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Volume 17, Number 1,
Issue of January 1, 1997
pp. 152-159
Copyright ©1997 Society for Neuroscience
Essential Role for dlg in Synaptic Clustering of
Shaker K+ Channels In Vivo
Francisco J. Tejedor2,
Amr Bokhari1,
Oscar Rogero2,
Michael Gorczyca1,
Jiangwen Zhang1,
Eunjoon Kim3,
Morgan Sheng3, and
Vivian Budnik1
1 Department of Biology, University of Massachusetts,
Amherst, Massachusetts 01003, 2 Unidad Asociada-Consejo
Superior de Investigaciones Científicas, Instituto de
Neurociencias, Universidad de Alicante, San Juan 03080 Alicante, Spain,
and 3 Howard Hughes Medical Institute, Massachusetts
General Hospital, Department of Neurobiology, Harvard Medical School,
Boston, Massachusetts 02114
ABSTRACT
- INTRODUCTION
- MATERIALS AND METHODS
- RESULTS
- DISCUSSION
- FOOTNOTES
- REFERENCES
ABSTRACT 
The assemblage of specific ion channels and receptors at synaptic
sites is crucial for signaling between pre- and postsynaptic cells.
However, the mechanisms by which proteins are targeted to and clustered
at synapses are poorly understood. Here we show that the product of the
Drosophila discs-large gene, DLG, is colocalized with
Shaker K+ channels, which are clustered at glutamatergic
synapses at the larval neuromuscular junction. In heterologous cells,
DLG can cluster Shaker-type K+ channels, and, in the yeast
two-hybrid system, the DLG PDZ1-2 domains bind directly to the
C-terminal tail of Shaker proteins. We also demonstrate that DLG-Shaker
interactions are required in vivo for Shaker clustering
at the neuromuscular junction. Synaptic clustering of Shaker channels
is abolished not only by mutations in dlg but also by a
mutation in Shaker that deletes its C-terminal DLG
binding motif. Analyses of various dlg mutant alleles
suggest that channel clustering and synaptic targeting functions depend on distinct DLG domains. These studies demonstrate for the first time
that DLG plays an important role in synaptic organization in
vivo that correlates with its ability to bind directly to
specific membrane proteins of the synapse.
Key words:
discs-large;
Drosophila;
glutamatergic synapse;
IA;
ion channel
clustering;
MAGUK;
neuromuscular junction;
PDZ;
potassium channels;
PSD-95/SAP90;
Shaker;
synapse;
synapse targeting
INTRODUCTION
Signal transmission between neurons and their
targets depends on the appropriate organization of proteins involved in
neurotransmitter release and postsynaptic signal transduction (for
review, see Froehner, 1993
; Hall and Sanes, 1993). For example,
localization of presynaptic Ca2+ channels in close
proximity to active zones allows rapid vesicle exocytosis after
Ca2+ influx. At the postsynaptic membrane, clustering of
ionotropic neurotransmitter receptors in regions apposed to the
presynaptic terminal allows the generation of postsynaptic
depolarizations sufficient to trigger action potentials (Froehner,
1993). The mechanisms by which proteins become so precisely organized
in synapses, however, are mainly unknown. At the vertebrate
neuromuscular junction (NMJ), clustering of postsynaptic acetylcholine
receptors (AChR) seems to be mediated by direct interactions with the
protein rapsyn (Apel and Merlie, 1995). In the vertebrate CNS a
clustering protein, gephyrin, which is required for aggregation of
glycine receptors at inhibitory synapses, also has been identified
(Khuse et al., 1995).
The diversity of synaptic proteins and the heterogeneity of neuronal
synapses, however, predict that components in addition to rapsyn and
gephyrin are required to assemble a functional synapse (Hall and Sanes,
1993). Recent studies suggest that a family of mammalian
membrane-associated guanylate kinases (MAGUKs), including PSD95/SAP90,
also may subserve such a role (Cho et al., 1992; Kistner et al., 1993)
(for review, see Budnik, 1996; Garner and Kindler, 1996; Gomperts,
1996). PSD95/SAP90 is localized at synapses, and in vitro it
interacts directly with NMDA receptors and Shaker-type K+
channels (Kim et al., 1995, 1996; Kornau et al., 1995; Kim and Sheng,
1996; Niethammer et al., 1996).
