Department of Cell and Molecular Physiology and Curriculum in Neurobiology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7545
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Abstract |
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Membrane scaffolding complexes are key features of many cell types, serving as specialized links between the extracellular matrix and the actin cytoskeleton. An important scaffold in skeletal muscle is the dystrophin-associated protein complex. One of the proteins bound directly to dystrophin is syntrophin, a modular protein comprised entirely of interaction motifs, including PDZ (protein domain named for PSD-95, discs large, ZO-1) and pleckstrin homology (PH) domains. In skeletal muscle, the syntrophin PDZ domain recruits sodium channels and signaling molecules, such as neuronal nitric oxide synthase, to the dystrophin complex. In epithelia, we identified a variation of the dystrophin complex, in which syntrophin, and the dystrophin homologues, utrophin and dystrobrevin, are restricted to the basolateral membrane. We used exogenously expressed green fluorescent protein (GFP)-tagged fusion proteins to determine which domains of syntrophin are responsible for its polarized localization. GFP-tagged full-length syntrophin targeted to the basolateral membrane, but individual domains remained in the cytoplasm. In contrast, the second PH domain tandemly linked to a highly conserved, COOH-terminal region was sufficient for basolateral membrane targeting and association with utrophin. The results suggest an interaction between syntrophin and utrophin that leaves the PDZ domain of syntrophin available to recruit additional proteins to the epithelial basolateral membrane. The assembly of multiprotein signaling complexes at sites of membrane specialization may be a widespread function of dystrophin-related protein complexes.
Key words: syntrophin; utrophin; dystrobrevin; Madin-Darby canine kidney; green fluorescent protein ![]() |
Introduction |
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THE cytoskeleton plays an important role in establishing structural and functional specializations at
sites of cell-cell contact. For instance, at the postsynaptic density, cytoskeletal proteins participate in a
complex that localizes receptors and ion channels for efficient signal transduction (Cho et al., 1992; Kornau et al.,
1995
; Kim et al., 1996
; Dong et al., 1997
; Srivastava et al.,
1998
). In polarized epithelia, membrane specializations
form tight junctions (TJs)1 which separate the apical and
basolateral membranes, and function as barriers to diffusion across the epithelium (Gumbiner, 1987
). Proteins
such as ZO-1 are highly localized at the TJ (Stevenson et al.,
1986
) where they are thought to anchor transmembrane
proteins, such as occludin (Furuse et al., 1994
; Fanning
et al., 1998
). Similar multiprotein complexes have been described for the apical and basolateral membranes where
transmembrane proteins, such as the cystic fibrosis transmembrane conductance regulator, and the proteoglycan,
syndecan, are linked to the actin cytoskeleton (Cohen et
al., 1998
; Short et al., 1998
). Finally, in skeletal muscle, the
cytoskeletal protein, dystrophin, anchors transmembrane
and signaling proteins at the neuromuscular junction (Colledge and Froehner, 1998
). Although expressed in different cell types, these complexes share the common ability
to serve as a scaffold on which transmembrane and peripheral membrane proteins assemble.
A scaffolding complex common to neurons, epithelia,
and muscle is the dystrophin-associated protein complex
(DAPC; Lidov et al., 1990; Kim et al., 1992
; Ahn and
Kunkel, 1993
; Ervasti and Campbell, 1993
; Schmitz et al.,
1993
; Montanaro et al., 1995
; Durbeej et al., 1998
). Dystrophin is a 427-kD flexible rod-like protein consisting of an
NH2-terminal actin binding domain, 24 spectrin-like repeats, a cysteine-rich region, and a COOH-terminal domain (Koenig et al., 1988
; Koenig and Kunkel, 1990
; Ahn
and Kunkel, 1993
; Rybakaova et al., 1996
). The DAPC has
been studied most extensively in skeletal muscle, where
mutations in the dystrophin gene result in the severe muscle-wasting of Duchenne and Becker muscular dystrophies
(Straub and Campbell, 1997
). Dystrophin belongs to a
large family of proteins, including short forms of dystrophin and the dystrophin homologues, utrophin, dystrophin-related protein 2 (DRP2), and
- and
-dystrobrevins
(Tinsley et al., 1992
; Blake et al., 1996
, 1998
; Roberts et al.,
1996
; Sadoulet-Puccio et al., 1996
; Peters et al., 1997b
;
Puca et al., 1998
).
Two possible functions have been ascribed to dystrophin family members. First, dystrophin binds a transmembrane protein, -dystroglycan, which is linked to
extracellular laminin through
-dystroglycan (Ibraghimov-Beskrovnaya et al., 1992
; Gee et al., 1993
). Dystrophin also binds actin, thereby providing a link between the
extracellular matrix (ECM) and the cytoskeleton (Ahn and
Kunkel, 1993
; Ervasti and Campbell, 1993
; Rybakaova et
al., 1996
). This structural role may protect muscle from the
shearing forces of contraction (Petrof et al., 1993
; Pasternak
et al., 1995
). In addition, the dystrophin complex has
emerged as a potential signaling complex, due in part to a
family of modular adapter proteins, the syntrophins. The three isoforms of syntrophin (
1,
1, and
2) interact directly with dystrophin family members, are encoded by
separate genes, and consist primarily of protein-protein
interaction domains (Ahn et al., 1994
, 1996
; Adams et al.,
1995
). Syntrophins have two pleckstrin homology (PH)
domains, which are often found in cytoskeletal or signaling
proteins where they mediate protein-protein or protein- lipid interactions (Shaw, 1996
). Syntrophins also contain a
single PDZ domain, named for the three proteins in which
they were first recognized (PSD-95, discs large, ZO-1).
