. The
GFP·mGluR7 fusion protein (GFP-mGluR7) has an
extracellular GFP incorporated inframe at a position 10-amino acids
distal to the signal sequence. PLAP-mGluR7 tail has the
mGluR7 cytoplasmic tail incorporated inframe with a single spanning
membrane protein containing the extracellular placental alkaline
phosphatase domain and the VSVG transmembrane domain. GFP-mGluR7
tail minus represents the GFP fusion receptor protein with its
cytoplasmic tail deleted.
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R7 wt and GFP·R7 Fusion Protein Distribution in Polarized and
Nonpolarized Cells--
Constructs were transiently expressed in COS-1
cells. Both R7 wt (Fig. 2a)
and GFP·R7 (Fig. 2b) attain a cell surface distribution in
COS-1 cells. Stably transfected MDCK cells also expressed mGluR7 at the
plasma membrane. MDCK cell lines expressing mGluR7 were grown to
confluence and examined by indirect immunofluorescence confocal
microscopy (Figs. 3 and 4) using the anti-mGluR7 antiserum for R7 wt
and either native GFP fluorescence or anti-GFP for
GFP·R7. GFP·R7 displayed a polarized
steady-state distribution at the basolateral surface of MDCK epithelial
cells (Fig. 3, d,
e, and f). To assess biochemically the polarized
distributions of the mGluR7 proteins, we employed the cell surface
biotinylation technique to differentially isolate MDCK apical and
basolateral proteins. Steady-state cell surface biotinylation supported
the immunofluorescent observation, by confirming GFP·R7 distribution
at the basolateral surface (Fig.
5A).

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Fig. 2.
Cellular distribution of mGluR7 receptor
constructs transiently expressed in COS-1 cells determined by
immunofluorescence. COS-1 cells were transiently transfected with
mGluR7 receptor cDNAs constructed in the pCB6 mammalian expression
vector, and exogenous protein localization was determined by
fluorescence microscopy. Cell surface distributions were demonstrated
for three of the mGluR7 constructs: mGluR7 wt as determined with an
anti-mGluR7 antibody (panel a), GFP·mGluR7 as
determined by intrinsic GFP fluorescence signal (panel
b), and PLAP·mGluR7-tail as determined with an
anti-PLAP antibody (panel c). However, an intracellular
(noncell surface) perinuclear distribution was observed for the
GFP·mGluR7-tail-minus receptor construct as determined by either
anti-GFP antibody staining (panel d) or via the intrinsic
GFP fluorescence signal.
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Fig. 3.
Cellular distribution of mGluR7-wt
and GFP·mGluR7 stably expressed in
polarized MDCK cells determined by immunofluorescence. MDCK cells
were stably transfected with each mGluR7 construct in the pCB6
mammalian expression vector. Confocal en face (a, c, d, f, g, i,
j, l) and confocal xz cross-section (b, e, h,
k) immunofluorescence images of MDCK cells were obtained.
a-c illustrate an intracellular distribution of mGluR7 wt
as determined by staining with anti-mGluR7-wt. c, the
intracellular distribution of mGluR7 is highlighted
(arrowheads). The GFP·mGluR7 receptor construct displays a
mainly basolateral cell surface distribution (d-f) in MDCK
cells as determined by native GFP fluorescence. f, minimal
intracellular mGluR7 is observed (arrowheads). In contrast,
however, immunofluorescence labeling of GFP·mGluR7 with the
cytoplasmic tail-directed anti-mGluR7-wt antibody (g-i)
shows a primarily intracellular distribution (i,
arrowheads) as observed in c. Immunofluorescence
labeling of GFP·mGluR7 with anti-GFP (j-l) illuminates
both extracellular and additional intracellular labeling as compared
with (d-f). The intracellular mGluR7 is highlighted
(l, arrowheads).
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Fig. 4.
Cellular distribution of PLAP·mGluR7
and GFP·mGluR7-tail-minus receptor
constructs in polarized MDCK cells as determined by
immunofluorescence. MDCK cells were stably transfected with
PLAP·mGluR7-tail and GFP·mGluR7-tail-minus in the pCB6 mammalian
expression vector. Confocal en face immunofluorescence images of MDCK
cells are presented. In a and b,
immunofluorescence of PLAP·mGluR7-tail shows the construct to be
contained in a punctate intracellular compartment as determined by
staining with anti-PLAP. In c and d,
immunofluorescence of GFP·mGluR7-tail-minus illustrates the
construct to be contained in a different intracellular compartment,
which shows a perinuclear distribution similar to that seen in Fig.
