Department of Physiology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7545
The syntrophins are a multigene family of intracellular dystrophin-associated proteins comprising
three isoforms, 1,
1, and
2. Based on their domain
organization and association with neuronal nitric oxide
synthase, syntrophins are thought to function as modular adapters that recruit signaling proteins to the membrane via association with the dystrophin complex. Using sequences derived from a new mouse
1-syntrophin
cDNA, and previously isolated cDNAs for
1- and
2-syntrophins, we prepared isoform-specific antibodies to
study the expression, skeletal muscle localization, and
dystrophin family association of all three syntrophins.
Most tissues express multiple syntrophin isoforms. In
mouse gastrocnemius skeletal muscle,
1- and
1-syntrophin are concentrated at the neuromuscular junction
but are also present on the extrasynaptic sarcolemma.
1-syntrophin is restricted to fast-twitch muscle fibers,
the first fibers to degenerate in Duchenne muscular
dystrophy.
2-syntrophin is largely restricted to the
neuromuscular junction.
The sarcolemmal distribution of 1- and
1-syntrophins suggests association with dystrophin and dystrobrevin, whereas all three syntrophins could potentially
associate with utrophin at the neuromuscular junction.
Utrophin complexes immunoisolated from skeletal
muscle are highly enriched in
1- and
2-syntrophins, while dystrophin complexes contain mostly
1- and
1-syntrophins. Dystrobrevin complexes contain dystrophin and
1- and
1-syntrophins. From these results,
we propose a model in which a dystrophin-dystrobrevin complex is associated with two syntrophins. Since
individual syntrophins do not have intrinsic binding
specificity for dystrophin, dystrobrevin, or utrophin, the
observed preferential pairing of syntrophins must depend on extrinsic regulatory mechanisms.
Syntrophins are intracellular peripheral membrane
proteins of 58-60 kD originally identified as proteins enriched at the postsynaptic apparatus in Torpedo electric organ (17). More recently, syntrophins in
mammalian skeletal muscle have been shown to be part of
a complex of proteins that associate with dystrophin, the
product of the Duchenne/Becker muscular dystrophy gene
(4, 28, 50, 54). Many of the dystrophin-associated proteins
(DAPs)1 are transmembrane proteins. Thus, the dystrophin
complex as a whole is thought to link cortical actin to the
extracellular matrix, thereby stabilizing the sarcolemma
during repeated cycles of contraction and relaxation (3).
At the neuromuscular junction (NMJ), the DAPs have been
implicated in agrin-stimulated clustering of nicotinic acetylcholine receptors (for review see reference 46). Dystrophin and DAPs are also found at synapses in the brain and
retina (29, 33, 45). Thus, the syntrophins and other DAPs
may participate in synaptogenesis as well as in sarcolemmal stabilization.
The three syntrophin isoforms, Differential association of dystrophin with certain syntrophin isoforms and/or DAPs may play a role in tailoring
the complex for a particular membrane specialization. Indeed, the protein complexes assembled by muscle dystrophin should be functionally distinct from those organized
by retinal dystrophin. Likewise, each of the dystrophin-related proteins, utrophin, dystrophin-related protein 2 (DRP-2), and dystrobrevin, may differentially associate with particular DAPs in different cell types. All of these
dystrophin family members contain amino acid sequences
homologous to the dystrophin carboxy terminus, the region in dystrophin shown to bind syntrophins and the
DAPs. Dystrophin, utrophin, and dystrobrevin have been
shown to be capable of binding all three syntrophin isoforms in vitro (4, 6). With the exception of dystrobrevin, each dystrophin family member also has a WW domain
postulated to bind the transmembrane DAP complex.
Since each of the dystrophin family members is expressed
in a wide range of cell types, their association with specific
subsets of syntrophins/DAPs may be critical for cell-specific function.
Among all the DAPs, the syntrophins appear particularly well suited for differentially associating with the dystrophin family members. Each of the three syntrophins
is postulated to function as a modular adapter recruiting
signaling proteins to dystrophin membrane complexes.