PDZ domains bind specific sequences on the COOH-terminal tails of ion channels and receptors, perhaps serving
to cluster these proteins at specialized sites (reviewed in
Sheng, 1996
; Kornau et al., 1997
). In addition, PDZ domains form homo- or heterodimers (Brenman et al., 1996
;
Srivastava et al., 1998
; Xu et al., 1998
). Using both mechanisms, syntrophin PDZ domains bind the extreme COOH-terminal tail of voltage-gated sodium channels (Gee et al.,
1998
; Schultz et al., 1998
), or the PDZ domain of neuronal nitric oxide synthase (nNOS; Brenman et al., 1996
),
thereby recruiting these signaling proteins to the dystrophin complex.
The DAPC is often concentrated at sites of membrane
specialization, such as central nervous system synapses
(Lidov et al., 1990; Kim et al., 1992
; Schmitz et al., 1993
;
Montanaro et al., 1995
) and the neuromuscular junction
(Ohlendieck et al., 1991b
; Bewick et al., 1992
; Peters et
al., 1997a
, 1998
), where the complex may serve both structural and signaling functions. Interestingly, the precise
composition of proteins in the DAPC varies by cell type or
subcellular localization. For instance, at the crests of the
neuromuscular postjunctional folds, rapsyn and utrophin are concentrated with nAChRs (Fertuck and Salpeter,
1974
; Sealock et al., 1984
; Ohlendieck et al., 1991b
; Bewick
et al., 1992
), while in the troughs of the folds dystrophin,
2-syntrophin, and voltage-gated sodium channels predominate (Flucher and Daniels, 1989
; Kramarcy, N., and
R. Sealock, personal communication). In addition, forms
of
-dystrobrevin generated by alternative splicing are distributed differentially at the neuromuscular junction (Balasubramian et al., 1998
; Peters et al., 1998
). Presumably,
the functional properties of the DAPC are determined by
its protein composition.
The expression of syntrophins, utrophin, and some dystrobrevin isoforms in tissues such as lung, kidney, testis,
and intestine (Khurana et al., 1990; Love et al., 1991
; Adams et al., 1993
; Blake et al., 1996
; Peters et al., 1997a
,b)
suggests that these proteins may also play a role in epithelial tissues. Expression of dystroglycans in epithelia in vivo
(Durbeej et al., 1998
) and the demonstration that antibodies that block laminin binding to dystroglycan disrupt epithelial cell differentiation (Durbeej et al., 1995
) indicate
that the dystrophin complex may play a critical functional
role in epithelial cells. We have explored the localization
and targeting of the DAPC using the MDCK epithelial cell line. Our data define a new variation of the DAPC
that may be involved in the generation or maintenance of
cell polarity and the formation of signaling complexes in epithelia.
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Materials and Methods |
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Antibodies
Antisyntrophin Antibodies.
mAb SYN1351 recognizes all syntrophin isoforms (Froehner et al., 1987). Syntrophin isoform-specific polyclonal antibodies SYN17 (
1-syntrophin), SYN28 (
2-syntrophin), and SYN37 (
1-syntrophin; all used at 30 nM IgG) have been described previously (Peters et al., 1997a
).
Antiutrophin Antibody.
mAb MANCHO-3 (a gift of G.E. Morris) is
described elsewhere (Nguyen et al., 1991; Morris et al., 1998
).
Antidystrobrevin Antibodies.
Antibody 13H1 (a gift of J.B. Cohen;
Carr et al., 1989) recognizes both
- and
-dystrobrevins (Peters et al.,
1997b
).
Antidystrophin Antibodies.
Antibody NCL-DYS2 (epitope in the COOH
terminus of dystrophin) is known to cross-react with the canine protein
(Novacastra). mAb 1808 raised against Torpedo dystrophin recognizes the
rod domain of dystrophin (Sealock et al., 1991).
Antidystroglycan Antibody.
Monoclonal anti--dystroglycan antibody,
VIA4-1 (Upstate Biotechnology Inc.), is described elsewhere (Ohlendieck
et al., 1991a
).
Other Antibodies.
Monoclonal and polyclonal anti-GFP (green fluorescent protein) antibodies (Clontech Laboratories, Inc.), anti--catenin
(Santa Cruz Biotechnology), anti-ZO-1 (Zymed Labs, Inc.), and anti-Na/K ATPase (Chemicon International, Inc.) were used according to
manufacturers' specifications.
Transfections
The following mouse 2-syntrophin constructs were subcloned using common restriction sites into pEGFPC2 expression vector (Clontech Laboratories, Inc.): full-length (FL; aa 1-520); PH1 (aa 1-94 and 175-288); PDZ
(aa 90-185); PH2 (aa 296-425); and syntrophin unique (SU; aa 421-520).
1-syntrophin PDZ (aa 75-171) and
1-syntrophin PDZ (aa 105-200)
were also subcloned into the same vector. The PH2SU (aa 296-520) and
PH1PDZ (aa 1-288) constructs were amplified by PCR and were subcloned using engineered restriction sites. All constructs were sequenced
before use. Type II MDCK cells were transfected using lipofectamine
(GIBCO BRL) according to manufacturer's recommendations. After 10-
14 d of growth in selection medium (DME + 5% FBS + 400 µg/ml G418),
individual colonies were isolated and stable lines were established. Wild-type MDCK cells were maintained in DME + 5% FBS.