2.
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Fig. 5.
GFP·mGluR7 is expressed at the basolateral
cell surface whereas the PLAP·mGluR7-tail and
GFP·mGluR7-tail-minus are not expressed at the
surface of MDCK cells. Steady-state cell surface biotinylation
analysis of mGluR7 receptor constructs in stably expressing MDCK cell
lines was carried out using either anti-GFP or anti-PLAP antibodies to
detect the respective constructs. A,
(GFP·mGluR7) is detected as a protein of ~130 kDa predominantly at
the basolateral cell surface (lanes 3, 4). Only a weak
signal is detected at the apical cell surface (lanes 1, 2).
Lane 5 illustrates the absence of the construct at the
surface of untransfected MDCK cells. The GFP·mGluR7-tail-minus
construct was not detected at either the apical (lanes 6,
7) or the basolateral (lanes 8, 9) cell surface.
B, lane 1, consistent with its
intracellular distribution (Fig. 4), cell surface biotinylation
analysis did not detect the PLAP·mGluR7-tail construct at either the
apical (lanes 2, 3) or basolateral (lanes
4, 5) cell surfaces. To ensure that the transfected
MDCK cells express the PLAP·mGluR7-tail protein, Western blot
analysis was performed on total cell lysates, revealing the presence of
a protein of ~65 kDa corresponding to the construct.
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To examine how neurons might sort and target GFP·R7, we determined
whether the construct is appropriately targeted and polarized at steady
state in cultured neurons. Use of the GFP fusion construct allowed
detection of only exogenously expressed mGluR7. Whereas the majority of
the immunofluorescence signal for GFP·R7 was intracellular or
perinuclear, GFP·R7 was found at the cell surface throughout both
axons and dendrites and with no obvious clustering. Exogenously expressed mGluR7 did not achieve a polarized distribution in cultured hippocampal neurons and was found at apparently equal intensity at the
cell surfaces of both axons and dendrites (see Fig. 8, a and
b).
Masking of the Cytoplasmic Tail of mGluR7 in MDCK
Cells--
Comparison of R7 wt and GFP·R7 fusion protein
distributions suggests that the C-terminal tails of these proteins
participate in a macromolecular complex. In MDCK cells stably
expressing R7 wt, the protein distribution appears to be intracellular
(Fig. 3, a-c) when immunolabeling is performed using an
antisera directed against the final 18 amino acids of the mGluR7
carboxyl terminus. Importantly, when examined using the same
C-terminal-directed antisera, the distribution of GFP·R7 also appears
to be intracellular (Fig. 3, g-i). In contrast, observation
of the GFP·R7 intrinsic GFP signal in the same field (Fig. 3,
d-f), together with cell surface biotinylation (see Fig.
5A), clearly illustrates a cell surface distribution (at the
basolateral plasmalema, Fig. 3, d-f). These data suggest
that in MDCK cells, the C-terminal portion of cell surface mGluR7 is
interacting with a protein or proteins that interfere sterically with
access of the C-terminal antibody to its epitope. According to this
hypothesis, the C termini of mGluR7 intracellular populations do not
participate in these interactions, thus permitting their detection in
immunohistochemical experiments employing the C-terminal antibody.
Observation of the GFP·R7 cell line with anti-GFP is consistent with
this conclusion, revealing intracellular labeling (Fig. 3,
j-l) in addition to the population detected at the cell
surface. It would appear, therefore, that incorporation of mGluR7 into
the basolateral plasma membrane of MDCK cells is most likely associated
with the formation of protein-protein interactions involving its
C-terminal tail.
Importance of the mGluR7 Cytoplasmic Tail for Sorting--
Because
the mGluR7 C-terminal tail appears to participate in protein-protein
interactions at the MDCK cell basolateral membrane, we wondered whether
the tail embodies information needed to establish protein localization.
To test this possibility, we examined the distributions of the
GFP·R7-tail-minus and the PLAP·R7-tail constructs.