MAGUKs are multidomain proteins that contain three PDZ domains, a
src homology 3 (SH3) domain, and a guanylate kinase-like domain (Woods and Bryant, 1991; Doyle et al., 1996) (for review, see
Woods and Bryant, 1993; Budnik, 1996). Direct binding occurs between
specific PDZ domains of PSD95/SAP90 and the amino acid motif ET/SXV (in
which E denotes glutamate, T/S threonine or serine, X any amino acid,
and V, valine) at the C terminus of NMDA receptors (Kornau et al.,
1995; Niethammer et al., 1996) and mammalian Shaker-type channel
subunits (Kim et al., 1995). Coexpression of Shaker-type K+
channels or NMDA receptors with PSD-95 in cultured heterologous cells
results in the formation of K+ channel or NMDA receptor
clusters (Kim et al., 1995, 1996; Kim and Sheng, 1996). These results
have led to the speculation that PSD95 mediates the aggregation of
specific membrane proteins at synaptic sites. However, in
vivo studies are, so far, lacking that would prove this hypothesis
and demonstrate the specificity of such a role.
discs-large (dlg) is a Drosophila homolog of the
PSD-95 MAGUK. It also contains three PDZ domains and is localized in
the fly CNS and in glutamatergic synapses at the larval NMJ (Woods and
Bryant, 1991; Lahey et al., 1994; Budnik et al., 1996). Moreover, Drosophila Shaker channel subunits also contain a C-terminal
ETDV sequence like their mammalian Shaker counterparts (Pongs et al., 1988; Schwarz et al., 1988). Because a variety of mutations in both
genes exist, the Drosophila system offered an opportunity to
determine whether DLG actually is involved in the synaptic localization
of Shaker K+ channels in the intact organism.
MATERIALS AND METHODS
Fly stocks. Flies were raised at 22-25°C in
standard cornmeal/molasses media. The following stocks were used for
these studies: y f dlgX1-2/Basc, y w
dlgm52/Bnsn, y f dlgv59/FM7/Y y+
dlg+, y f dlg1P20/FM7, y f
dlgsw/FM7, and Df(1)N71/FM7; Dp (1,2)
65v/+ is a deficiency that uncovers the dlg locus
(referred to as Df in the text). These dlg mutant stocks are
described in Perrimon (1989), Woods and Bryant (1991), and Woods et al.
(1996). All anatomical experiments were done in dlg mutants
over Df. Males from the stock
B55D/W32P/C(1)M3 are deficient in
the Sh genomic region and were used as controls for the
specificity of the anti-Shaker antibody (Ferrús et al., 1990;
Rogero and Tejedor, 1995). Sh102 is a mutant
that produces a truncated form of Shaker protein (Gisselman et al.,
1989). BG487 is a Gal-4 P-element insertion strain isolated
in a previous enhancer trap screen, and UAS-dlg transformants are described elsewhere (Budnik et al., 1996). As wild-type control, the strain Canton-S was used. Genetic
markers and balancer chromosomes are described in Lindsley and Zimm
(1992).
Immunocytochemistry and Western blot analysis. For
anti-Shaker immunocytochemistry, body wall muscles were dissected on
ice and fixed at 4°C for 15 min in fresh Bouin's and for 15 min in Bouin's containing 0.2% Triton X-100. After being washed [0.1 M phosphate buffer, pH 7.2, and 0.1% Triton X-100 (PBT)],
samples were incubated overnight at 4°C and then for 1 hr at room
temperature with 1:20 affinity-purified rabbit anti-Sh antiserum
(Rogero and Tejedor, 1995) diluted in PBT containing 0.2% Triton X-100
and 5 mg/ml BSA (PBTS). Samples were washed and then incubated with 1:200 FITC-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA), washed, and incubated overnight at 4°C with a mouse
anti-FITC monoclonal antibody (Sigma, St. Louis, MO) at 1:500 dilution.
After washes, samples finally were incubated in 1:200 FITC-conjugated
donkey anti-mouse IgG (Jackson ImmunoResearch), washed again, mounted
in Vectashield mounting medium (Vector Laboratories, Burlingame, CA),
and visualized under epifluorescence or confocal microscopy as in Lahey
et al. (1994). For double-labeling experiments, anti-DLG antibody
(Woods and Bryant, 1991; Lahey et al., 1994) was applied simultaneously
with anti-FITC monoclonal at 1:250 dilution, followed by simultaneous
incubation with Texas Red-conjugated donkey anti-rabbit and
FITC-conjugated donkey anti-mouse. No specific staining was observed in
controls in which one or both primary antibodies were omitted.
The procedures for the isolation of membrane and cytosolic fractions of
Drosophila CNS and Western blot analysis of these proteins
using anti-Shaker antiserum were described previously (Rogero and
Tejedor, 1995). Isolation and Western blot analysis of body-wall muscle
proteins with anti-DLG are described in Lahey et al. (1994), using
anti-DLG antibody at a 1:10,000 dilution and HRP-conjugated anti-rabbit
IgG at 1:2000 dilution.