Yet, syntrophin isoforms share only ~50% amino acid
identity, suggesting that each may recruit a distinct set of
proteins. Like members of the dystrophin family, the syntrophins are expressed in a wide range of tissues (1, 5).
We have shown previously that in rat skeletal muscle, Here, we examine the associations of all three syntrophin isoforms with dystrophin family members expressed
in skeletal muscle. We have previously isolated mouse
cDNAs encoding Mouse A Antibodies
Antisyntrophin.
mAb SYN1351 raised against Torpedo syntrophin has
been described previously (17). Polyclonal antibodies (Abs) specific for each syntrophin isoform were prepared by immunizing rabbits with peptides according to standard methods (36). The Ab SYN37 was prepared
against the peptide C-RLGGGSAEPLSSQSFSFHRDR, corresponding to
amino acids 220-240 of mouse
Antidystrobrevin.
mAb 13H1 (a gift of J.B. Cohen, Harvard Medical
School) was raised against Torpedo dystrobrevin (12). Abs DB308 and
DB433 were prepared against peptides corresponding to mouse dystrobrevin, as previously described (8).
Antidystrophin.
Ab DYS3669 was prepared against COOH-terminal
10 amino acids of mouse dystrophin (22) plus a terminal cysteine (C-PGKPMREDTM) according to standard methods (36). mAb Mandys-8 (34)
was purchased from Sigma Chemical Co. (St. Louis, MO).
Antiutrophin.
Ab UTR3165 was previously described (28). mAb DRP-1
was purchased from Novacastra Laboratories (Newcastle upon Tyne, UK).
Anti-myosin heavy chain (MHC).
mAbs BF-F3 (diluted 1:30) (44) and
NCL-MHCf (Novacastra Laboratories, diluted 1:30) are specific for MHC
expressed in type IIB fibers and all type II fibers, respectively. mAb A4.74
(diluted 1:30) (52) strongly labels rat MHC in type IIA and labels type IID
fibers to a lesser extent.
Immunoaffinity Purification of Protein Complexes
Tissues from control (C57BL10 SNJ; Jackson Laboratory, Bar Harbor,
ME), mdx (Jackson Laboratory), and
Immunoblotting
Samples were resolved by electrophoresis on 8% acrylamide/0.8% bis-acrylamide gels buffered with tris-tricine (43). Proteins were transferred
to Immobilon-P (Millipore Corp., Bedford, MA) in 192 mM glycine, 25 mM
Tris, 20% methanol using a Trans-Blot apparatus (BioRad Labs). After
blocking in 5% milk/TBS, 0.1% tween-20 (TBST), membranes were incubated with primary antibodies (30 nM) in 1% milk/TBST. After three
5-min washes in TBST, bound primary antibodies were detected with appropriate secondary antibodies conjugated to horseradish peroxidase (Jackson Immunoresearch, West Grove, PA). Because the heavy chain of polyclonal antibodies that leaked from resins comigrated with syntrophins,
selected blots were probed with biotinylated antisyntrophin polyclonal antibodies and detected with peroxidase-avidin detection reagent (Vectastain
ABC elite; Vector Laboratories, Burlingame, CA). Bands were visualized
by enhanced chemiluminescence (Pierce) and exposed to film (Dupont/
NEN, Wilmington, DE). In some cases, blots were stripped for 1 h at 50°C in
2% SDS, 0.1 M Immunohistochemistry
The gastrocnemius, plantaris, and soleus were removed intact from mice,
flash-frozen in liquid nitrogen-cooled isopentane, and cryosectioned (7 µm).
