From the Department of Neurobiology, University of
Heidelberg, Im Neuenheimer Feld 364, Heidelberg 69120, the
Unit of Molecular Cell Biology, Department of Zoology,
University of Mainz, Bentzelweg 3, Mainz 55099, and the
§ Max Planck Institute of Experimental Medicine,
Hermann-Rein-Strasse 3, Göttingen 37075, Germany
Received for publication, September 30, 2002, and in revised form, November 14, 2002
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ABSTRACT |
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The proteoglycan NG2 is expressed by
immature glial cells in the developing and adult central nervous
system. Using the COOH-terminal region of NG2 as bait in a yeast
two-hybrid screen, we identified the glutamate receptor interaction
protein GRIP1, a multi-PDZ domain protein, as an interacting partner.
NG2 exhibits a PDZ binding motif at the extreme COOH terminus which
binds to the seventh PDZ domain of GRIP1. In addition to the published
expression in neurons, GRIP1 is expressed by immature glial cells.
GRIP1 is known to bind to the GluRB subunit of the AMPA glutamate
receptor expressed by subpopulations of neurons and immature glial
cells. In cultures of primary oligodendrocytes, cells coexpress GluRB and NG2. A complex of NG2, GRIP1, and GluRB can be precipitated from
transfected mammalian cells and from cultures of primary oligodendrocytes. Furthermore, NG2 and GRIP can be coprecipitated from
developing brain tissue. These data suggest that GRIP1 acts as a
scaffolding molecule clustering NG2 and AMPA receptors in immature
glia. In view of the presence of synaptic contacts between neurons and
NG2-positive glial cells in the hippocampus and the close association
of NG2-expressing glial cells with axons, we suggest a role for the
NG2·AMPA receptor complex in glial-neuronal recognition and signaling.
The NG2 proteoglycan is as a large transmembrane
glycoprotein expressed by oligodendrocyte progenitor cells but
down-regulated upon differentiation into mature oligodendrocytes (1,
2). The AN2 proteoglycan (3) is the mouse homolog of NG2 (4). In the
developing and adult nervous system many NG2-positive cells abound in
both white and gray matter whose classification as oligodendrocyte or
astrocyte lineage cells remains unclear but whose electrophysiological properties are typical of immature glial cells (5). Recently, Bergles
and colleagues (6) identified a novel type of synapse in the developing
and adult rat hippocampus where pyramidal neurons of the CA3 area form
morphologically identifiable synaptic boutons on NG2-positive cells.
Furthermore, stimulation of the neurons resulted in a Ca2+
signal in the NG2-positive glial cells which was dependent on the
activity of the AMPA1 class
of glutamate receptors. AMPA receptors are known to be expressed by
subclasses of glial cells including oligodendrocyte precursor cells (7)
where their function is thought to include an inhibitory influence on
proliferation and lineage progression of oligodendrocyte progenitors
(8).
NG2 is a single pass transmembrane proteoglycan with a large
extracellular domain and two LAM G (LNS) domains near the
NH2 terminus, suggesting a role as an adhesion molecule.
NG2 has a short intracellular tail of 76 amino acids. The PDZ protein
MUPP1 was identified recently by yeast two-hybrid screening of a
library from E9.5/10.5 mouse embryo as an intracellular binding partner of rat NG2 (9), although the biological significance of this finding
remains unclear. Because the COOH terminus of NG2 exhibits a putative
PDZ binding motif and PDZ domain proteins are adaptor proteins that
target and cluster protein complexes including neurotransmitter receptors to the cell surface (10), we sought to identify intracellular partners of NG2 in glial progenitor cells. The cytoplasmic domain of
murine NG2 was used as bait in yeast two-hybrid analysis to screen a
library from early postnatal mouse brain. This screen revealed the
glutamate receptor interaction protein
(GRIP) 1 as a binding partner. We show
that GRIP1 acts as a direct molecular link between AMPA receptors and
the glycoprotein NG2 in immature glial cells. This protein complex,
together with as yet to be identified additional members, may
contribute to a postsynaptic microdomain in immature glial cells and
contribute to glial-neuronal signaling.
Animals--
NMRI mice were obtained from the central animal
facilities of the University of Heidelberg.
Antibodies--
The following primary antibodies were used:
monoclonal (mc) GRIP1 (Becton Dickinson), polyclonal (pc) antibodies
against GRIP1 (kind gift from Dr. R. Klein), and pc
GluRB2 (Chemicon,
Hofheim, Germany). The antibodies recognize specifically the
short GluRB form (COOH-terminal peptide: SVKI*), which binds to GRIP1.
We also used pc AN2 antibodies (3), anti-GST880S16 (pc AN2 against a
GST fusion protein consisting of amino acids 1255-1545, mc AN2
antibodies (3), pc antibodies against NCAM (11), mc
anti-Myc (Sigma), and mc 8-18-C5 against MOG (a kind gift from
Dr. C. Linington).
Isolation of GRIP1 by Yeast Two-hybrid Screen--
The
COOH-terminal region of mouse NG2 (NH2-RKRNKT ...
NGQYWV-COOH, GenBank accession number AF352400) was fused to the GAL4 binding domain by cloning it into the pGBT9 vector
(Clontech) with EcoRI/BamHI.