Immunofluorescence Analysis of MDCK Cells
Cells were grown to confluence on glass coverslips (5-7 d), fixed in 2%
paraformaldehyde for 15 min, and either analyzed for GFP expression immediately, or prepared further for immunofluorescence. Cells were permeabilized for 15 min in PBS/0.5% Triton X-100, blocked in PBS/1% fish
gelatin/0.8% BSA for 30 min, and incubated with primary antibodies at
the appropriate dilution for 1 h at room temperature or overnight at 4°C.
After washing in PBS/0.5% Triton X-100, cells were incubated with Alexa
488-conjugated secondary antibody (Molecular Probes, Inc.) and either
Texas red-conjugated (Jackson ImmunoResearch Laboratories, Inc.) or
Alexa 594-conjugated (Molecular Probes, Inc.) secondary antibody for 1 h
at room temperature. Cells were washed with PBS/0.5% Triton X-100 and
mounted onto slides in glycerol with n-propyl gallate to minimize fading
(Giloh and Sedat, 1982). Staining was analyzed using confocal microscopy (Leica TCS-NT). Results were similar when MDCK cells were grown for
5-7 d on transwell filters (Corning Costar).
Coimmunoprecipitations
Wild-type or GFP-expressing MDCK cell lines were grown on 100-mm
plates for 5-7 d. Cells were rinsed in ice-cold homogenization buffer (HB;
10 mM sodium phosphate, 0.4 M NaCl, 5 mM EDTA, pH 7.8) and lysed
for 30 min on ice in HB/1% Triton X-100 (750 µl/plate) with protease inhibitors (2 mM PMSF, 1 µM bestatin, and 1 µg/ml each of aprotinin, leupeptin, antipain, and pepstatin A). Insoluble proteins were pelleted at
39,000 g for 30 min. The soluble extracts were then incubated with 1 µg of
specific antibody or control IgG for 1 h at 4°C. Protein A- or G-agarose
(Sigma Chemical Co.; 25 µl of 50% slurry) was added and incubated overnight at 4°C. Beads were washed extensively in HB containing 1 M NaCl/
1% Triton X-100. Proteins eluted in SDS sample buffer were separated by SDS-PAGE and transferred to nitrocellulose for immunoblotting as described previously (Peters et al., 1997a). In brief, after blocking in TBS/
0.1% Tween 20 (TBST) with 5% milk for 1 h at room temperature, nitrocellulose was incubated with primary antibody diluted in TBST/1% milk
for 1 h at room temperature. Blots were washed 3 times for 15 min each in
TBST, and were then incubated with an HRP-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, Inc.) for 1 h at room temperature. Blots were washed as before and signal was detected using enhanced chemiluminescence (Pierce Chemical Co.) and exposed to film.
Nitrocellulose blots were occasionally reprobed after stripping with stripping buffer (Chemicon International, Inc.).
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Results |
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Syntrophin, Utrophin, and Dystrobrevin Are Expressed in Polarized Epithelial Cells
Syntrophins are found in many tissues in which epithelia
are a major cell type, including lung and kidney (Adams et
al., 1993; Peters et al., 1997a
). However, these tissues contain a mixture of epithelial and nonepithelial cells. To
study the expression and distribution of syntrophins in polarized epithelial cells, we used MDCK cells. Expression
of components of the DAPC was determined by immunoblotting of Triton X-100 soluble and insoluble fractions from MDCK cells. An mAb that recognizes all three syntrophin isoforms (SYN1351) detected a single band of 60 kD (the size expected for syntrophins; Fig. 1). Most of this
protein was found in the Triton X-100 soluble fraction.
Utrophin, a dystrophin homologue, was also found (Fig. 1)
and, like syntrophins, was detected primarily in the Triton
X-100 soluble fraction. Syntrophin and utrophin were also
found in two additional polarized epithelial cell lines: HBE, a human bronchial epithelia cell line; and LLCPK, a
pig renal epithelia cell line (data not shown).
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The expression of syntrophin isoforms differs among tissue types. For instance, skeletal muscle contains all three
syntrophin isoforms, while 1-syntrophin is the predominant form in liver (Peters et al., 1997a
). To identify the
syntrophin isoforms expressed in polarized epithelia, samples enriched for syntrophins by immunoprecipitation with SYN1351 were analyzed by immunoblotting with antibodies specific for each of the three syntrophin isoforms.
2-Syntrophin was the only syntrophin isoform consistently detected (Fig. 2), although in some experiments
small amounts of
1-syntrophin were also found. Given its
abundance in the epithelial cell lines tested and in tissues
rich in epithelial cells,
2-syntrophin appears to be the
predominant form of syntrophin in epithelia.
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In skeletal muscle, syntrophins associate directly with
dystrophin family members (Kramarcy et al., 1994; Yang
et al., 1994
; Ahn and Kunkel, 1995
; Dwyer and Froehner,
1995
; Ahn et al., 1996
). This also appears to be the case in
MDCK cells. Utrophin and a form of dystrobrevin were
specifically enriched in SYN1351 preparations, but were
absent from control preparations (Fig. 2). These results indicate that syntrophin, utrophin, and dystrobrevin exist in a stable complex in polarized epithelial cells.
Syntrophin and Utrophin Are Expressed on the Basolateral Membrane of Polarized Epithelia
Polarized epithelial cells have two distinct membrane domains that differ greatly in lipid and protein content (Simons and Fuller, 1985; Rodriguez-Boulan and Nelson,
1989
). To determine the localization of the syntrophin/
utrophin complex, we compared the distribution of syntrophin with ZO-1,
-catenin, and Na/K ATPase, markers of
distinct membrane specializations in polarized MDCK
cells. The distribution of syntrophin (Fig. 3 A) was very similar to that of Na/K ATPase, which is largely basolateral. Syntrophin outlined the cell on its basal and lateral
surfaces, but was absent from the apical membrane.