Before labeling, sections were fixed with 0.5% paraformaldehyde, permeabilized with 0.5% Triton X-100/PBS/0.2 M NH4Cl, and blocked with PBS
containing 2% fish gelatin/0.08% BSA. Sections were incubated with primary antibodies (30 nM). After several PBS washes, sections were incubated with a cocktail containing either goat anti-rabbit IgG or anti-mouse
IgM conjugated to Texas red (Jackson Immunoresearch; 1:200) mixed
with either donkey anti-mouse IgG conjugated FITC or Isolation of Mouse The cDNA encoding the mouse form of Specificity of Syntrophin Antibodies
To investigate the expression of syntrophins and their association with dystrophin and dystrophin-related proteins,
we prepared and characterized a series of antisyntrophin
antibodies. Polyclonal antibodies were generated against
synthetic peptides corresponding to syntrophin sequences
from a region poorly conserved among the three isoforms
(see boxed region in Fig. 1 B). To test the specificity of these
antibodies, syntrophin isoforms were partially purified with
the appropriate syntrophin peptide antibody from mouse
tissues rich in either In addition to proteins of the size expected for full-length syntrophins, smaller proteins were recognized by
the anti-
Differential Expression of Syntrophin Isoforms
To examine the relative amounts of the three syntrophin
isoforms expressed in different tissues, syntrophins were
partially purified from detergent-solubilized tissue extracts
using the pan-specific mAb SYN1351. Equal amounts of
total syntrophins from each tissue (as judged by immunoblotting with SYN1351) were subjected to immunoblotting
with the isoform-specific antibodies (Fig. 3). The relative
tissue distribution of each syntrophin isoform is consistent
with previous Northern blot analysis (1, 5, 6). Thus, skeletal muscle contains the highest levels of Previously, we proposed that dystrophin and related
proteins might each be associated with a particular syntrophin isoform (1). This proposal was based largely on the
common expression patterns between dystrophin family
members and individual syntrophin isoforms. In vitro
binding studies have demonstrated, however, that all three
syntrophins are able to bind to a region in the COOH-terminal domain of dystrophin encoded by exon 74, and to the analogous regions in utrophin and dystrobrevin (6, 15), with no intrinsic binding specificity. However, the associations that occur in vivo might be regulated by additional
factors. To examine this possibility, we have used a combination of immunofluorescence microscopy and biochemical analysis to determine which syntrophin isoforms are
associated with dystrophin, utrophin, and dystrobrevin complexes.
Skeletal muscle was chosen for these studies for several
reasons. First, skeletal muscle expresses all three syntrophins, although We performed immunofluorescence microscopy on sections of adult mouse muscle with the syntrophin isoform-specific antibodies. As reported previously (38, 55), labeling for
We have previously shown that Multiple Syntrophin Isoforms in Dystrophin and
Utrophin Complexes
Previous biochemical studies showed that purified dystrophin complexes contain a triplet of syntrophin bands (55).
However, the isoform identity of these syntrophin bands
was not determined. Likewise, the syntrophin isoforms
associated with utrophin in skeletal muscle have not been
established. To determine which syntrophin isoforms are
present in dystrophin and utrophin complexes, we partially purified dystrophin and utrophin from skeletal
muscle extracts using immunoaffinity purification. When
isolated in this way, dystrophin preparations contain no
detectable utrophin, and utrophin preparations are free of
detectable dystrophin (Fig. 6 A). Given the relative paucity of NMJ membrane (the site of highest concentration
of utrophin) in skeletal muscle fibers, it is likely that much
of the utrophin complex originated from nerve and blood
vessels. In addition to the full-length utrophin (predicted
to be 395 kD), a smaller utrophin-immunoreactive protein was identified. This ~140-kD protein appears to be larger
than G-utrophin and thus may either result from proteolysis of full-length utrophin or represent a previously undescribed utrophin homologue of Dp140. Aliquots of dystrophin and utrophin complexes containing approximately
equal amounts of total syntrophin were analyzed by immunoblotting, thereby allowing comparison of the relative amounts of individual syntrophin isoforms. As shown in
Fig. 6 A, dystrophin complexes were highly enriched in
To corroborate these results, syntrophin isoform complexes were isolated by immunopurification. Samples containing approximately equal amounts of total syntrophin
were then tested for relative levels of dystrophin and utrophin. As shown in Fig. 6 B, Syntrophin Distribution in mdx Skeletal Muscle
Previous studies with the pan-specific syntrophin mAb
SYN1351 showed that the loss of dystrophin from the sarcolemma causes a dramatic decrease of syntrophin staining (11). This decrease occurred despite the fact that total
syntrophin, as measured by immunoblot analysis, was essentially unchanged in mdx muscle. To determine which
isoforms are affected, we compared syntrophin isoform
staining in normal and mdx gastrocnemius muscle. In wild-type mice, fibers of the gastrocnemius muscle, which are
predominantly fast fiber type, have high levels of
At some sites in mdx muscle, intense syntrophin staining
was retained, and in each case, its distribution paralleled
that of utrophin. As previously shown (38, 55), labeling for
In nonsynaptic regions, utrophin labeling was frequently
seen on small caliber myofibers with centrally located nuclei,
the hallmark of regenerated fibers (Fig. 7 B). In adjacent
serial sections, staining for Two Syntrophin-binding Sites in the
Dystrophin Complex
Results from other laboratories suggest that the syntrophin content in purified dystrophin complexes is approximately twice that of other dystrophin-associated proteins
(16, 56). While only a single syntrophin-binding region has
been clearly identified on dystrophin, a second syntrophin-binding protein, dystrobrevin, is known to be associated
with the dystrophin complex in skeletal muscle (50, 51).