The resulting bait construct was designated pGBT9cyto. Using the
lithium acetate method, the yeast strain CG1945 was transformed
sequentially with pGBT9cyto and a postnatal mouse brain MATCHMAKER
cDNA library in pACT2 (Clontech). 33 × 106 transformants were screened. Transformants were grown
on SD medium-Leu-Trp-His plates; 5 mM
3-amino-1,2,4-triazole was added to the medium to suppress leaky HIS3
reporter gene expression. Positive clones were tested for
GRIP1 Deletion Analysis--
The yeast two-hybrid system was
used to map the interaction site of GRIP1 with NG2. Results from the
sequencing of GRIP1 revealed the seventh PDZ domain as putative
interaction site. Mouse GRIP1 deletion constructs in pACT2 were used as
resulting from the screen: mouse GRIP1 4p-7 (LEIEFD ... EPTNTL),
mouse GRIP1 5-7 (KHSVEL ... EPTNTL), mouse GRIP1, 7 (ATIMSG ... EPTNTL). Further control constructs of rat GRIP1 were
kind gifts from Dr. R. Klein: rat GRIP1 4-6 (TSPRGT ... KLSDVY),
rat GRIP1, 6 (EDNSDE ... KQTDAQ), rat GRIP1 6-7 (GAIIYT ...
EPTNTL). The NH2- and COOH-terminal sequences above correspond to sequences from mouse GRIP1 library plasmids or to the
published rat GRIP1 sequence (12). All constructs were verified by DNA
sequencing. Cotransformed yeast cells were grown on double dropout
medium and assayed for GRIP2 Interaction Analysis--
The yeast two-hybrid system was
used to test the interaction of NG2 and the seventh PDZ domain of
GRIP2. The GRIP2 cDNA sequence was generated by reverse
transcription PCR, which introduced EcoRI/BamHI sites and then cloned into the pACT2 vector: rat GRIP2, 7 (RSREVGT ... SSPQMI); the sequence corresponds to the published
rat sequence (13) and was verified by sequencing.
Identification of PDZ Binding Motif--
The yeast two-hybrid
system was used to map the PDZ binding motif at the extreme COOH
terminus of NG2. Individual mutations of the 0, Transfection and Expression Constructs--
COS7 cells were
transfected by electroporation (0.25 kV, 250 microfarads). Plasmids
were used at 15 µg/300 µl of cell suspension (4 × 106 cells/ml, Dulbecco's modified Eagle's medium and 10%
fetal calf serum). The NG2 deletion mutant was generated by
trimolecular ligation of two PCR-amplified regions of NG2 with
artificially introduced restriction sites in the pRK5 vector (14). The
deletion mutant consists of the signal sequence, one-fourth of the very NH2-terminal extracellular portion (including both LNS
domains), the transmembrane domain, and the complete intracellular
region. Mouse GRIP1/PDZ7 and mouse GRIP1/PDZ5-7 expression constructs were generated by cloning murine sequences into pRK5 vector with EcoRI/BamHI and
EcoRI/HindIII sites introduced by PCR,
respectively. The constructs contain a translation initiation sequence
(15) and encode an NH2-terminal Myc tag. GluRB full-length,
flop, short form was a kind gift from Dr. H. Monyer. Constructs were
verified by sequencing.
Coimmunoprecipitation--
Transfected COS7 cells were washed
with phosphate-buffered saline (PBS) 24 h after transfection,
starved for 1 h in methionine/cysteine-free medium, then
metabolically labeled with 100 µCi/ml [35S]Met/Cys for
4 h. Cells were washed twice with PBS and lysed (1% Triton X-100,
50 mM Tris, pH 7.5, 150 mM NaCl, protease
inhibitors), and the lysates were chilled for 30 min and centrifuged at
3,000 rpm for 5 min to remove nuclei. For immunoprecipitation the
following antibodies were used: mouse mc Myc (Sigma), rabbit pc AN2
(3), pc GluRB (Chemicon). Lysates were preabsorbed with protein
A-Sepharose (Amersham Biosciences) for 1 h at 4 °C, then
subjected to immunoprecipitation overnight at 4 °C. Precipitation
was performed with protein A-Sepharose. Precipitates were washed three
times with radioimmune precipitation assay buffer (0.1% SDS, 1%
Nonidet P-40, 1% sodium deoxycholate, 150 mM NaCl, 50 mM Tris, pH 7) and once with PBS before adding sample
buffer and resolving the proteins by SDS-PAGE. Gels were dried, exposed
to screens, and evaluated with a PhosphorImager.
Total mouse brains (P7) were homogenized in buffer (50 mM
Tris, pH 7.8, 3 mM MgCl2, 320 mM
sucrose, protease inhibitors) using an Ultra Turrax. The homogenate was
centrifuged for 10 min at 1,000 rpm, 4 °C, the resulting supernatant
was centrifuged for 1 h at 100,000 × g, 4 °C.
The pellet was extracted in buffer (50 mM Tris, pH 7.8, 150 mM NaCl, 1 mM EDTA, 0.5% SDS, 0.05% sodium deoxycholate, 1% Triton X-100, protease inhibitors) for 1 h at 4 °C and centrifuged afterward for 1 h, 100,000 × g, 4 °C. The supernatant was preabsorbed with protein
A-Sepharose and subjected to immunoprecipitation as described above.
Precipitates were washed three times with 1% Triton X-100 buffer and
once with PBS and analyzed by SDS-PAGE and Western blotting.
Primary oligodendrocytes were lysed in 1% Triton X-100, 50 mM Tris, pH 7.5, 150 mM NaCl, protease
inhibitors, and the lysates were chilled on ice, centrifuged, and
subjected to immunoprecipitation. Precipitates were washed three times
with lysis buffer and once with PBS and analyzed by SDS-PAGE and
Western blotting.
Western Blot Analysis--
SDS-PAGE was performed according to
Laemmli (16). Proteins were blotted onto polyvinylidene difluoride
membrane (Hybond P, Amersham Biosciences), blocked with 4% milk powder
in PBS and 0.1% Tween 20, and incubated with primary antibodies for
1 h. The blots were washed with PBST and incubated with
appropriate secondary horseradish peroxidase-conjugated anti-mouse
immunoglobulin (Dianova, Hamburg, Germany), horseradish
peroxidase-conjugated anti-rabbit immunoglobulin (Dianova). They were
washed twice with PBST and once with PBS and subsequently developed by
enhanced chemiluminescence (Amersham Biosciences).