Yeast two-hybrid assay. The coding sequence for the last 10 amino acids of Shaker B1 (Schwarz et al., 1988) was synthesized as
complementary oligonucleotides and fused to the LexA DNA binding domain
[13 of the last 14 amino acids are identical in all
Drosophila Shaker splice variants, including the C-terminal
four amino acids-ETDV (Schwarz et al., 1988)]. The LexA-Kv1.4
C-terminal tail constructs (wild-type-ETDV, and mutant-ETDE) have
been described previously (Kim et al., 1995). PDZ domains of DLG
[PDZ1-2 (amino acids 20-265); or PDZ3 (amino acids 483-570)] were
fused to the GAL-4 activation domain of the vector pGAD10. Analogous
constructs of PDZ domains from PSD95 have been described (Kim et al.,
1995). Various combinations of these were transformed into the L40
yeast strain harboring the reporter genes HIS3 and 
-galactosidase
(-gal) (Bartel et al., 1993; Kim et al., 1995). HIS3 activity was
determined by the percentage of colonies growing on histidine-lacking
medium, and -gal activity was determined by the time required for
colonies to turn blue in X-gal filter lift assay at room
temperature.
Transfection experiments. Kv1.4, Shaker B1, and
dlg cDNAs were subcloned into the mammalian expression
vector GW1-CMV (British Biotechnology, Oxford, UK) and transfected into
COS7 cells by the lipofectamine method (Life Technologies,
Gaithersburg, MD). Cells were fixed with 2% formaldehyde 2 d
after transfection and visualized by immunocytochemistry using
anti-Kv1.4 (Sheng et al., 1992) or anti-DLG antibodies (Lahey et al.,
1991; Woods and Bryant, 1991).
RESULTS
Shaker and DLG are colocalized at Type I synaptic boutons
Colocalization of Shaker and DLG proteins is a prerequisite for
their direct interaction in vivo. To determine whether
there was spatial overlap between DLG and Shaker, we examined the
distribution of these proteins in Drosophila larval body
wall muscles using anti-DLG (Woods and Bryant, 1991; Lahey et al.,
1994) and anti-Drosophila Shaker antibodies (Rogero and
Tejedor, 1995). The Shaker antibodies equally recognize all Shaker
channel splice isoforms (Rogero and Tejedor, 1995).
Electrophysiological and genetic studies have demonstrated previously
that a Shaker-mediated K+ current
(IA) is expressed in the larval body wall
muscles (Wu and Haugland, 1985; Haugland and Wu, 1990).
We found that Shaker immunoreactivity was concentrated around Type I
synaptic boutons at the larval NMJ (Fig.
1A). This immunoreactive pattern is
highly reminiscent of anti-DLG immunoreactivity (Lahey et al., 1994).
Four types of potassium currents, including a delayed rectifier
(IK), two Ca2+-dependent potassium
currents with different kinetic properties, and
IA, which are mediated by channels coded in
different genes, are found in the body wall muscles (Singh and Wu,
1989). Therefore, it was important to determine the
specificity of the anti-Shaker staining, although the antibody used was
generated against a region of the Sh sequence with low
homology to the other K+ channel genes (Rogero and Tejedor,
1995). That the immunoreactivity observed at Type I boutons represented
specific Shaker channel distribution was demonstrated by using
Shaker-deficient larvae (Ferrús et al., 1990), which were devoid
of anti-Shaker immunoreactivity (Fig. 1B).
Fig. 1.
Distribution of Shaker channels in wild-type
Drosophila larval neuromuscular junctions and
colocalization with DLG. A, Anti-Shaker immunoreactivity
at Type I boutons in a wild-type third instar larva. Muscle number
designations are indicated. Arrows indicate Type I
boutons. B, Absence of immunoreactive signal in a
deficiency of Shaker larva, demonstrating the specificity of the
staining. C-E, Synaptic colocalization of DLG and
Shaker visualized in a high magnification view of Type I synaptic
boutons double-labeled with anti-Shaker (C, green
channel) and anti-DLG (D, red
channel). E, Merged red and green
channels. At this magnification, intense punctuate Shaker
immunoreactivity is revealed within the area of the synaptic bouton
that is stained with anti-Shaker antibodies. Scale bars: A,
B, 80 µm; C-E, 4 µm.
[View Larger Version of this Image (87K GIF file)]
At least three types of synapses, with different morphologies and
containing different neurotransmitters, have been described in
Drosophila NMJs (Johansen et al., 1989; Gorczyca et al.,
1993; Monastirioti et al., 1995). Of these, only the glutamatergic Type I synapses have been shown to contain DLG protein (Lahey et al., 1994).
This DLG expression at Type I synapses is regulated developmentally, being most prominent at presynaptic regions in the late embryo and at
postsynaptic sites during larval stages (Guan et al., 1996). Significantly, Shaker immunoreactivity was found to be associated only
with Type I synapses, and the Shaker staining pattern colocalized with
that of DLG on those synapses (Fig. 1C-E). Thus, in
wild-type flies, Shaker channels are concentrated at the same synaptic
regions in which DLG is localized, consistent with a potential
interaction of these two proteins in vivo. In contrast to
anti-DLG immunoreactivity, which appears homogeneously distributed
around Type I boutons, Shaker immunoreactivity exhibited more intense
"hot spots" within the synaptic staining (Fig.