Originally identified as a Torpedo phosphoprotein of 87 kD (51), dystrobrevin is homologous to the COOH-terminal region of dystrophin and contains a syntrophin-binding
site (4, 15). This site is followed by two tandem heptad repeats of leucines predicted to form coiled-coils (7). Ozawa
and colleagues have shown that dystrophin binds in vitro
to a protein called A0, and they have mapped its binding
site to the first heptad repeat of dystrophin (50). Subsequently, A0 was shown to be equivalent to dystrobrevin
(58). A weaker binding site was found in the region of dystrophin that contains the second heptad repeat (50). Thus,
we propose a model in which dystrophin and dystrobrevin associate via coiled-coil interactions (7) and could thus recruit two syntrophins to the dystrophin complex.
In complexes that contain two syntrophin-binding proteins (dystrophin and dystrobrevin), it is difficult to determine the type of syntrophin associated with either protein
independently. In this regard, the
As an additional test of association of We did note one important difference in the dystrobrevin complexes isolated with Abs DB308 and DB433. The
dystrophin content in Ab DB433 preparations was dramatically reduced in comparison with the amounts obtained when Ab DB308 was used (Fig. 8 C, lanes DB433).
It appears that Ab DB433, which recognizes the linker sequence between the coiled-coils of dystrobrevin, disrupts
the interaction between dystrophin and dystrobrevin. Alternatively, it is possible that the epitope for Ab DB433 is
more accessible in dystrobrevins that are not bound to
dystrophin. Both of these possibilities are consistent with
the idea that interaction between dystrobrevin and dystrophin is mediated by their coiled-coil regions.
The syntrophins are a multigene family of modular
adapter proteins thought to recruit signaling proteins to
the membrane via association with dystrophin and other
members of the dystrophin family. The existence of three
isoforms derived from distinct genes makes the syntrophins unique among dystrophin-associated proteins. In
skeletal muscle, we find that the syntrophins have distinct
but overlapping distributions. These differential localizations imply that each syntrophin has a unique function,
probably derived in part from association with either dystrophin or utrophin, in combination with dystrobrevin.