Lipid Raft Preparation--
12 × 106 cells
(primary oligodendrocytes) were extracted in 750 µl of 1% Triton
X-100 buffer, chilled for 30 min, and centrifuged (see
"Coimmunoprecipitation"). 750 µl of extract was adjusted to 40%
sucrose with 750 µl of 80% sucrose. The extract was overlaid with
1.75 ml of 30% sucrose and 1.5 ml of 5% sucrose in an SW60 tube
(Beckman). After centrifugation (4 h at 218,000 × g,
4 °C) eight fractions were collected (17) and analyzed by 4-10%
SDS-PAGE and Western blotting.
Cell Culture and Immunofluorescence Staining--
Primary
oligodendrocytes were cultured according to Trotter and colleagues (11,
18), shaken-off oligodendrocytes were cultured on
poly-L-lysine-coated glass coverslips for 2 days in SATO medium containing 1% horse serum, 10 ng/ml platelet-derived growth factor, and 5 ng/ml basic fibroblast growth factor. Cells were
washed with PBS, fixed for 10 min with 4% paraformaldehyde, washed
with PBS, permeabilized for 5 min with 0.05% Triton X-100, washed with
PBS, and blocked with NG2 Binds to the Seventh PDZ Domain of GRIP1 and GRIP2--
The
complete mouse NG2 cytoplasmic region consisting of 76 amino acids
(RKRN ... QYWV*, * = translation stop codon) was used as a bait in
a yeast two-hybrid analysis to screen a postnatal mouse brain cDNA
library. 33 × 106 transformants were screened. Ten
independent library plasmids represented fragments of the same
sequence, which was identical to murine GRIP1, a multi-PDZ domain
protein (Fig. 1a). GRIP1 was originally identified as an interacting protein of AMPA receptor subunits GluRB and GluRC (12) The GRIP1 clones isolated in the screen
were grouped according to their PDZ domain composition: group1 encodes
a partial PDZ domain 4 through PDZ domain 7 (GRIP1/4p-7), group2 PDZ
domain 5 through 7 (GRIP1/5-7), group3 a partial PDZ domain 6 through
7 (GRIP1/6p-7), group4 encodes PDZ domain 7 only (GRIP1/7). This
analysis already identified the seventh PDZ domain as the putative
interaction site.
Yeast two-hybrid technology was used to verify the interaction of GRIP1
with the cytoplasmic region of NG2 and to confirm the interaction site
by excluding binding of NG2 to GRIP1 PDZ domains other than PDZ7. Yeast
was cotransformed with isolated GRIP1 plasmids and pGBT9cyto, grown on
double dropout medium, and subsequently assayed for
GRIP2, a GRIP1 homolog, also consists of seven PDZ domains and is
expressed in the central nervous system (13, 19). Because the sequences
of the corresponding PDZ domains of GRIP1 and GRIP2 are very well
conserved, we tested whether the interaction with NG2 was similarly
conserved. The results in Fig. 1c demonstrate that NG2 also
interacts with the seventh PDZ domain of GRIP2, even though no clone
for GRIP2 was isolated in the original screen.
NG2 Has a PDZ Binding Motif at the Extreme COOH Terminus--
PDZ
domains bind to COOH-terminal peptides of the interacting protein. The
COOH-terminal tetrapeptide of NG2 (QYWV*) is conserved among rat,
mouse, human, and Drosophila. It is similar to the PDZ
binding motifs of ephrin B1 (YYKV*), which binds to PDZ6 of GRIP1/2
(19) and neurexins (EYYV*), which bind to CASK (20), with a valine at
position 0 and a tyrosine at the
PDZ7 of GRIP1/2 thus belongs to class II PDZ domains, which bind to
motifs with hydrophobic amino acids at positions 0 and
NG2-GRIP1 and NG2-GRIP2 associations were further confirmed by
coimmunoprecipitation of NG2 together with GRIP1 or GRIP2 expressed in
COS7 cells. First, cells were analyzed after transfection with an NG2
construct (NG2del, Fig. 3a),
comprising the signal sequence, approximately one-fourth of the
extracellular portion, the transmembrane domain, and the complete
intracellular region. The protein was incorporated into the membrane
and recognized by pc AN2 antibodies (Fig. 3b). Subsequently
the cells were transfected with the NG2del construct together with
Myc-tagged GRIP1/PDZ7 or GRIP2/PDZ7, respectively (Fig. 3a).
Anti-Myc antibodies precipitated GRIP1 and associated NG2del from
cotransfected COS7 cells (Fig. 3c, lane 2) and pc AN2 antibodies recognizing mouse NG2 precipitate NG2del and associated GRIP1 (Fig. 3c, lane 3). Anti-Myc antibodies also
precipitate GRIP2 and associated NG2del from transfected COS7 cells
(Fig. 3c, lane 4) expressing both these
constructs.
To confirm the NG2-GRIP1 interaction in vivo, extracts from
P7 total mouse brain were subjected to immunoprecipitation using AN2
antibodies followed by Western blot analysis of coprecipitated GRIP1.
The results (Fig. 3d) demonstrate that GRIP1 associates with
NG2 in vivo. Pc AN2 antibodies precipitate NG2 (lane
1) and associated GRIP1 as revealed by Western blotting with
antibodies against GRIP1 (lane 2). Lanes 3 and
4 are controls showing the presence of GRIP1 and NG2 in the
brain extracts before precipitation.