1D).
Direct in vitro interaction between Shaker and DLG
To examine whether direct protein-protein interactions occur
between DLG and fly Shaker as previously demonstrated for PSD95 and the
mammalian Shaker channel subunit Kv1.4 (Kim et al., 1995), we used the
semiquantitative yeast two-hybrid interaction assay (Fields and Song,
1989; Bartel et al., 1993), based on the level of induction of the
reporter genes HIS3 and -gal. PDZ1-2 or PDZ3 domains from DLG and
PSD95 were tested for binding to the last 10 amino acids of the
C-terminal tail of Drosophila Shaker B1 and Kv1.4.
The C-terminal Shaker tail was found to bind strongly to the first two
PDZ domains of DLG (PDZ1-2; Table 1). In contrast, no
binding was observed between the Shaker C-terminal tail and the PDZ3
domain of DLG (Table 1). This is identical to the binding specificity
of Kv1.4 for the PDZ domains of PSD95. In fact, the C-terminal tail of
Drosophila Shaker also bound to PDZ1-2 from PSD95, and
similarly, mammalian Kv1.4 bound readily to DLG as well as PSD95 (Table
1). A mutant Kv1.4 carrying the C-terminal sequence-ETDE was unable to
interact with either PSD95 or DLG in the two-hybrid assay (Table 1),
confirming that the binding is dependent on the C-terminal PDZ binding
motif. These results demonstrate that, as in mammals,
Drosophila DLG and Shaker establish direct protein
interactions via the binding of PDZ1-2 domains and the C terminus of
the K+ channel.
To examine the cell biological correlates of such an interaction, we
cotransfected DLG with Shaker channel subunits into cultured heterologous cells (Fig. 2). When Kv1.4 or Shaker B1
cDNAs were singly transfected into COS7 cells, the K+
channel immunoreactivity was distributed diffusely throughout the cell
(Fig. 2A,C). DLG expressed alone also exhibited a
diffuse intracellular distribution pattern (Fig. 2B).
However, when Kv1.4 was cotransfected with dlg, both the
mammalian Shaker protein and DLG showed a redistribution into
plaque-like clusters on or near the cell surface (Fig.
2D,E). The appearance of these Kv1.4 clusters was
essentially identical to those resulting from Kv1.4 coexpression with
PSD95 (Kim et al., 1995). Thus Drosophila DLG can function
like its mammalian homolog to cluster Shaker K+ channels in
heterologous cells. However, in these COS7 clustering assays, DLG was
consistently less efficient than PSD-95 in clustering Kv1.4 or
Drosophila Shaker K+ channels (5-10%, compared
with 40-70%, respectively, as measured by the percentage of
transfected cells with clear plaque-like clusters of K+
channels).
Fig. 2.
Clustering of Shaker-type K+ channels
by DLG in heterologous cells. A, COS7 cell singly
transfected with Kv1.4 and stained with anti-Kv1.4 antibodies.
B, COS7 cell singly transfected with DLG and stained
with anti-DLG antibodies. C, COS7 cell singly transfected with Shaker and stained with anti-Shaker
antibodies. D, E, COS7 cells cotransfected with Kv1.4
and DLG and stained with anti-Kv1.4 (D) or with anti-DLG
(E) antibodies. F, COS7 cells cotransfected with Shaker and dlg and
stained with anti-Shaker antibodies. When expressed alone, Kv1.4, DLG,
and Shaker are distributed diffusely in the cell with some perinuclear
accumulation. On coexpression of DLG and Kv1.4, or DLG and Shaker, both
proteins are redistributed into plaque-like clusters
(D-F) that are essentially identical to those
seen with PSD-95 and Kv1.4 (Kim et al., 1995). Scale bar, 10 µm.
[View Larger Version of this Image (104K GIF file)]
Much weaker clustering effect also was observed with cotransfection of
dlg and Drosophila Shaker (Fig.
2F). Because clustering efficiency depends critically
on the absolute and relative expression levels of PSD95 and Kv1.4 (E. Kim, unpublished observations), it is possible that poor expression of
Drosophila DLG and Shaker in monkey COS7 cells could account
for the quantitative difference. Alternatively, some facilitating
factor required by Drosophila DLG/Shaker may be absent in
COS7 cells.