Following transient expression in COS-1 cells, GFP·R7-tail-minus
localized to perinuclear intracellular compartments whose morphology
and distribution are reminiscent of the Golgi complex (Fig.
2d). When these constructs are expressed in MDCK cells, the
GFP·R7-tail-minus construct recapitulated its COS cell distribution and was found in a perinuclear intracellular compartment (Fig. 4, c and d). To
further identify the intracellular compartment in which the
GFP·R7-tail-minus construct accumulates; we performed double labeling
with an antibody specific to a Lupus auto-antigen expressed in the TGN
(provided by A. Gonzalez; Ref. 24). In both COS-1 cells (Fig.
6, a-c) and in MDCK cells
(Fig. 6, d-f and g-i) we found that
GFP·R7-tail-minus colocalizes with the TGN marker. In addition, the
Golgi population of this tail-minus construct does not seem to be able
to cycle to the cell surface and return to the TGN via endosomes, as
has been noted for TGN38 and furin. In the case of furin and TGN38,
return to the TGN requires endosomal acidification and is inhibited by
agents that raise endosomal pH (25). Incubation with chloroquine (100 micromolar, 4 h) did not alter GFP·R7-tail-minus distribution in
MDCK cells (data not shown). The perinuclear Golgi-like distribution
did not change and did not disperse to a characteristic punctate
endosomal distribution during treatment. Thus, the tail-minus construct appears to be a fairly stable resident of the TGN that does not traffic
from the cell surface via endosomes.

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Fig. 6.
Intracellular distribution of
GFP·mGluR7-tail-minus in polarized and nonpolarized
cells colocalizes with the trans-Golgi network. Confocal en face
immunofluorescence images of COS-1 (a-c) and MDCK cells
(d-i) stably expressing GFP·mGluR7.
a-c, comparison of the distribution of
GFP·mGluR7-tail-minus and a TGN marker in COS cells.
a, distribution of GFP·mGluR7 as determined with anti-GFP.
b, TGN as visualized with an anti-human TGN lupus
auto-antibody. c, superimposition of a
(green) and b (red), illustrating
colocalization within the trans-Golgi network. d-f and
g-i, comparison of the distribution of
GFP·mGluR7-tail-minus with a trans-Golgi network marker in MDCK
cells. d and g, distribution of GFP·mGluR7 as
determined with anti-GFP. e and h, TGN
distribution as determined with an anti-human TGN lupus
auto-antibody. f and i,
superimposition of d or g (green) and
e or h (red), illustrating
colocalization within the trans-Golgi network.
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When transiently expressed in COS-1 cells, the PLAP·R7-tail construct
attained a cell surface distribution (Fig. 2c). However, the
PLAP·R7-tail construct did not attain a cell surface distribution in
MDCK cells. Instead it was concentrated in a dispersed, punctate intracellular distribution (Fig. 4, a and b).
This compartment is not labeled by antibodies to the lysosomal membrane
protein LAMP-1 (Fig. 7, a-c).
Subsequent experiments also indicated that it does not become labeled
by internalized fluorescein isothiocyanate-dextran (Fig. 7,
d-f). It would appear, therefore, that this compartment is
neither lysosomal nor endosomal in origin. Steady-state cell surface
biotinylation of MDCK monolayers supported immunofluorescent observations, confirming that neither PLAP·R7-tail nor
GFP·R7-tail-minus were present at the cell surface (Fig.
5, A and B).

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Fig. 7.
The intracellular distribution of
PLAP·mGluR7-tail in polarized cells does not overlap
with lysosomal or endosomal compartments. Confocal en face
immunofluorescence images were generated of MDCK cells stably
expressing PLAP·mGluR7-tail. a-c, comparison of the
distribution of PLAP·mGluR7-tail and a lysosomal marker (LAMP-1).
a, distribution of PLAP·mGluR7-tail as determined by
anti-PLAP. b, distribution of LAMP-1 as determined with
anti-LAMP-1. c, superimposition of a
(green) and b (red), illustrating the
absence of colocalization of PLAP·mGluR7-tail and lysosomal
compartments. d-f, comparison of the distribution of
PLAP·mGluR7-tail and endosomes, as revealed by uptake of fluorescent
dextran. d, distribution of PLAP·mGluR7-tail.
e, distribution of endosomal compartments as determined
by fluorescent dextran uptake into MDCK cells. f,
superimposition of d (green) and e
(red), illustrating the absence of colocalization of
PLAP·mGluR7-tail with endosomal compartments.