NG2-positive Cultured Oligodendrocyte Progenitors Express GRIP and
AMPA Receptors--
Biochemical studies of GRIP1 have largely focused
on neuronal expression, where GRIP1 is enriched in the postsynaptic
density and at synaptic plasma membranes (13, 23). Because NG2 is expressed by immature glial cells including oligodendrocyte progenitors (3), it was important to demonstrate that the interaction partner GRIP1
is also expressed in these cells. A Western blot analysis of lysates of
the murine oligodendrocyte progenitor cell line Oli-neu (24)
and cultures of mouse primary oligodendrocytes consisting of a range of
differentiation stages with antibodies against GRIP1 demonstrated a
band of 130 kDa (Fig. 4a,
lanes 1 and 2). A total lysate from P9 mouse
brain exhibited a band at the same molecular mass (lane
3) as in the control blot with rat cerebrum (lane 4).
These results demonstrate that GRIP1 is indeed expressed in
oligodendrocyte lineage cells.
Because GRIP also binds to the GluRB subunit of AMPA receptors (12) and
AMPA receptors are expressed by glial cells (7), we examined whether
NG2-positive cells also express GluRB. First, a Western blot analysis
was performed using antibodies to GluRB (Fig. 4b). A band of
108 kDa was detected in lysates of primary oligodendrocytes (lane
2) and in the control lysate (P9 total mouse brain, lane
3). No expression was detected in the Oli-neu cell line
(lane 1). To define GluRB expression in individual cells, mixed cultures of primary oligodendrocytes were costained with AN2
antibodies and GluRB antibodies (Fig. 4c). All
oligodendrocyte lineage cells staining with antibodies to GluRB
coexpressed NG2; however, not all NG2-positive cells expressed GluRB.
GluRB staining was restricted to more immature cells with a few
processes and revealed a punctate distribution of the protein along the
major processes in addition to an intense intracellular staining. More mature oligodendrocytes characterized by a more complex morphology no
longer expressed GluRB (see also Fig. 6d, white
star).
GRIP Expression Is Down-regulated Later in Maturing
Oligodendrocytes Than GluRB--
NG2 expression shows a partial
overlap with the expression of the O4 marker (3), which characterizes a
late stage of oligodendrocyte progenitor cells (25) and is retained on
more mature oligodendrocytes (26). In contrast, there is no overlap of
NG2 and proteins expressed by more mature oligodendrocytes (3). To
study the timing of GRIP1 and GluRB expression in oligodendrocyte
lineage cells (Fig. 5), mixed cultures of
primary oligodendrocytes were used as an in vitro model for
oligodendrocyte maturation (27) and analyzed after 2, 8, and 14 days in
culture by Western blotting. These cultures represent a dynamic
population of different developmental stages that initially consists
(div2) of predominantly immature cells. GRIP1 expression is high in
immature cultures and decreases slowly as oligodendrocytes mature. In
contrast, GluRB expression drops dramatically as oligodendrocytes
mature. Maturation of oligodendrocytes is shown by increasing
expression of MOG, a marker of mature oligodendrocytes. Staining of
such cultures demonstrates that NG2 and MOG are never coexpressed and
that GluRB-expressing cells are always MOG-negative. GRIP1 expression
persists over a longer period of maturation than GluRB expression,
implying that GRIP1 may have multiple binding partners during the
course of oligodendroglial development.
NG2 Forms a Trimeric Complex with GRIP1 and the AMPA Receptor
Subunit GluRB--
Transfected COS7 cells were subjected to
radiolabeling and immunoprecipitation to investigate whether a complex
consisting of NG2, GRIP1, and GluRB can be isolated. Cells were
transfected with the NG2del, Myc-tagged GRIP1 and the AMPA subunit
GluRB (flop, short form). NG2 as well as the GluRB subunit were
incorporated into the plasma membrane. The GRIP1 construct encodes PDZ
domains 5-7 (Fig. 6a). GluRB
was reported to bind to PDZ4-5 of GRIP (12), and we have shown that
NG2 binds to GRIP/PDZ7. Fig. 6b shows immunoprecipitation of
the complex: pc AN2 antibodies precipitate NG2del together with
associated GRIP1/PDZ5-7 and GluRB (third lane from
left), pc GluRB antibodies precipitate GluRB with
associated NG2del and GRIP1 (fourth lane), and anti-Myc
antibodies precipitate GRIP1 with associated NG2 and GluRB
(second lane). Although it was reported that PDZ4 and 5 and
the NH2-terminal residues of GRIP1 are required for
interaction in the yeast two-hybrid system, the GRIP1 (PDZ5-7) protein
was sufficient to bind to GluRB in mammalian cells. In cells
transfected with NG2del and GluRB but without GRIP1, pc GluRB
antibodies precipitate only GluRB with no associated NG2del (first lane, control).
Mixed cultures of primary oligodendrocytes (div2) were also subjected
to coimmunoprecipitation to demonstrate the interaction of endogenously
expressed proteins. Two different pc GRIP antibodies precipitated GRIP1
with associated NG2 and GluRB. Fig. 6c shows a Western blot
of GRIP immunoprecipitates analyzed with mc AN2 antibodies (first
lane), mc GRIP1 antibodies (second lane), and pc GluRB
antibodies (third lane). The specificity of the
coprecipitated proteins was tested by probing the blot with antibodies
to NCAM, which is highly expressed by oligodendrocytes. No signal for
NCAM could be detected in the GRIP1 precipitates, although this protein is present at a high level in the lysates (Fig. 6c,
fourth and fifth lanes, respectively), thus
demonstrating the specificity of the coprecipitation.
Confocal analysis of immature oligodendrocytes costained with AN2
antibodies and antibodies to GRIP (Fig. 6d) showed that GRIP
is highly expressed in the cell body and in the proximal regions of the
processes. NG2 staining outlines the cell body plasma membrane into the
very tips of the processes. There is an overlap of GRIP and NG2
staining at the somal cell membrane. NG2 also overlaps with GluRB
staining at the plasma membrane, even though there are additionally
high levels of intracellular GluRB.