DLG is required in vivo for both Shaker K+
channel clustering and synaptic targeting
Taken together with the in vivo colocalization data
(Fig. 1), the two-hybrid (Table 1) and heterologous transfection
experiments (Fig. 2) suggest that a direct interaction between DLG and
the C-terminal-ETDV sequence in Shaker may mediate the clustering of
the K+ channel in synaptic regions. One prediction of this
model is that specific deletion of the Shaker C-terminal sequence would disrupt synaptic clustering of Shaker channels at synapses in vivo. To test this hypothesis, we used a Sh mutant
allele, Sh102, which lacks the C-terminal-ETDV
motif (Gisselman et al., 1989). In Sh102 mutant
flies, which contain a normal copy of dlg, Shaker channels failed to cluster and Type I boutons were devoid of Shaker
immunoreactivity, in agreement with our model (Fig.
3A). In contrast, the distribution of
DLG immunoreactivity at these boutons was normal (Fig. 3B). The Shaker immunostaining in Sh102 mutants
appeared diffusely distributed at the muscle membrane, barely above
background levels. This apparent decrease of the Shaker immunostaining
is most likely attributable to the dilution of Shaker K+
channels in the whole muscle cell membrane. An alteration in the
expression of the truncated Sh102 protein attributable to
anomalous insertion in the cellular membrane is very unlikely, because
adequate expression of the mutant Shaker protein is observed in Western
blot analyses of a membrane fraction (Fig. 3C).
Fig. 3.
Lack of Shaker clustering in
Sh102 mutants. A, Anti-Shaker
immunoreactivity in a third instar Sh102 larva,
showing the absence of immunoreactivity at synaptic regions. B, Anti-DLG immunoreactivity in a different
Sh102 preparation, showing normal DLG
distribution at type I synapses. Scale bar, A, B, 20 µm. C, Western blot analysis of CS and
Sh102 cytosol (c) and membrane
(m) fractions. Molecular weights (kDa) are indicated to
the right of the blot. Multiple bands in the immunoblots
are attributable to different Shaker isoforms produced by alternative
splicing, which are detected by the antiserum (Rogero and Tejedor,
1995). Note the absence of proteolysis products and the very low levels
of Sh102 protein in the cytosolic fraction,
suggesting normal insertion of the truncated Sh protein
in the plasma membrane.
[View Larger Version of this Image (86K GIF file)]
Additional genetic evidence that DLG is required for Shaker channel
clustering at synapses was obtained by examining dlg mutants (Fig. 4A) (Woods and Bryant, 1991;
Woods et al., 1996). dlgX1-2 is a severe
hypomorphic allele, and <5% of DLG protein, as determined by Western
Blot analysis, is observed during larval stages (Fig. 4B). Recent molecular analysis of this allele
suggests that, in addition to the reduced protein levels, a stop codon
is introduced after the end of exon 8, predicting a truncated protein
that lacks the GUK domain (Woods et al., 1996). However, in our studies
we detected no size changes in DLGX1-2 protein
(Fig. 4B). In dlgX1-2
mutant larvae, Shaker immunoreactivity was absent at Type I boutons, and very low levels were observed throughout the muscle as with the
Sh102 mutant (Fig. 3A). This
did not seem to be the result of a lack of Shaker protein expression in
dlgX1-2, but rather to a mislocalization
or lack of clustering of the protein, because Shaker immunoreactivity
was still quite intense in the larval brain (data not shown).
Fig. 4.
Genetic analysis of Shaker channel clustering and
localization by DLG. A, Schematic diagram of the domain
organization of the DLG protein. Bars in the bottom of
A indicate defects in several dlg mutant
alleles (Woods and Bryant, 1991; Woods et al., 1996). Black, Intact protein region; gray,
deleted protein region; asterisk, amino acid
substitution. The vertical black bar between SH3 and GUK
represents localization of the putative band 4.1 binding site. B, Western blot of body-wall muscle proteins stained
with anti-DLG antibodies. Two bands of 97 and 108 kDa are observed in
both wild-type and mutants, but the levels in
dlgX1-2 are decreased to <5% of
wild-type levels. Each lane was loaded with 97 µg of
body-wall muscle protein. C, Anti-Shaker
immunoreactivity in dlgX1-2 body wall
muscles showing lack of clustering at synaptic boutons. D, dlgv59 mutant body wall
muscles labeled with anti-Shaker antibodies showing normal clustering
of Shaker channels at Type I boutons. Scale bar, C, D,
40 µm.
[View Larger Version of this Image (47K GIF file)]
Several domain-specific mutations in the dlg gene have been
isolated, providing the opportunity to test whether Shaker channel localization is altered in these mutants and which regions of DLG may
be required for this function. In dlgv59,
dlg1p20, and
dlgsw, different extents of the GUK
domain region are deleted, but SH3 and PDZ domains are normal (Woods
and Bryant, 1991; Fig. 4A). In
dlgm30, a point mutation results in the
substitution of a highly conserved leucine to proline in the SH3
domain, without altering PDZ and GUK domains. Although it is not clear
whether this point mutation disrupts the SH3 domain,
dlgm30 mutant animals develop large
neoplastic tumors and die at the beginning of metamorphosis. This
observation suggests that dlg function is abnormal in this
mutant allele. In dlgm52 a splicing
defect reveals a stop codon in intron 5, resulting in a truncated
protein containing PDZ1, PDZ2, and the beginning of PDZ3 (Woods at al.,
1996; Fig. 4A). Normal clustering of Shaker channels
at the NMJ was observed in dlgv59 (Fig.