Our results suggest that pairs of syntrophin isoforms associate with dystrophin and utrophin complexes. Previous
stoichiometric analyses of purified dystrophin complexes
is in good agreement with two syntrophins per complex
(16, 56). Several features of the dystrophin complex could
account for the presence of two syntrophins. In addition to
the known syntrophin-binding site in dystrophin encoded
by the first half of exon 74 (4, 50), a second syntrophin-binding site has been proposed. A peptide corresponding
to the latter half of 74 and exon 75 of dystrophin binds an
~60-kD DAP (50). Although this protein was thought to
be The model we favor incorporates a previous proposal (7)
that dystrophin and dystrobrevin interact via their coiled-coils. Considerable evidence, including new results that we
present here, supports an association of dystrophin with dystrobrevin. Dystrophin and dystrobrevin colocalize in skeletal
muscle (8, 12), copurify biochemically (51), and associate
directly in vitro via the coiled-coil region of dystrophin
(50). We now find that dystrobrevin complexes isolated
with an antibody to the coiled-coil region of dystrobrevin
contain only small amounts of dystrophin when compared
to complexes isolated with an antibody directed to another site. Thus, antibody binding to the coiled-coil region is incompatible with dystrophin-dystrobrevin association. Finally, purified dystrophin complexes can be partially dissociated into three groups, a dystroglycan subcomplex, a
sarcoglycan subcomplex, and a dystrophin-dystrobrevin- syntrophin subcomplex by treatment with n-octyl
In skeletal muscle, which expresses all three syntrophin
isoforms, particular pairs of syntrophins preferentially associate with dystrophin or utrophin complexes. These
preferential associations are not likely to result from intrinsic selectivity of either dystrophin or utrophin for particular syntrophins. Previous studies have shown that dystrophin, utrophin, and dystrobrevin are each able to bind
any of the three syntrophin isoforms in vitro (5, 6). From
our studies of native complexes from control, mdx, and
mdx transgenic mice, we conclude that these associations
are selective but not absolutely exclusive. Nevertheless,
the pairings of syntrophin isoforms with dystrophin family
members appear to be more highly regulated than suggested by in vitro studies. Potential mechanisms that could
account for this selectivity include cell-specific expression of
particular syntrophin isoforms and posttranslational modifications, such as phosphorylation, that alter the binding affinity of syntrophins for dystrophin and related proteins.
Since the pairing of syntrophin isoforms is thus unlikely
to be determined by intrinsic selectivity for dystrophin
family members, several possible pairings within an individual dystrophin-dystrobrevin complex can be envisioned.
For example, the observed pairing of Syntrophins in Duchenne Muscular Dystrophy
A role for the syntrophins in muscular dystrophies or
other myopathies has not received much attention, despite
the fact that sarcolemmal expression of syntrophins, like
that of the other proteins of the dystrophin complex, is
dramatically reduced in Duchenne muscular dystrophy
(DMD). Defects in other proteins of the dystrophin complex are the primary causes of other muscular dystrophies
in which dystrophin is normal. For example, mutations in
the genes encoding the sarcoglycans have been implicated
in severe childhood autosomal recessive muscular dystrophy and in several forms of limb-girdle muscular dystrophy (for review see reference 37). Although no myopathies or other diseases have been linked to primary defects
in syntrophins, our finding that Identifying other proteins associated with The physiological role of nNOS in normal skeletal muscle remains unknown, although some results suggest that it
promotes muscle relaxation through a cGMP signaling
pathway (26). The mechanism by which nNOS is activated
in skeletal muscle is also poorly understood, although it is
known that activation requires calcium/calmodulin and
nNOS homodimerization (25). Neither the source of activating calcium nor the requirements for dimerization has
been determined. Syntrophin association may play a role
in both aspects of nNOS regulation. For example, close
pairing of two syntrophins (discussed below) may facilitate
nNOS dimerization, while syntrophin-mediated membrane
localization may target nNOS to an appropriate Ca2+
source. Furthermore, loss of the syntrophin-nNOS complex from the membrane in dystrophic muscle may disrupt
the nNOS-mediated relaxation pathway in favor of inappropriate cytoplasmic nNOS activation. This combination
of loss of a potentially protective activity and acquisition
of a potentially cytotoxic function may be central to DMD
pathogenesis.