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Expression in neurons was performed to test how neurons would
distribute the tail-containing and tail-minus constructs at steady
state. Immunostaining with anti-PLAP and anti-GFP antibodies enabled
detection of only exogenously expressed constructs. In contrast to
results in MDCK cells, the immunofluorescence signal for PLAP indicated
that PLAP·R7-tail was expressed at the neuronal cell surface
throughout both axons and dendrites. It was not distributed in a
polarized manner and exhibited no obvious clustering (Fig. 8, e and f). Once
again, when exogenously expressed in neurons, GFP·R7-tail-minus was
observed in a perinuclear intracellular distribution consistent with a
Golgi-like pattern (Fig. 8c), with no observable label at
the cell surface (Fig. 8d).

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Fig. 8.
Cellular distribution of mGluR7 fusion
proteins in cultured hippocampal neurons determined by
immunofluorescence. Cultured hippocampal neurons were transfected
with mGluR7 constructs in the pCB6 mammalian expression vector, and the
distributions of the exogenous proteins were determined by
immunofluorescence. a and b illustrate the
presence of GFP·mGluR7 at the surface of the axonal growth cone
(a), and at the surface of both axons and dendrites
(b) as detected using an anti-GFP antibody.
GFP·mGluR7-tail-minus was detected in an intracellular perinuclear
distribution by native GFP fluorescence (c) and was not
found at the cell surface by staining with anti-GFP (d).
Immunofluorescence labeling of PLAP·mGluR7-tail with anti-PLAP
antibody shows the construct to be present at the surface of both axons
and dendrites (e, × 40; f, × 100).
|
|
 |
DISCUSSION |
Neuronal function relies upon the polarized distribution of
synaptic proteins. The mGluR7 subtype is a protein distributed with
polarity in neurons, being found only in highly selective locations at
cell surface synaptic sites. In different brain regions, mGluR7 can be
found selectively distributed in either pre- or postsynaptic locations.
In particular, mGluR7 distributes to selective subpopulations of axon
terminals within individual neurons of the hippocampus. Recently,
expression studies have examined the distribution of mGluR7
heterologously expressed in cultured hippocampal neurons (15). Results
indicated that full-length mGluR7 was present at the cell surface of
both axons and dendrites, suggesting that the exogenously expressed
protein did not achieve a polarized distribution. The authors of this
study proposed that the C terminus of mGluR7 contains an axonal
targeting signal, because it permitted somatodendritic mGluR2 to enter axons.
The molecular nature of the mGluR7 sorting signal remains undefined. We
wished therefore, to further examine the signals and mechanisms
involved in mGluR7 sorting. Toward this end, we expressed mGluR7 and a
variety of receptor constructs in neurons and epithelial cells. These
constructs included full-length mGluR7, a GFP fusion construct of
full-length mGluR7, mGluR7 without its cytoplasmic tail, and a
construct where the cytoplasmic tail of mGluR7 was appended to a single
pass transmembrane protein. Consistent with the findings of Stowell and
Craig (15), we find that in cultured hippocampal neurons, mGluR7 is
expressed in a nonpolarized distribution at the surfaces of both
somatodendritic and axonal domains. However in MDCK epithelia,
exogenously expressed mGluR7 is polarized at the basolateral cell
surface. Further experiments illustrated that the cytoplasmic tail of
mGluR7 was necessary for surface delivery in both epithelia and in
neurons but was not sufficient to mediate a polarized distribution in
either setting.
It has been suggested that epithelial cells and neurons can use similar
signals and mechanisms to sort membrane proteins to polarized
distributions at the cell surface, because some neuronal proteins found
in axons can sort to the apical surface of epithelia, whereas dendritic
proteins can sort to the basolateral surface (22, 26, 27). In the case
of mGluR7 this is clearly not the case. There must exist, therefore,
mechanisms in neurons that interpret the mGluR7 sorting information
differently from the mechanisms that interpret this information in MDCK
cells. Similar discrepancies have been seen with a number of proteins,
including norepinepherin and serotonin transporters (28, 29).