4D), dlgsw,
dlg1p20, and
dlgm30, although anti-Shaker
immunoreactivity was consistently lower in
dlgv59.
In contrast, the clustering of Shaker protein around synaptic boutons
was disrupted in dlgm52 (Fig.
5). In these mutants Shaker immunoreactivity was absent from Type I synapses. However, bright Shaker immunoreactive clusters were observed at nonsynaptic locations, often near the one or two
muscle nuclei most proximal to the NMJ, but away from synaptic boutons
(Fig. 5A,B). These results indicate that in
dlg mutants the formation of Shaker clusters, which depend
on PDZ1-2, is still present but that the targeting of channel clusters
to the appropriate synaptic regions is disrupted. This model is
summarized in Figure 6.
Fig. 5.
Targeting of Shaker clusters to Type I synapses is
altered in dlg mutants containing only PDZ1-2 but can
be rescued by postsynaptic dlg targeting.
A, Anti-Shaker immunoreactivity in
dlgm52. In these mutants Shaker clusters
(arrow) are formed at ectopic muscle regions.
B, High-magnification view of ectopic clusters in muscles 12 and 13 of a dlgm52
sample. Arrows indicate the localization of synaptic
boutons determined by double labeling with anti-HRP antibodies (data
not shown). C, Anti-Shaker immunoreactivity in a
dlgm52 strain carrying the
P[Gal-4] element BG487, which expresses Gal-4 in a
subset of muscles, and UAS-dlg. Note that in this strain clustering of Shaker channels around Type I synapses is normal and that
only small ectopic clusters are observed (arrow). Scale bars: A, 90 µm; B, 25 µm;
C, 20 µm.
[View Larger Version of this Image (81K GIF file)]
Fig. 6.
Model of Shaker clustering and Shaker synapse
targeting by DLG. A, In wild-type, Shaker channels are
clustered at the muscle junctional region by the interaction of their
carboxyl-ETDV motif and PDZ1-2 of DLG. B, In the
absence of DLG, Shaker channels fail to aggregate at the junction and
remain diffusely distributed along the muscle membrane.
C, When only PDZ1-2 domains are intact in DLG, Shaker
channels are clustered, but at extrajunctional regions of the muscle
membrane. D, Lack of ET/SXV motif in Shaker channels
prevents their clustering and their localization at the junction even
when DLG is normally localized. E, Abnormal SH3 or GUK
domains do not prevent Shaker clustering at the junction.
[View Larger Version of this Image (61K GIF file)]
To determine whether the abnormal distribution of Shaker in
dlgm52 mutants could be restored by the
presence of normal DLG protein, we used the Gal-4 enhancer
trap system to drive DLG expression in the muscle cells (Brand and
Perrimon, 1993). For this experiment we used a Gal-4 P-element
insertion strain, BG487, which shows strong Gal-4 expression in muscles
6 and 7 throughout the larval period, as determined in
BG487/UAS-LacZ larvae. Besides a few sensory cell bodies
and salivary glands, no other tissue expresses detectable levels of
Gal-4 during embryonic and larval stages (Budnik et al., 1996). To
drive DLG expression in muscles 6 and 7, we crossed
dlgm52 mutants containing the BG487
insertion to the deficiency stain transformant containing a
UAS-dlg element (Budnik et al., 1996). We found
that in the progeny
(dlgm52/Df;BG487/UAS-dlg)
Shaker immunoreactivity was normally distributed at Type I boutons and
that only small ectopic clusters could be observed (Fig.
5C). This result shows that targeting DLG to muscle cells
rescues the abnormal Shaker clustering phenotype of
dlgm52 mutants.
DISCUSSION
In this paper we show that in Drosophila DLG interacts
directly with Shaker K+ channels and that this interaction
is essential for clustering and targeting these channels to proper
junctional domains in the intact animal. The clustering function had
been suggested previously by in vitro studies with mammalian
dlg homologs (Kim et al., 1995; Kim and Sheng, 1996).
However, this is the first study to demonstrate that this clustering
activity is significant for the organization of a synaptic component
in vivo. In addition, our genetic dissection of the process
of Shaker channel organization at the synapse provides evidence for an
additional role of DLG
the targeting of channels to synaptic
regions.