Syntrophins at the Neuromuscular Synapse
All three syntrophins are concentrated at the NMJ, but
only Paired Syntrophins: Implications for Function
The discovery of two syntrophin isoforms in dystrophin/
utrophin complexes has clear implications for the mechanism of syntrophin function within membrane specializations. Specifically, the pairing of two PDZ-containing
proteins in a submembrane complex resembles the membrane-associated guanylate kinase (MAGUK) complex. Like the syntrophins, the MAGUKs are a multigene family of
PDZ-containing proteins that form submembranous protein scaffolds. The best characterized MAGUKs are the
neuronal forms: PSD-95, Chapsyn-110, SAP97, and SAP102,
which when expressed in COS cells combine to form homotypic and heterotypic multimers (24). MAGUKs have
recently been shown to cluster membrane proteins such as
K+ channels and NMDA-type glutamate receptors (for
review see reference 48). The PDZ domains of certain
MAGUKs mediate binding to the extreme COOH terminus of the cytoplasmic tail of membrane proteins (14, 49).
Because this binding is dependent on the sequence of the
COOH-terminal tail of the membrane protein, different
MAGUK isoforms appear to bind different sets of membrane proteins. Thus, the combination of MAGUK isoforms at a particular membrane site may determine the
composition of membrane proteins clustered there.
In contrast to the MAGUKs, which have three PDZ domains, syntrophins have only a single PDZ motif. This
would appear to limit the ability of syntrophins to act as
adapters in membrane organization by binding simultaneously multiple types of membrane proteins (such as ion
channels) or a single membrane protein and an effector enzyme (such as nNOS). However, an attractive feature of
the model in which two syntrophins are present in a single
dystrophin complex is that each simultaneously could bind
different proteins. Furthermore, a single complex might
bind different combinations of proteins, depending on the
specificity of the syntrophin PDZ domains. Thus, by regulating the syntrophin isoforms associated with dystrophin and dystrobrevin, or utrophin and dystrobrevin, the associated membrane or signaling protein might be different.
The models shown in Fig. 9 show hypothetical combinations that might confer distinct functions on utrophin and
dystrophin complexes, depending on the binding partners
for the syntrophin PDZ domains.
Further studies will be required to determine if syntrophins play a role similar to that of the MAGUKs in membrane organization. Identifying the syntrophin PDZ domain binding partners and understanding the mechanisms
that regulate the combinations of syntrophin isoforms in
dystrophin complexes will be important for understanding
the role of syntrophins in muscular dystrophy and in synapse formation.
1,
1, and
2, are encoded by different genes but have similar domain organizations. All known syntrophins contain two pleckstrin homology (PH) domains (2, 19), which are modules of ~100
amino acids found in a wide array of signaling proteins.
PH domains in other proteins bind phosphatidylinositol lipids and proteins, such as the
-subunits of trimeric G
proteins (for review see reference 47). Thus, PH domains
may mediate signal-dependent membrane association. Inserted within the first syntrophin PH domain is a PDZ domain (originally identified in postsynaptic density-95, discs
large, ZO-1), a 90-amino acid domain found in more than
40 proteins, many of which are restricted to membrane
specializations such as tight junctions or synapses (48). A
trend emerging from study of other PDZ-containing proteins suggests that PDZ domains bind the cytoplasmic carboxy-terminal tails of transmembrane proteins (examples
of which include NMDA receptors, K+ channels, Fas [42],
and EGF receptors (for review see reference 48]). Finally,
the COOH-terminal 57 amino acids of syntrophins are
highly conserved among the three isoforms but are otherwise unique. This region, termed the syntrophin-unique
(SU) domain, may contain the binding site for dystrophin
family members (2, 6). Thus, the syntrophins are a family
of multidomain proteins that likely function as modular
adapters in recruiting signaling proteins to dystrophin
complexes and the membrane.
1-syntrophin is localized on the sarcolemma with dystrophin,
whereas
2-syntrophin is restricted to the NMJ, similar to
utrophin (38). In contrast, the transmembrane dystroglycans are expressed in many tissues and are the products of
a single gene (for review see reference 21). The sarcoglycans are comprised of multiple forms, but these are
highly restricted to muscle (35, 53). In addition, most sarcoglycans are unrelated in amino acid sequence (32, 35), suggesting that they have distinct functions. Thus, among
the known DAPs, syntrophins are leading candidates for
associating with different dystrophin family members and
could be involved in targeting of dystrophin family members to distinct membrane sites, or in conferring different
functions to DAP complexes.