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The lateral membrane of polarized epithelial cells contains two specialized structures, the TJ and the adherens
junction. The TJ is located at the intersection of the apical
and basolateral membrane, where it acts to regulate diffusion across the epithelium and as a barrier between the
apical and basolateral membrane (reviewed in Gumbiner,
1987). The adherens junction is a cadherin-based specialization believed to be involved in adhesion of epithelial cells (Gumbiner, 1996
; reviewed in Yap et al., 1997
). In addition to providing useful markers for subdomains within
the lateral membrane, the TJ and adherens junction are
key structures of epithelial cells where numerous PDZ-containing proteins have been shown to be important
(Stevenson et al., 1986
; Beatch et al., 1996
; reviewed in
Kim, 1997
; Mandai et al., 1999
; Haskins et al., 1998
; Izumi et al., 1998
; Satoh et al., 1998
). Therefore, we were interested in the relationship between the syntrophin complex
and these junctions.
-Catenin is found enriched at the adherens junction and in a cytosolic pool (Hinck et al., 1994
;
Nathke et al., 1994
). As shown in Fig. 3 B, syntrophin and
-catenin are colocalized on the lateral membrane. Anti-
ZO-1 specifically labeled TJs (Stevenson et al., 1986
), and
appeared to represent the upper limit of syntrophin expression (Fig. 3 C). The distribution of utrophin was indistinguishable from that of syntrophin, having the same limits of expression when double labeled with anti-ZO-1
antibodies (Fig. 3 D).
GFP-tagged 2-Syntrophin Targets to the Basolateral
Membrane of MDCK Cells
While PDZ-containing proteins have been shown to be
important at synapses and cell-cell junctions (reviewed in
Kim, 1997), only the targeting of Dlg/SAP97 (Lue et al.,
1996
; Hough et al., 1997
; Wu et al., 1998
) and ZO-1 (Fanning et al., 1998
) has been studied extensively. As a first
step in determining which domains of syntrophin are responsible for its localization on the basolateral membrane,
we studied the distribution of exogenously expressed GFP
fusion proteins of syntrophin (Fig. 4). MDCK cells were
transfected with a plasmid encoding GFP fused to the NH2
terminus of full-length
2-syntrophin (GFP-FL) and multiple stable cell lines were established. GFP-FL was found
exclusively on the basolateral membrane, in a distribution
indistinguishable from that of endogenous syntrophin (Fig. 5 A). In contrast, GFP alone was found only in the
cytoplasm.
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Syntrophins contain four distinct domains: two PH domains, a PDZ domain, and an SU domain (Adams et al.,
1995; Ahn and Kunkel, 1996). Any of these domains could
be involved in targeting to the basolateral membrane.
Since PH domains in other proteins have been shown to be
capable of binding phospholipids (Shaw et al., 1996), the
PH domain(s) of syntrophin may recruit syntrophin to the
basolateral membrane through binding to specific types of lipids. This model is especially attractive since the apical
and basolateral membranes differ in lipid content (Simmons and Fuller, 1985; Rodriguez-Boulan and Nelson,
1989
). Alternatively, the PDZ domain could direct syntrophin to the basolateral surface by binding a ligand that is
restricted to the basolateral membrane. This could be a
general mechanism by which PDZ-containing proteins are
recruited to specialized sites. Finally, the SU domain,
which is unrelated to any known protein domain, may be
responsible for targeting syntrophin to specific subcellular compartments.
To examine these possibilities, we established stable
MDCK cell lines expressing individual 2-syntrophin
domains fused with GFP (Fig. 4) and compared the distribution of the GFP fusion proteins with endogenous
syntrophin. The PH1, PDZ, PH2, and SU domains of
2-syntrophin all failed to accumulate at the basolateral
membrane (Fig. 5 B). The PDZ domains of
1-syntrophin
and
1-syntrophin also failed to target (data not shown).
Each individual domain was distributed diffusely throughout the cell and was identical to the distribution of GFP
alone (Fig. 5 B). In the PH1 and PH2 cell lines, labeled
cells were very sparse. We counted >200 positive cells in
three independent cell lines for each construct. In all cases,
the GFP-tagged domain was diffusely distributed throughout the cell.
The failure of individual domains to localize preferentially to the basolateral membrane may be due to their inability to bind a partner, perhaps utrophin, which resides
on this membrane. The COOH-terminal 37 kD of 1-syntrophin, consisting of part of the PH1 domain, along with
the PH2 and SU domains, is sufficient to bind to dystrophin family members in vitro (Ahn and Kunkel, 1995
). Individual domains of syntrophin may not retain their ability to bind utrophin, and therefore cannot be recruited to or
retained at the basolateral membrane. To test this hypothesis, we made two additional GFP constructs which consisted of tandemly linked PH2 and SU domains (PH2SU),
or the linked PH1 and PDZ domains of
2-syntrophin
(Fig. 4). In multiple stable cell lines, GFP-PH2SU was
found on the basolateral membrane (Fig. 5 C), in a distribution indistinguishable from endogenous syntrophin or
expressed GFP-FL. In contrast, GFP-PH1PDZ failed to
accumulate at the basolateral membrane (Fig. 5 C), indicating that the PH2SU construct is necessary and sufficient for basolateral sorting. Recently, the targeting of
some GFP-tagged proteins was shown to be substratum
dependent (Moyer et al., 1998
). However, we obtained
similar syntrophin domain targeting results whether transfected MDCK cells were plated on glass coverslips or on
transwell filters (data not shown).