The combined genetic and molecular data provide compelling evidence
that the clustering of Shaker channels depends on the integrity of PDZ1-2 in DLG and the C-terminal-ETDV motif in Shaker; these are the domains that mediate the direct interaction between the
two proteins. However, in dlg mutants in which only PDZ1-2 domains remain intact (dlgm52), Shaker
clusters apparently still can form, but these clusters are localized at
ectopic sites, away from their normal synaptic location (Fig. 6). The
ectopic clustering could be rescued by targeting DLG expression to the
muscle cells during the postembryonic period, demonstrating that this
phenotype is dependent on the presence of intact DLG protein.
Thus, the dlgm52 phenotype suggests that
a region of the DLG protein C-terminal to PDZ1-2 plays a role in
targeting Shaker clusters to synapses. This synaptic targeting is
unlikely to involve the SH3 and GUK domains, as evidenced by the
results with dlgv59,
dlg1p20,
dlgsw, and
dlgm30 alleles (Fig. 6). A well conserved
cytoskeletal binding motif that binds to protein 4.1 has been
identified in several dlg homologs (Lue et al., 1994). Band
4.1 has been associated with the function of linking membrane proteins
to the underlying actin/spectrin cytoskeleton (Marchesi, 1985). The 4.1 binding motif is also present in Drosophila DLG (Lue et al.,
1994) and is a potential candidate for the anchoring of Shaker clusters
to synapses. A gene encoding for a band 4.1 homolog is encoded by the
coracle gene. Like DLG, COR is localized at septate
junctions in epithelial tissues (Fehon et al., 1994). However, COR is
not colocalized with DLG at the larval NMJ (V. Budnik, unpublished
observations).
The genetic analysis of Shaker channel clustering also provides
intriguing evidence for segregation of other in vivo
functions among different domains of the DLG protein. Although our
results indicate that the SH3 and GUK domains are not necessary for DLG to cluster Shaker channels at synapses, the GUK domain is essential for
normal development of Type I bouton postsynaptic morphology (Lahey et
al., 1994; Budnik et al., 1996; Guan et al., 1996). Moreover, both SH3
and GUK domains are required for the tumor suppressor activity of DLG
in the CNS and imaginal disk epithelial cells (Woods and Bryant, 1991;
Woods et al., 1996). Although potential signaling roles of SH3 and GUK
domains have been speculated on, the functions of these domains remain
to be determined, both in epithelial cell and synaptic junctions (Woods
and Bryant, 1993).
The protein rapsyn associates with nicotinic AChR and is involved in
the mechanism of postsynaptic AChR clustering at the mammalian
neuromuscular junction (Apel and Merlie, 1995). Because PSD95 family
proteins bind and cluster the NMDA subclass of ionotropic glutamate
receptors in addition to Shaker K+ channels (Kim et al.,
1995, 1996; Kornau et al., 1995; Kim and Sheng, 1996; Niethammer et
al., 1996), they have been suggested to play a role similar to rapsyn
at glutamatergic synapses in the mammalian CNS (Kim et al., 1995, 1996;
Budnik, 1996; Gomperts, 1996). In this regard, it is pertinent that DLG
seems to have an important channel clustering function at the fly
neuromuscular junction, which also uses glutamate as its main
excitatory neurotransmitter. Interestingly, the two-hybrid and
heterologous cell transfection experiments show that PDZ1-2 domains of
both PSD95 and DLG can interact with either the mammalian or the fly
Shaker channel. This observation shows that PDZ1-2 domains have
retained their ligand-binding specificities despite some divergence in
sequence (65% identity between PDZ1-2 of rat PSD95 and
Drosophila DLG, as compared with 82-87% identity in this
region among different members of the mammalian PSD95 family).
Our in vivo observations indicate that DLG and DLG-like
proteins provide one mechanism by which the precise localization of ion
channels and other proteins at synaptic regions is regulated. For
example, studies of synapse structure in dlg mutants show that postsynaptic specializations at Type I boutons are underdeveloped (Lahey et al., 1994; Guan et al., 1996). This phenotype may be related
to the clustering of synaptic components, such as cytoskeletal and
membrane elements, required to modify synapse structure during development. Alternatively, changes in the clustering of ion channels may modify the normal synaptic activity patterns required for a correct
development of NMJs (Budnik et al., 1990; Broadie and Bate, 1993;
Jarecki and Keshishian, 1996). However, different domains of the DLG
protein seem to be required for Shaker channel clustering and the
development of postsynaptic structure. For example, postsynaptic
structure is altered in both dlgv59 and
dlgm52 (Lahey et al., 1994; Guan et al.,
1996), whereas Shaker channel clustering appears normal in
dlgv59.
The diversity of channels and receptors in the central and peripheral
nervous systems predicts that a large number of molecules are necessary
for the proper molecular architecture of synapses. The study of
DLG-like proteins, rapsyn, and gephyrin, is forming the framework by
which we are beginning to understand the mechanisms of synapse
assembly.