We have demonstrated that the cytoplasmic tail of mGluR7 is clearly
necessary for surface delivery, because the mGluR7-tail-minus construct
remained in a perinuclear intracellular pool and did not reach the cell
surface in either MDCK cells or neurons. This compartment colocalizes
with a trans-Golgi network antigen in both COS and MDCK cells. It would
appear therefore, that the cytoplasmic tail-minus construct seems
unable to exit the Golgi. The mechanism of this Golgi retention is not
clear. We can state, however, that the protein does not appear to cycle
between Glogi and cell surface via endosomes. This cycling behavior is
observed for other proteins concentrated in the TGN, including furin
and TGN 38. Membrane protein traffic from the TGN to the cell surface
has been theorized to involve carbohydrate moieties as either targeting
signals (30) or for modulation of the state of protein aggregation
(31). In the case of mGluR7-tail-minus, glycosylation sites remain
intact. Thus, the cause of retention may be more related to an
inability to participate in protein-protein interactions important for
stable surface expression. In addition, the cytoplasmic tail of mGluR7 is clearly not sufficient to mediate polarized sorting in epithelia, where the PLAP·mGluR7-tail construct remained within the cell in a
cytoplasmic pool distinct from endosomal or lysosomal compartments. In
neurons however, this construct was present at the surface of both
axons and dendrites, as was found for exogenously expressed mGluR7, but
did not achieve a polarized distribution. Thus, the capacity of mGluR7
constructs to attain cell surface expression is highly dependent upon
the cell type in which they are expressed. The mGluR7 cytoplasmic tail
is sufficient for cell surface delivery in neurons, but not in MDCK
cells. Importantly, this portion of the protein is not by itself
sufficient to specify polarized targeting in either cell type.
It is interesting to note that the cytoplasmic tails of a number of
GPCRs interact with PDZ-containing cytoskeletal proteins (32).
Cell surface machinery may be vital to polarized sorting (33).
Interaction with cytoskeletal elements, particularly PDZ-containing proteins, have been implicated not only in cell surface interactions that retain proteins at specific locations (34), but also at earlier
stages of protein targeting at the TGN (35). This is consistent with
our observation that the cytoplasmic tail epitope is obscured in MDCK
cells, where mGluR7 is distributed in a polarized fashion, but not in
COS cells, which lack polarized membrane domains. Physical interaction
of putative PDZ-containing cytoskeletal proteins with the C-terminal
tail of mGluR7 could explain our inability to observe the construct at
the basolateral surface with an antiserum directed to the cytoplasmic
tail. In fact, the three final C-terminal residues of mGluR7 may
represent a possible hydrophobic class II PDZ interaction motif (36).
However, the absence of observable clustering or detergent insolubility
for mGluR7 (data not shown) do not support a stable or continuous
interaction with the MDCK cytoskeleton, as has been correlated with
proper localization of some membrane proteins in MDCK (37, 38).
In summary, the cytoplasmic tail of mGluR7 is clearly necessary for the
surface delivery of the receptor but not sufficient to mediate its
polarized distribution in epithelia or in neurons. Other domains of the
mGluR7 molecule must be involved in contributing to sorting
information. The nature of these domains, the signals they encode, and
the interactions that interpret them remain to be determined.
 |
ACKNOWLEDGEMENTS |
We thank Drs. J. P. Conn, A. Gonzalez, and
S. Nakanishi for providing the various cDNAs and antibodies.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grant GM42136 and National Eye Institute Grant EY09534.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.
§
To whom correspondence should be addressed. Tel.: 212-570-2900;
Fax: 212-988-3672; E-mail: jbm2001@med.cornell.edu.
Published, JBC Papers in Press, December 5, 2000, DOI 10.1074/jbc.M008290200
 |
ABBREVIATIONS |
The abbreviations used are:
RER, rough
endoplasmic reticulum;
TGN, trans-Golgi network;
MDCK, Madin-Darby
canine kidney epithelial cells;
PCR, polymerase chain reaction;
GFP, green fluorescent protein;
wt, wild type;
DMEM, Dulbecco's modified
Eagle's medium;
mGluR7, metabotropic glutamate receptor subtype 7a;
PLAP, placental alkaline phosphatase;
VSVG, vesicular stomatitis virus
G protein.
 |
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