Syntrophin Domain Interactions with Utrophin
To test biochemically whether GFP-PH2SU retains the
ability to bind utrophin while individual syntrophin domains and PH1PDZ do not, we immunoprecipitated each
GFP fusion protein and determined whether utrophin was
specifically coisolated. Detergent extracts from cell lines
expressing GFP, or fusion proteins of full-length 2-syntrophin, PH2SU, PH1PDZ, or individual syntrophin
domains were subjected to immunoprecipitation with
anti-GFP or control antibody. To confirm the size and expression level of each fusion protein, immunoprecipitates
were analyzed by immunoblotting with a monoclonal anti-GFP antibody. Sample loadings were then adjusted to obtain comparable amounts of each fusion protein (Fig. 6). The top halves of the same blots were incubated with a
monoclonal antiutrophin antibody. As expected, utrophin
was specifically coimmunoprecipitated with GFP-FL, indicating that fusion with GFP did not interfere with the interaction between syntrophin and utrophin (Fig. 6 A).
Utrophin also coimmunoprecipitated with the PH2SU
tandemly linked domains, but not with PH1PDZ or individual syntrophin domains (Fig. 6 A). This ability of the
GFP-PH2SU fusion protein to bind utrophin may underlie
its localization on the basolateral membrane.
|
A current model of the stoichiometry of the DAPC in
muscle predicts one utrophin, one dystrobrevin, and two
syntrophins per complex: one syntrophin binds directly to
utrophin or dystrophin and another binds to a dystrobrevin family member (Fig. 7; Peters et al., 1997a; Sadoulet-Puccio et al., 1997
). To test whether two syntrophins are
present in epithelial syntrophin/utrophin complexes, we
determined whether endogenous syntrophin copurified with exogenously expressed GFP-syntrophin (Fig. 6 B).
We used an antibody directed against the syntrophin PDZ
domain (SYN1351) to detect endogenous syntrophin in
samples immunoprecipitated with GFP antibodies. Although SYN1351 also detects GFP-FL, GFP-PH1PDZ, and GFP-PDZ constructs (Fig. 6 B, thin arrows), they are
well separated from the endogenous syntrophin on our immunoblots (except in the case of the PH1PDZ construct,
which is the same size as endogenous syntrophin). In these
immunoprecipitation experiments we find that endogenous syntrophin (Fig. 6 B, thick arrow) copurifies with
GFP-FL and GFP-PH2SU, but not with GFP-PH1, GFP-PDZ, GFP-PH2, or GFP-SU fusion proteins. Although we
cannot determine whether endogenous syntrophin copurifies with GFP-PH1PDZ, we believe it does not since it
fails to copurify utrophin and dystrobrevin. To accommodate two syntrophin binding sites, a dystrobrevin must also
be present in the complex. When blots were probed with a panspecific dystrobrevin antibody, we observed a 65-kD
dystrobrevin isoform in samples immunoprecipitated from
GFP-FL and GFP-PH2SU cell lines (Fig. 6 C). Dystrobrevin was not detected when GFP antibody was used to immunopurify individual GFP-tagged syntrophin domains or
the PH1PDZ fusion protein. The failure of the PDZ fusion proteins to associate with endogenous syntrophin suggests
that PDZ homodimerization does not occur with syntrophins as it does with other PDZ proteins (Srivastava et al.,
1998
; Xu et al., 1998
).
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Discussion |
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In this study, we have characterized an epithelial utrophin-associated protein complex expressed in MDCK cells. Our
data demonstrate that this complex is restricted to the basolateral cell surface of epithelial cells, and includes 2-syntrophin and an isoform of dystrobrevin. We find that
utrophin and dystrobrevin copurify with syntrophin, providing strong evidence that these proteins are associated in
MDCK cells as they are in other tissues (Kramarcy et al.,
1994
; Ahn and Kunkel, 1995
; Dwyer and Froehner, 1995
; Yang et al., 1995).
The dystrobrevins are divided into two families, and
,
which undergo extensive alternative splicing (Blake et al.,
1996
, 1998
; Sadoulet-Puccio et al., 1996
; Peters et al.,
1997b
; Puca et al., 1998
). The molecular mass of dystrobrevin found in MDCK cells is ~65 kD (Figs. 2 and 6 C)
and, thus, could correspond to either
-dystrobrevin-2 or
-dystrobrevin. Although our isoform-specific antibodies
are not reactive with the canine proteins, therefore preventing a direct test of this question, we believe that this MDCK protein is likely
-dystrobrevin for two reasons.
First,
-dystrobrevin is enriched in epithelial tissues (Peters et al., 1997b
; Blake et al., 1998
), while
-dystrobrevin-2 is restricted to brain, skeletal, and cardiac muscle
(Peters et al., 1997b
). In addition,
-dystrobrevin-2 associates preferentially with dystrophin (Peters et al., 1998
), and
therefore is unlikely to be found in a utrophin complex.
Taken together, these data suggest that
-dystrobrevin is
the isoform present in MDCK cells.
While multiple syntrophins are often expressed in the
same tissue, a single isoform often predominates. For instance, 1-syntrophin is the major form in skeletal and cardiac muscle,
1-syntrophin in liver and smooth muscle,
and
2-syntrophin in epithelial-rich tissues like kidney and
lung (Peters et al., 1997a
). In tissues where
2-syntrophin
is not the dominant form, such as brain and muscle, it is often restricted to sites of membrane specialization, such as
the neuromuscular junction (Peters et al., 1994
, 1997a
) and
retinal synapses (Peters, M.F., C. Houlihan, and S.C.