GRIP Is Not Localized in Lipid Raft Microdomains in Immature
Oligodendroglia--
GRIP and other PDZ domain proteins play an
important role in targeting to and clustering proteins at the membrane
(10, 28-30). Bruckner and colleagues (19) described the association of
GRIP proteins with detergent-insoluble membrane microdomains (lipid rafts). Lipid rafts are implicated in cell signaling, and
raft-associated proteins can be isolated by their insolubility in
Triton X-100 at low temperature and flotation in sucrose density
gradients (31, 32). Because lipid rafts were shown to be important for signaling and sorting of proteins in oligodendrocytes (33, 34), we
analyzed oligodendroglial rafts for the presence of NG2 and GRIP. Fig.
7 shows that neither GRIP1 (a)
nor NG2 (b) is raft-associated in oligodendroglial cells.
Both proteins are located in bottom fractions (6-8) of a density
gradient and not in the lipid raft fraction floating in low density
gradient fractions. NCAM 120, a lipid raft-associated
glycosylphosphatidylinositol-anchored protein, is in fraction 2, whereas NCAM 140 and 180, both transmembrane isoforms, are in the
bottom fractions (6-8, Fig. 7c). This suggests that NG2
binds to GRIP in a lipid raft-independent membrane domain of
oligodendroglial progenitors, whereas in neurons raft-associated proteins recruit GRIP into lipid rafts.
NG2 Is a Constituent of a Protein Complex in Immature Glial
Cells--
Using the yeast two-hybrid approach, we identified GRIP1, a
multi-PDZ domain protein, as an interaction partner of NG2 in the
central nervous system. GRIP1 was originally identified as binding to
the AMPA receptor subunits GluRB and GluRC (12). In addition, it was
reported to interact with ephrin B1 ligand (19), Eph receptors (35),
GRASP-1, a Ras guanylate exchange factor (36), liprin- Immature Glia Form a Complex of NG2, GRIP, and AMPA
Receptors--
GRIP1 was originally defined as a neuron-specific
protein, but our results show that is also expressed by glial cells,
implying that GRIP1 can act as a scaffolding molecule in this cell type and cluster protein complexes at the cell surface. GRIP1 binds to the
AMPA receptor subunits GluRB and C (12), and it has been shown that
AMPA receptor subunits are expressed not only by neurons but also by
glia (6, 39-41), including cells of the oligodendroglial lineage (7)
and hippocampal glial cells in situ (42-44). In this report
we have shown the coexpression of GRIP1 and GluRB in NG2-positive
cultured oligodendroglial precursor cells. NG2 and GRIP have several
intracellular partners in glial cells
(9),4 and thus visualization
of the complex as spots at the cell membrane and a complete overlap of
staining of NG2, GRIP, and GluRB would not be expected.
A triple complex of these proteins can be precipitated from transfected
COS7 cells and can be isolated from immature glial cells in culture.
NG2 isolated from brain extracts is bound to GRIP 1, and thus the
complex of NG2, GRIP, and AMPA receptor exists in vivo.
GRIP1 functions as an adaptor molecule and binds to both NG2 and
GluRB/C, whereby the PDZ5-7 domains are sufficient for the
interaction. In reality the complex may indeed be a lot larger than
these three components and may include proteins that have been shown to
interact with GRIP in neurons, such as the ephrins. Furthermore there
are studies of GRIP1 interacting with itself and GRIP2 (13) via a
PDZ-PDZ interaction, thus magnifying the complexity of the
associations. Such typical multiprotein clusters determine the pre- and
postsynaptic density (for review, see Refs. 10, 30, and 45).
NG2-positive Glial Cells Expressing AMPA Receptors Are Situated at
Sites of Glial-Neuronal Contact and Receive Neuronal
Signals--
NG2-expressing cells are found throughout the developing
central nervous system in both white and gray matter. The function and
lineage assignment of these cells are under intense discussion. NG2
cells include the precursors for oligodendrocytes; NG2-positive cells
isolated from P3 brain behave like classic O2A cells, developing into
oligodendrocytes or astrocytes according to the composition of the
culture medium (5), and NG2 cells from optic nerve and cerebellum have
also been proposed to represent O2A progenitor cells (46, 47). However,
NG2 cells, especially in the adult central nervous system, most likely
represent a heterogeneous population.
The functional role of these AMPA receptors in immature
oligodendrocytes is thought to include a regulation of cell
proliferation and differentiation (8, 48, 49). The receptors are
down-regulated when the glial progenitor cells mature into
oligodendrocytes or astrocytes. In the adult rat hippocampus many
NG2-positive cells surround synapses (50), and it has been shown
recently that in the developing and adult rat hippocampus, neurons of
the CA3 area make synapses on NG2-positive glial cells visualized by
electron microscopy (6). Stimulation of the neurons results in
activation of the glial AMPA receptors via the released glutamate,
yielding a change in membrane potential and a Ca2+ signal
in the glia (6). The NG2·GRIP1·AMPA receptor complex that we have
isolated may be part of this glial postsynaptic domain. In the adult
central nervous system, NG2-positive cells make close contact to the
node of Ranvier (51). Early work by Fulton and co-workers (52) showed
that in the adult nervous system cells taking up cobalt in response to
quisqualate stimulation (acting on AMPA receptors) are also localized
at the nodes and would suggest that these paranodal NG2-positive cells
may also have AMPA receptors.
The AMPA GluRC subunit can also bind GRIP, suggesting that NG2 can
complex with both calcium-permeable (GluRB-containing) and
calcium-impermeable (lacking GluRB) glutamate receptors. Activation of
glial AMPA receptors and controlled variation of their calcium permeability may be important regulators of the morphology of glial
processes and regulate signaling at neuron-glial synapses or
neuron-neuron synapses where glial cells ensheath the synapse (50,
53-55).