FOOTNOTES
Received Sept. 4, 1996; revised Oct. 18, 1996; accepted Oct. 22, 1996.
This work was supported by National Institutes of Health Grants RO1
NS30072 and KO4 NS01786 to V.B. and DGICYT Grant PB93-0148 and
Generalitat Valenciana GV-3117/95 to F.J.T. M.S. is Assistant Investigator of the Howard Hughes Medical Institute, and V.B. is an
Alfred P. Sloan Fellow.
Correspondence should be addressed to Dr. Vivian Budnik, Department of
Biology, Morrill Science Center, University of Massachusetts, Amherst,
MA 01003.
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M Sone, E Suzuki, M Hoshino, D Hou, H Kuromi, M Fukata, S Kuroda, K Kaibuchi, Y Nabeshima, and C Hama
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R. B. Nehring, E. Wischmeyer, F. Doring, R. W. Veh, M. Sheng, and A. Karschin
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P. J. Mohler, S. M. Kreda, R. C. Boucher, M. Sudol, M. J. Stutts, and S. L. Milgram
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L. L. Mitic, E. E. Schneeberger, A. S. Fanning, and J. M. Anderson
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T. M. Boeckers, M. R. Kreutz, C. Winter, W. Zuschratter, K.-H. Smalla, L. Sanmarti-Vila, H. Wex, K. Langnaese, J. Bockmann, C. C. Garner, et al.
Proline-Rich Synapse-Associated Protein-1/Cortactin Binding Protein 1 (ProSAP1/CortBP1) Is a PDZ-Domain Protein Highly Enriched in the Postsynaptic Density
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M. Wyszynski, J. G. Valtschanoff, S. Naisbitt, A. W. Dunah, E. Kim, D. G. Standaert, R. Weinberg, and M. Sheng
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A. W. McGee and D. S. Bredt
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K. Jo, R. Derin, M. Li, and D. S. Bredt
Characterization of MALS/Velis-1, -2, and -3: a Family of Mammalian LIN-7 Homologs Enriched at Brain Synapses in Association with the Postsynaptic Density-95/NMDA Receptor Postsynaptic Complex
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K. W. Roche, C. D. Ly, R. S. Petralia, Y.-X. Wang, A. W. McGee, D. S. Bredt, and R. J. Wenthold
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N. Ullrich
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D. Lin, G. D. Gish, Z. Songyang, and T. Pawson
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T. Tezuka, H. Umemori, T. Akiyama, S. Nakanishi, and T. Yamamoto
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PNAS,
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Y.-P. Hsueh and M. Sheng
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W. Zhang, L. Vazquez, M. Apperson, and M. B. Kennedy
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D. Zhou, S. Lambert, P. L. Malen, S. Carpenter, L. M. Boland, and V. Bennett
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J. E. Brenman, J. R. Topinka, E. C. Cooper, A. W. McGee, J. Rosen, T. Milroy, H. J. Ralston, and D. S. Bredt
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A. Bordey and H. Sontheimer
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J. W. Lin, M. Wyszynski, R. Madhavan, R. Sealock, J. U. Kim, and M. Sheng
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A. Rao, E. Kim, M. Sheng, and A. M. Craig
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L. Y. Jan and Y. N. Jan
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C. D. Hough, D. F. Woods, S. Park, and P. J. Bryant
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M. Kim, D. J. Baro, C. C. Lanning, M. Doshi, J. Farnham, H. S. Moskowitz, J. H. Peck, B. M. Olivera, and R. M. Harris-Warrick
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S. N. MacFarlane and H. Sontheimer
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S. Ranta, A.-E. Lehesjoki, M. d. F. Bonaldo, J. A. Knowles, A. Hirvasniemi, B. Ross, P. J. de Jong, M. B. Soares, A. de la Chapelle, and T. C. Gilliam
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D. J. Baro, R. M. Levini, M. T. Kim, A. R. Willms, C. C. Lanning, H. E. Rodriguez, and R. M. Harris-Warrick
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S. Naisbitt, E. Kim, R. J. Weinberg, A. Rao, F.-C. Yang, A. M. Craig, and M. Sheng
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O. Rogero, B. Hammerle, and F. J. Tejedor
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E. Kim, S. Naisbitt, Y.-P. Hsueh, A. Rao, A. Rothschild, A. M. Craig, and M. Sheng
GKAP, a Novel Synaptic Protein That Interacts with the Guanylate Kinase-like Domain of the PSD-95/SAP90 Family of Channel Clustering Molecules
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C.S. Goodman, G.W. Davis, and K. Zito
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G. Jefford and R. R. Dubreuil
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M. E. Adams, H. A. Mueller, and S. C. Froehner
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(no significant growth)], and