Froehner, unpublished results). Thus,
2-syntrophin may
play a unique role at membrane specializations. Here, we
find that
2-syntrophin is the dominant syntrophin isoform expressed in MDCK cells. The predominance of
2-syntrophin could be explained by the failure of the
1-syntrophin and two
1-syntrophin isoform specific antibodies to recognize canine syntrophins. However, the abundance
of
2-syntrophin in epithelial-rich rodent tissues and in a
human bronchial epithelial cell line (data not shown), in
which our antibodies are known to be reactive, suggests
that
2-syntrophin is the major isoform in epithelia.
The syntrophin/utrophin complex of MDCK cells probably includes the dystroglycans. We find -dystroglycan
on the basolateral membrane of MDCK cells (data not
shown) consistent with report of dystroglycans on the
basal surface of epithelial cells in vivo (Durbeej et al.,
1998
). These data support a model (Fig. 7) in which the
syntrophin/utrophin complex serves as a link between the
ECM and the actin cytoskeleton in epithelia, as it does in skeletal muscle.
There have been reports of a dystrophin short form,
Dp140, on the basal surface of tubule epithelial cells in
kidney (Durbeej et al., 1997; Lidov and Kunkel, 1998
).
While we did detect some dystrophin by immunofluorescence in MDCK cells (data not shown), it was not found at
the basolateral membrane. Thus, dystrophin may be important in certain epithelia, but we do not believe it is part
of the basolateral complex in MDCK cells.
Interaction of Syntrophin Domains with Utrophin
The region on dystrophin that binds syntrophins has been
mapped to a short segment encoded by exon 74 (and to
homologous regions in utrophin and dystrobrevin; Yang et
al., 1994; Ahn and Kunkel, 1995
; Suzuki et al., 1995
). However, identification of the region of syntrophin responsible
for binding to dystrophin family members has not been reported. Binding studies using in vitro translated proteins
showed that a 37-kD fragment containing the COOH terminus of
1-syntrophin is sufficient for association with
dystrophin family members (Ahn and Kunkel, 1995
). We
find that individual PH2 or SU domains fail to bind utrophin, but when these domains are tandemly linked (as they
are in the native protein) binding to utrophin is restored.
These data suggest that the combined PH2 and SU domains function as a unit. Perhaps the binding site for utrophin is formed by noncontiguous partial binding sites in
the PH2 and SU domains. Alternatively, the region of syntrophin responsible for binding utrophin may bridge sequences in the PH2 and SU domains. Although the individual constructs used for expression of PH2 and SU
domains overlapped by four amino acids, it is possible that
proper folding of this bridge region requires a longer
polypeptide. Finally, it is possible that the utrophin binding site is contained within a single domain, but that both
domains are required for proper folding of the interacting
site. While our data do not discriminate between these
possibilities, they do provide in vivo evidence that an intact PH2SU domain is necessary and sufficient for syntrophin binding to utrophin.
Two Syntrophins per Complex
In muscle, the current model of the DAPC includes dystrophin or utrophin directly associated with a dystrobrevin
family member through a coiled-coil interaction (Peters et
al., 1997a; Sadoulet-Puccio et al., 1997
). The presence of
syntrophin binding sites in both dystrophin or utrophin,
and dystrobrevin allows for the binding of two syntrophins
per complex (Fig. 7). Support for this model comes from
the demonstration that dystrophin or utrophin complexes contain pairs of syntrophin isoforms (Peters et al., 1997a
)
and a stoichiometry of two syntrophins per complex
(Yoshida and Ozawa, 1990
; Ervasti and Campbell, 1991
).
In this study, we tested the hypothesis that two syntrophins are contained within the epithelial syntrophin/
utrophin complex by expressing GFP-tagged syntrophin
(GFP-FL) in MDCK cells. In experiments in which GFP-tagged syntrophin was immunoprecipitated with anti-GFP, we detected not only utrophin and dystrobrevin, but
also endogenous syntrophin. A single syntrophin binding
site on utrophin and dystrobrevin likely accounts for the
two syntrophins in the purified complex. The amount of
endogenous syntrophin is approximately half that of the
GFP-tagged syntrophin, suggesting that some purified
complexes contain two GFP-tagged syntrophins, while
others contain one GFP-tagged syntrophin and one endogenous syntrophin. Whether GFP-tagged syntrophin
displaces endogenous syntrophin or instead binds to unoccupied sites on utrophin and dystrobrevin is unclear. Alternatively, our results could also be explained by dimerization of syntrophins within the complex. Although
evidence for syntrophin dimerization has been reported (Yang et al., 1994; Madhavan and Jarrett, 1995
), alternative explanations for these findings have been suggested
(Ahn et al., 1996
; Peters et al., 1997). Our data indicate
that if syntrophin dimerization does occur, it must be via
the PH2 and SU domains, since none of the other constructs were able to copurify endogenous syntrophin.
Targeting of the Syntrophin/Utrophin Complex in Polarized Epithelia
Epithelial cells contain distinct apical and basolateral cell
surfaces with unique protein and lipid compositions (Simon and Fuller, 1985; Rodriguez-Boulan and Nelson,
1989). The epithelial syntrophin/utrophin complex is restricted to the basolateral cell surface. As a first step in understanding the sorting of the syntrophin/utrophin complex to the basolateral membrane of epithelial cells, we
investigated the targeting of syntrophin in MDCK cells.
Our results show that the tandemly linked PH2 and SU
domains are necessary and sufficient for directing GFP-tagged syntrophin to the basolateral membrane (Fig. 5
C). The PH domain and additional COOH-terminal sequences are also required to target cytohesion-1 and Bruton's tyrosine kinase to the plasma membrane (Nagel et
al., 1998
). However, it is not simply the presence of a PH
domain which confers membrane targeting of syntrophin
since the PH1PDZ construct failed to accumulate at the
basolateral membrane. Interestingly, replacement of the cytohesion-1 PH domain with the PH domain of the
1-adrenergic receptor abolished membrane targeting (Nagel
et al., 1998
).