The Ménage à Trois Involving GluRB, GRIP, and NG2 May
Position Glial AMPA Receptors toward Neurons--
The NG2 molecule
when first cloned was found to have little homology to known proteins
but limited homology to cadherins (56). However, NG2 has been found to
possess two LAM G (LNS) domains at the amino terminus (57). These
domains are found in the neurexins and are thought to be independently
folding domains (58, 59). Neurexins bind protein ligands with high
affinity (60) and are thought play a role as cell adhesion molecules;
The close apposition of NG2-positive glial cells with the nodes of
Ranvier in the central nervous system (51), the deposition of NG2 at
nodes in the peripheral nervous system (63), and the expression of NG2
by immature oligodendrocytes as well as Schwann cells in close
association with neurons (4) support the existence of a neuronal
receptor. A neuronal receptor for NG2 has been postulated before in the
context of axonal growth inhibition (64). We propose the following
functions of the NG2·AMPA receptor complex. First, NG2 may function
to position glial AMPA receptors toward glutamergic neurons and may be
instructive for the formation of the glial-neuronal synapses (Fig.
8a) described by Bergles and
colleagues (6). Second, NG2-positive glial cells may surround classical
neuronal synapses (Fig. 8b) (50) and could thus influence
synaptic formation and signaling (53-55, 65, 66). The complex may play
a role in sensing neuronal activity. Third, NG2 expressed by
oligodendroglial progenitor cells may play a role in axonal recognition
during the early phases of myelination: the associated AMPA receptor could thus sense neuronal activity, which would in turn regulate the
proliferation and differentiation of the glial cells (Fig. 8c). Fourth, NG2-positive glia at the node of Ranvier may
play a regulatory role at the node (Fig. 8d). It will be
important to determine the role of NG2 cells in glial-neuronal
signaling and to elucidate the functional implications of the elicited
glial signals in this network.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase gene activity: yeast colonies were grown on
SD-Leu-Trp-His, transferred onto reinforced nitrocellulose membrane,
submerged in liquid nitrogen, and placed on a Z-buffer/X-gal-soaked Whatman (Z-buffer: 16.1 g/liter Na2HPO4*7H2O,
5.5 g/liter NaH2PO4*H2O, 0.75 g/liter KCl,
0.246 g/liter MgSO4*7H2O, pH 7; Z-buffer/X-gal solution:
100 ml of Z-buffer, 0.27 ml of
-mercaptoethanol, 1.67 ml of 20 mg/ml
X-gal stock solution). Blue color was allowed to develop for 30 min-3
h. DNA-sequencing results revealed 48 isolates and 10 independent
clones of GRIP1. All clones included the seventh PDZ domain and the
COOH terminus of GRIP1. The specificity of the NG2-GRIP1 interaction
was confirmed by a
-galactosidase test and growth selection of
cotransformed yeast cells with pGBT9cyto and isolated library plasmids.
Unspecific NG2 interactions were excluded by contransformation of yeast
cells with pGBT9cyto and GRIP1 constructs in pGADGH lacking the
seventh PDZ domain (kind gift from Dr. R. Klein). Unspecific
interactions of the seventh PDZ domain of GRIP1 were excluded by
cotransformation of yeast with a pGBT9 construct encoding the
cytoplasmic COOH terminus of an unrelated transmembrane protein
M6A.3
-galactosidase gene activity and additionally
selected for growth on triple dropout medium.
1,
2, and
3
positions of the COOH-terminal peptide QYWV* were introduced by PCR,
cloned into pGBT9, and designated as NG2 0G (Val mutated to Gly), NG2
1G (Trp mutated to Gly), NG2
2G (Tyr mutated to Gly), NG2
2F (Tyr
mutated to Phe), NG2
3G (Gln mutated to Gly). Mutant NG2 constructs
were contransformed with mouse GRIP1 PDZ7 and rat GRIP2 PDZ7. Yeast
cells were grown on double dropout medium and assayed for
-galactosidase gene activity and additionally selected for growth on
triple dropout medium.
-mercaptoethanol and 10% horse serum. Primary
antibodies were diluted in blocking buffer and incubated for 30 min.
Cells were washed three times with
-mercaptoethanol and 10% horse
serum and incubated for 30 min with appropriate secondary Cy2- and
Cy3-conjugated pc antibodies (Dianova), diluted in blocking buffer. The
washed coverslips were mounted in Moviol and analyzed by confocal
microscopy (Leica, Bensheim, Germany).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (29K):
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Fig. 1.
NG2 interacts with the PDZ domain 7 of GRIP1
and GRIP2. a depicts the protein structure of GRIP1 and
GRIP2; the seven PDZ domains are represented by ovals.
b, different GRIP1 constructs were used to verify the
interaction with the cytoplasmic region of NG2. Transformed yeast were
grown on SD-Trp-Leu plates, assayed for -galactosidase
(
gal) activity, and selected for growth on
SD-Trp-Leu-His. c shows the interaction of GRIP2 (PDZ7) with
NG2. m, mouse; r, rat; partial PDZ domain are
indicated by p, + indicates growth on selection plates,
and
indicates no growth. The resulting color reaction of
-galactosidase activity from growing yeast is shown.
-galactosidase
activity. The yeast colonies were additionally selected for growth on
triple dropout medium. As shown in Fig. 1b, yeast two-hybrid
analysis and subsequent
-galactosidase assay confirmed the binding
of NG2 to GRIP1/PDZ4p-7, GRIP1/PDZ5-7, GRIP1/PDZp6-7, and GRIP1/PDZ7.
When rat GRIP1 constructs consisting of PDZ domains 4-6 and the sixth
domain only were tested for interaction, the yeast cells were negative
for
-galactosidase activity, and failed to grow on triple dropout
medium. Rat constructs containing the seventh PDZ domain such as
GRIP1/PDZ6p-7 and GRIP1/PDZ6-7 restored the interaction of NG2 and
GRIP1 and clearly identified the seventh PDZ domain of GRIP as the
interaction site.