The amino acid residues responsible for targeting syntrophin to the basolateral membrane may also be necessary for binding to utrophin. Alternatively, syntrophin may contain two independent regions, one responsible for binding to utrophin and a second involved in binding structures at the basolateral membrane. Site-directed mutagenesis of the PH2SU domain may allow for the discrimination of utrophin-binding and basolateral targeting functions within this region of syntrophin.
In epithelia, the signals responsible for polarized sorting
are best characterized for transmembrane proteins. For instance, several integral membrane proteins that target to
the basolateral surface in epithelial cells contain di-leucine
or tyrosine-based basolateral sorting sequences (Ktistakis
et al., 1990; Thomas et al., 1993
; Hunziker and Fumey,
1994
; Matter et al., 1994
; Sheikh and Isacke, 1996
; Simonsen et al., 1998
). Therefore, we examined the sequence
of
-dystroglycan for potential targeting sequences and
found a conserved sequence, EDQATFI (amino acids
784-790 in mouse and human) in the COOH terminus
of
-dystroglycan, that is very similar to the consensus
sequence for the di-leucine basolateral sorting motif,
DDQxxLI (Matter et al., 1994
; Simonsen et al., 1998
). In
-dystroglycan, this motif is predicted to be cytoplasmic and does not overlap with the dystrophin binding site
(Jung et al., 1995
). The cytoplasmic targeting motifs found
in basolateral proteins are often followed by small clusters
of acidic residues (Matter et al., 1994
). The presence of the
sequence DELDD downstream of the putative di-leucine
motif in
-dystroglycan supports a role for this motif in basolateral sorting. It will be interesting to examine the importance of this sequence in the targeting of
-dystroglycan and other components of the syntrophin/utrophin complex in epithelial cells.
Role of Syntrophin/Utrophin Complex in Epithelia
At this time, we can only speculate as to the function of
the basolateral syntrophin/utrophin complex in polarized
epithelia. The syntrophin/utrophin complex, through its
ability to link ECM proteins to actin, may serve a structural role in epithelia. In skeletal muscle, the link between
the ECM and the cytoskeleton is thought to maintain cell
membrane integrity during contraction (Petrof et al., 1993;
Pasternak et al., 1995
). Some epithelia must also withstand
the forces of contraction (i.e., in the gastrointestinal tract).
Thus, a link between the ECM and actin provided by the syntrophin/utrophin complex, may maintain membrane integrity in epithelia. The syndecan/CASK complex also
links the ECM to the actin cytoskeleton (Cohen et al.,
1998
), and may play a role similar to the syntrophin/utrophin complex in epithelia.
The syntrophin/utrophin complex may also function to
recruit proteins to the basolateral surface of epithelial
cells. Syntrophins are modular adapter proteins made up
almost exclusively of protein-protein interaction domains
(Ahn et al., 1994, 1996
; Adams et al., 1995
). Our results indicate that the PH1 and PDZ domains do not play a role in
targeting syntrophin to the basolateral membrane. In contrast, the PDZ domains of Dlg/SAP97 are necessary for efficient subcellular targeting (Lue et al., 1996
; Hough et al.,
1997
; Wu et al., 1998
), indicating that modular adapter
proteins may be targeted via different mechanisms. The
PH1 and PDZ domains of syntrophin are also unnecessary
for the interaction of syntrophin with utrophin. Therefore,
these domains are free to interact with additional proteins
to generate a large multiprotein complex (Fig. 7). Binding
partners for the PH1 domain of syntrophin have not been
identified, but PH domains in other proteins are capable of binding proteins such as protein kinase C, and the
and
subunits of G proteins (Touhara et al., 1994
; Yao et al.,
1994
).
The ability of the PDZ domain of syntrophin to bind
nNOS in muscle (Brenman et al., 1996) suggests that one
function of syntrophin is to recruit signaling proteins to
the membrane. Interestingly, the presence of two syntrophins per complex (Fig. 7) may allow two different proteins to be brought in close apposition, allowing for one to
modulate the other. For instance, in skeletal muscle, syntrophin PDZ domains also bind voltage-gated sodium
channels (Brenman et al., 1996
; Gee et al., 1998
; Schultz et
al., 1998
). NO modulates the activity of certain sodium
channels (Li et al., 1998
), a process that may occur with
high efficiency and specificity if nNOS and sodium channels reside in the same complex. The recruitment of two
proteins into the DAPC in muscle has potential functional consequences: the formation of similar complexes containing signaling molecules, and effector proteins may also occur in epithelia. Thus, it will be important to identify binding partners for the PH1 and PDZ domains to gain further
understanding of the function of the syntrophin/utrophin
complex in epithelia.
PDZ domain-containing proteins are a common feature
of many scaffolding complexes (Cho et al., 1992; Kornau
et al., 1995
; Kim et al., 1996
; Dong et al., 1997
; Cohen et
al., 1998
; Short et al., 1998
; Srivastava et al., 1998
; Xu et al.,
1998
). Through their interactions with the COOH-terminal tails of receptors and ion channels, PDZ domains are
critical in the assembly of multiprotein complexes. Many
scaffolding proteins contain multiple PDZ domains, which
may tether multiple copies of the same ligand at a particular subcellular location. More often, the PDZ domains
within a single protein have distinct binding specificities,
allowing different proteins to be recruited to the same subcellular location. An elegant illustration of the efficiency
of such complexes comes from studies of INAD (inactivation no after potential D) where a single type of scaffold
links all (or most) proteins needed for Drosophila phototransduction (reviewed in Montell, 1998
). Interestingly,
homodimerization of INAD molecules generates a complicated network of proteins at the membrane (Xu et al.,
1998
). Furthermore, the potential for homodimerization
of proteins which contain 6-13 PDZ domains (Dong et al.,
1997
; Srivastava et al., 1998
; Ullmer et al., 1998
), raises the
complexity of scaffolding complexes to almost incomprehensible heights.