2 position. To test whether QYWV* of
NG2 is indeed a PDZ binding motif, positions 0,
1,
2, and
3 were
mutated individually to glycine (Fig. 2). The mutations 0G,
1G, and
2G eliminated the interaction of NG2 and
GRIP1/2, confirming that there is a PDZ binding motif at the extreme
COOH terminus of NG2. Mutating the
3 position was without effect. The
2 position has been reported to be critical for the interaction. To
characterize further the relevance of the amino acid at the
2
position, tyrosine was mutated to phenylalanine (Fig. 2), thus testing
whether the hydroxyl group was essential for the interaction as is the
case of PSD95 binding to the third PDZ domain of CRIPT (21), where a
hydrogen bond forms with the residue of the binding groove. However,
this mutation (
2F) did not interfere with the interaction of NG2 and
GRIP, showing that it is not the OH group that is essential but rather
the hydrophobic nature of the aromatic side chain.
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Fig. 2.
The COOH-terminal peptide of NG2 binds to the
PDZ domain of GRIP. The cytoplasmic region of NG2 was mutated at
the indicated positions; the resulting COOH-terminal sequence is shown
in parentheses. Wild type and mutated COOH termini were
tested for interaction with PDZ7 of GRIP1 and GRIP2. Transformants were
grown on double dropout selection plates, assayed for -galactosidase
(
gal) activity, and selected for growth on triple dropout
selection plates.
2:
X
* (where
is a hydrophobic amino acid, and
X is any amino acid (10, 22)). NG2 associates with GRIP1 or
GRIP2 in mammalian cells.
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Fig. 3.
NG2 associates with GRIP1 and GRIP2 in
transfected COS7 cells and with GRIP1 in the brain. a shows
the Myc-tagged GRIP1/2 construct and the NG2del construct giving rise
to a 77-kDa protein and a larger glycosylated form. b, to
control for correct membrane insertion of NG2del, COS7 cells were
transfected with the NG2del construct, cultured for 24 h, followed
by immunofluorescence staining of the live cells with pc AN2 antibodies
and fixation. Confocal analysis shows NG2del staining at the cell
surface. c, COS7 cells were cotransfected with NG2del and
GRIP1 or GRIP2, respectively. The cells were cultured for 24 h,
incubated for 1 h in Met/Cys-free medium, metabolically labeled
for 4 h with [35S]Met/Cys, lysed in 1% Nonidet P-40
buffer, and subjected to immunoprecipitation (IP).
Lane 1, protein A-Sepharose preclear; lane 2,
immunoprecipitated GRIP1 associates with NG2del; lane 3,
immunoprecipitated NG2del associates with GRIP1; lane 4,
immunoprecipitated GRIP2 associates with NG2del. The
asterisk indicates glycosylated NG2del. d, total
mouse brain (P7) was homogenized using an Ultra Turrax, nuclei were
removed by centrifugation, and the homogenate was centrifuged at
100,000 × g. The pellet was solubilized in
detergent-containing buffer, and insoluble material was removed by
centrifugation. Lysates were subjected to immunoprecipitation using pc
AN2 antibodies, resolved by SDS-PAGE on a 4-10% gel, and analyzed by
Western blotting (WB). Lane 1 shows an
immunoprecipitation with pc AN2 antibodies followed by Western blotting
with mc AN2 antibodies. Lane 2 shows immunoprecipitation
with pc AN2 followed by Western blotting with mc GRIP1. Lanes
3 and 4 are control lanes that confirm the presence of
GRIP1 and NG2 in the lysate used for immunoprecipitation.
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Fig. 4.
Cultures of mixed primary oligodendrocytes
and the Oli-neu cell line express GRIP1 and
NG2-positive oligodendrocyte lineage cells express the AMPA receptor
subunit GluRB. a, lysates from Oli-neu
(lane 1), primary oligodendrocytes (lane 2), P9
mouse brain (lane 3, control), and rat cerebrum (lane
4, control) were resolved by 10% SDS-PAGE, blotted onto membrane,
probed with mc GRIP1 antibodies, and developed with enhanced
chemiluminescence. A 130 kDa band is visible in lanes 1-4.
b, lysates from Oli-neu (lane 1),
primary oligodendrocytes (lane 2), and P9 mouse brain
(lane 3, control) were resolved by 10% SDS-PAGE, blotted
onto membrane, probed with pc GluRB antibodies, and developed with ECL.
There is no GluRB expression in Oli-neu, but cultures of
primary oligodendrocytes and the control (mouse brain, P6) demonstrate
the presence of GluRB. c, shaken off primary
oligodendrocytes were grown on poly-L-lysine-coated
coverslips for 2 days, fixed, permeabilized, and costained with mc AN2
antibodies and pc GluRB antibodies.
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Fig. 5.
Comparison of GRIP1 and GluRB expression in
developing oligodendrocytes in culture. a, cultures of
primary oligodendrocytes (div2, 8, 14)
were lysed, and equal amounts of protein were resolved by 7.5%
SDS-PAGE and immunoblotted with mc GRIP. Membranes were stripped and
reprobed with pc GluRB antibodies, mc MOG antibodies (marker for mature
oligodendrocytes), and -tubulin antibodies. b, primary
oligodendrocytes stained with either anti-GluRB and anti-MOG
antibodies, or anti-AN2 and anti-MOG antibodies, show mutually
exclusive immunofluorescent signals.
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Fig. 6.
NG2 associates with GRIP1 and GluRB in
transfected COS7 cells and primary cultures of oligodendrocytes.
a shows an NH2-terminally Myc-tagged GRIP1
construct consisting of PDZ 5-7. b, COS7 cells were triple
transfected with NG2del, GRIP1 (PDZ, m, 5-7) and full-length GluRB
(flop, short form, COOH-terminal peptide SVKI*). Cells were grown for
24 h, starved for 1 h in Met/Cys-free medium, metabolically
labeled with [35S]Met/Cys, lysed in 1% Nonidet P-40
buffer, and subjected to immunoprecipitation. First lane
from left (control), pc GluRB antibodies precipitate only
GluRB from COS7 cells transfected with GluRB and NG2del, but without
GRIP1 (PDZ5-7). Second lane, Myc antibodies precipitate
GRIP1 and associated NG2del and GluRB from cells transfected with all
three constructs. Third lane, pc AN2 antibodies precipitate
NG2del and associated GRIP (PDZ5-7) and GluRB. Fourth lane,
pc GluRB antibodies precipitate GluRB and associated NG2del and GRIP1
(PDZ5-7). The asterisk indicates glycosylated NG2del.
c, cultured primary oligodendrocytes were subjected to
immunoprecipitation using pc GRIP1 antibodies followed by Western blot
analysis. pc GRIP1 antibodies precipitate GRIP1 with associated NG2
(first lane), GRIP1 (second lane), and GluRB
(third lane). No NCAM could be detected in the GRIP1
precipitate (fourth lane), although it is present in the
total lysate (fifth lane). d, primary
oligodendrocytes were cultured for 2 days, fixed with paraformaldehyde,
permeabilized with 0.05% Triton X-100, and costained with mc AN2, pc
GRIP1 antibodies, and pc GluRB antibodies, respectively. Cells were
analyzed by confocal microscopy.
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Fig. 7.
GRIP1 is not raft-associated in
oligodendrocytes. Primary oligodendrocytes (div2) were lysed in
1% Triton X-100 buffer and adjusted to 40% sucrose followed by
overlayering with 30 and 5% sucrose. The gradient was centrifuged for
4 h at 218,000 × g, 4 °C. Eight fractions were
collected from the top, resolved by 4-10% SDS-PAGE, and immunoblotted
with different antibodies. a, mc GRIP antibodies;
b, pc AN2 antibodies (GST880S16); c, pc NCAM
antibodies. GRIP, NG2, and NCAM140/180 are located in the bottom
gradient fractions (6-8); glycosylphosphatidylinositol-anchored
NCAM120 is in the raft fraction (2).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(37), and
kinesin (38). PDZ domains function as scaffolding units clustering
multiprotein complexes at the cell surface (10) by binding to the COOH
termini of transmembrane proteins. NG2, a large transmembrane
proteoglycan, has a short cytoplasmic COOH terminus that binds to the
seventh PDZ of both GRIP1 and its homolog GRIP2.
-neurexins and their binding partners neuroligins have been
demonstrated to trigger cell-cell adhesion (61). It is very likely that
NG2 is an adhesion molecule. A putative association between adhesion
molecules and neurotransmitter receptors has been observed previously:
the N-methyl-D-aspartate receptor and adhesion
molecules N-cadherin and L1 are physically associated in large
multiprotein complexes isolated by immunoprecipitation of
N-methyl-D-aspartate type of glutamate receptors
from mouse brain (62). However, a direct link between these molecules
has not been demonstrated. Similarly, members of the ephrin family of
cell adhesion molecules (ligands and receptors) bind GRIP (19, 35),
thus potentially complexing AMPA receptors and ephrins.
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Fig. 8.
Model of interactions between NG2-positive
glial cells expressing AMPA receptors and neurons. Based on the
finding of Bergles and colleagues (6), where neuronal boutons make
synapses onto NG2 positive glial cells, the NG2·GRIP·AMPA receptor
complex could be involved in the alignment and formation of a
glial-neuronal synapse (a). b shows a modified
model after Benson and colleagues (55), which focuses on the importance
of adhesion molecules in shaping a synapse. This event may involve the
NG2·GRIP·AMPA receptor complex expressed on immature glial cells
surrounding the synapse. NG2-positive oligodendrocyte progenitor cells
also make contact to axons as shown in c, and NG2-positive
glial cells are present at the nodal region in the CNS (d)
(51).
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ACKNOWLEDGEMENTS |
---|
We gratefully acknowledge the critical comments of Dr. Eva-Maria Krämer and Dr. William Wisden and the excellent technical assistance of Iris Ehret-Bünzli and Carolin Stünkel.
![]() |
FOOTNOTES |
---|
* This work was supported by Deutsche Forschungsgemeinschaft Grants SFB 317 and 488 and by the Graduiertenkolleg Molecular and Cellular Neuroscience.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.
¶ Present address: Dept. of Cell Biology, Yale University School of Medicine, 295 Congress Ave., New Haven, CT 06510.
** To whom correspondence should be addressed: Dept. of Neurobiology, University of Heidelberg, Im Neuenheimer Feld 364, Heidelberg 69120, Germany. Tel.: 49-6221-548-319; Fax: 49-6221-546-700; E-mail: jtrotter@sun0.urz.uni-heidelberg.de.
Published, JBC Papers in Press, November 27, 2002, DOI 10.1074/jbc.M210010200
2 The AMPA receptor subunits GluRA, B, C, and D are sometimes referred to as GluR1, 2, 3, and 4, respectively.
3 H. Werner and K. Nave, unpublished data.
4 J. Stegmüller, H. Werner, K.-A. Nave, and J. Trotter, unpublished results.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
AMPA, -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid;
GRIP, glutamate
receptor interaction protein;
GST, glutathione
S-transferase;
mc, monoclonal;
MOG, myelin oligodendrocyte
glycoprotein;
NCAM, neural cell adhesion molecule;
PBS, phosphate-buffered saline;
pc, polyclonal;
X-gal, 5-bromo-4-chloro-3-indolyl-
-D-galactopyranoside;
PDZ, postsynaptic density 95/discs large/zona occludens 1;
LNS, laminin/neurexin/sex hormone binding globulin.
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