In epithelia, the asymmetric localization of proteins and
lipids results in functional differences between the apical
and basolateral membranes required for epithelial cell
function. MDCK cells sort secretory and membrane-associated proteins to apical and basolateral surfaces by several different mechanisms (reviewed in Simons and Wandinger-Ness, 1990; Caplan, 1997
). Some proteins are packaged
upon exit from the TGN into separate apical or basolateral transport vesicles (Wadinger-Ness et al., 1990). Other
proteins are targeted exclusively to the basolateral domain
but do not remain there; instead they are internalized into
endosomes and targeted via the transcytotic pathway to
the apical cell surface. Finally, some proteins are transported in a nonpolarized manner to both cell surfaces, but
are selectively stabilized at one surface. For example, the
Na/K ATPase is stabilized at the basolateral cell surface
by association with the actin cytoskeleton and ankyrin (Nelson and Veshnock, 1987
; Jordan et al., 1995
; Thevananther et al., 1997
). Syntrophin may play a similar role
and act to specifically anchor transmembrane proteins by
high affinity protein-protein interactions via the PDZ or
PH domains. However, since PDZ proteins are present on
both apical and basolateral cell surfaces, and at the TJs,
additional factors that define binding specificities must be involved.
In addition to a targeting role once a polarized monolayer is formed, the epithelial syntrophin/utrophin complex may be involved in the development of polarity in epithelial cells. Drubin and Nelson (1996) proposed that
extracellular cues, such as cell-cell adhesion, define discrete areas of membrane as sites of submembranous cytoskeletal assembly. Once assembled, the cytoskeleton
serves as a docking site for specific proteins, leading to further specialization of this region of membrane. In MDCK
cells, E-cadherin-mediated adhesion defines the site for
recruitment of the sec6/sec8 complex (Grindstaff et al.,
1998
). This protein complex then serves as a docking site
for additional proteins. By serving as a link between the
ECM and the submembranous cytoskeleton, the syntrophin/utrophin complex may also assist in defining basal
membranes during morphogenesis. Laminin, a component
of the basement membrane which binds dystroglycan (Ibraghimov-Beskrovnaya et al., 1992
; Gee et al., 1993
),
plays an important role in the differentiation of epithelia
in vivo and in vitro (Klein et al., 1988
; Ekblom et al., 1990
;
Durbeej et al., 1995
). Antibodies that block the binding of
laminin to dystroglycan in organ cultures inhibit epithelial
differentiation (Durbeej et al., 1995
). Perhaps the binding
of laminin serves to recruit the syntrophin/utrophin complex to the basal membrane, where it participates in the
specialization of this cell surface. In addition, the heparan
sulfate proteoglycan, agrin, is found in the basement membrane of epithelial tissues, where it binds dystroglycan
(Gesemann et al., 1998
; Raats et al., 1998
). It is important
to note that antibodies that disrupt the laminin-dystroglycan interaction may also block agrin binding (Ervasti and
Campbell, 1993
; Campanelli et al., 1994
; Gee et al., 1994
),
suggesting that agrin may also be involved in epithelial
morphogenesis via the syntrophin/utrophin complex. The
use of additional cell culture model systems and transgenic
or knockout animals will help in defining functions for the
epithelial syntrophin/utrophin complex.
![]() |
Footnotes |
---|
Address correspondence to S.L. Milgram, Department of Cell and Molecular Physiology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7545. Tel.: (919) 966-9792. Fax: (919) 966-6413. E-mail: milg{at}med.unc.edu or S.C. Froehner, Department of Cell and Molecular Physiology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7545. Tel.: (919) 966-1239. Fax: (919) 966-6413. E-mail: froehner @
Received for publication 30 October 1998 and in revised form 5 February 1999.
We thank Peter Mohler for advice on culturing and transfecting epithelial
cells, Stuart Krall for assistance with tissue culture, Dr. Michael Chua for
assistance with confocal microscopy, Dr. Raghavan Madhavan for pointing out the potential targeting sequence in -dystroglycan, and Dr. Marcie
Colledge and members of the Froehner and Milgram labs for helpful discussion. We also thank the following individuals for providing reagents:
Dr. Stephen Gee for plasmids encoding syntrophin domains, Dr. G.E.
Morris for donating the antiutrophin antibody, and Dr. J.B. Cohen for providing the antidystrobrevin antibody.
This work was supported by National Institutes of Health grant R29DK50744 and Cystic Fibrosis Foundation grant MILGRA9710 to S.L. Milgram, and National Institutes of Health grant NS33145 to S.C. Froehner.
![]() |
Abbreviations used in this paper |
---|
DAPC, dystrophin-associated protein
complex;
ECM, extracellular matrix;
GFP, green fluorescent protein;
GFP-FL, GFP fused to NH2 terminus of full-length 2-syntrophin;
HB, homogenization buffer;
nNOS, neuronal nitric oxide synthase;
PDZ, protein domain named for PSD-95, discs large, ZO-1;
PH, pleckstrin homology;
SU, syntrophin unique;
TJ, tight junction.
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