1- and
2-syntrophins (1) and examined the corresponding protein localization (38). However,
these studies did not consider the recently described mammalian dystrobrevins (8, 41) or
1-syntrophin (5). We began this study by isolating the mouse
1-syntrophin cDNA and generating and characterizing isoform-specific antibodies. These isoform-specific antibodies were then used
to define the distribution of each syntrophin and the association of syntrophins with dystrophin, utrophin, and dystrobrevin isoforms from normal, dystrophin-deficient, and
71-74 dystrophin transgenic mouse skeletal muscle. We
find that pairs of syntrophin isoforms selectively copurify
with dystrophin and utrophin. Based on these results, we
propose a model in which particular pairs of syntrophin
isoforms associate with dystrophin/dystrobrevin or utrophin/dystrobrevin complexes.
Materials and Methods
1-Syntrophin cDNA Isolation
gt11 mouse liver cDNA library (Clontech Labs, Palo Alto, CA) was
screened by hybridization with 32P-labeled human
1-syntrophin cDNA
(a generous gift of Dr. Louis Kunkel, Howard Hughes Medical Institute,
Boston, MA) by methods described previously (1). Five clones were isolated, but none of them contained the extreme 5
coding region. Therefore, this sequence was obtained by two consecutive rounds of 5
rapid
amplification of cDNA ends using a kit purchased from GIBCO BRL
(Gaithersburg, MD) and RNA isolated from C57Bl6 mouse liver (13). PCR
products were amplified with Vent polymerase (NEB) using the sequence-specific primers 5
-CCTAATCTTGGAGACTCAGGTGG (round 1) and
5
-TCCCGCAGGTCTGCTCCGTTC (round 2). Resulting DNA from
each round was cloned into Bluescript II (Stratagene, La Jolla, CA), and
multiple clones were sequenced by the University of North Carolina Automated DNA Sequencing Facility (Chapel Hill, NC) on a DNA sequencer (model 373A; Applied Biosystems, Inc., Foster City, CA). Sequence was analyzed with the aid of a DNAStar Lasergene computer
software package (Madison, WI).
1-syntrophin, plus an NH2-terminal cysteine (see boxed region in Fig. 1 B). The
2-syntrophin antibody, SYN28,
was prepared against C-SGSEDSGSPKHQNTTKDR as an alternative to
the weaker Ab SYN24 previously prepared against the same peptide (38).
The
1-syntrophin antibody, SYN17, was described previously (38). Each
antipeptide antibody used in this study was affinity purified with peptide
coupled to Affi-Gel-10 or -15 (BioRad Labs, Hercules, CA) as described
previously (28) and was used directly or after biotinylation with sulfo-NHS-biotin (Pierce, Rockford, IL) according to the manufacturer's protocol.
Fig. 1.
Cloning, sequence, and domain structure of murine
1-syntrophin sequence. (A) Strategy for cloning mouse
1-syntrophin and structure of the combined cDNAs, showing the coding region bounded by start and stop codons. (B) The deduced
amino acid sequence of mouse
1-syntrophin is aligned with
mouse
1- and
2-syntrophins (1, 2). Identical amino acids are
shaded. The boundaries of PH (6, 19), PDZ (2), and SU (2) domains are indicated by arrows. The boxed region denotes sequences used to generate synthetic peptides for production of isoform-specific antibodies. (C) Schematic diagram showing the
relative organization of PH, PDZ, and SU domains in syntrophins.
Mouse
1-syntrophin cDNA sequence data are available from
GenBank/EMBL/DBJ under the accession number U89997.
[View Larger Version of this Image (60K GIF file)]
71-74/mdx mice (J.S. Chamberlain, University of Michigan, Ann Arbor, MI) (39) were dissected and frozen in liquid nitrogen. Protein complexes were partially purified as described previously (28). Briefly, tissues (5 g) were homogenized in 10 vol
(wt/vol) of ice-cold homogenization buffer (HB; 10 mM Na phosphate, 0.4 M
NaCl, 5 mM EDTA, pH 7.8, plus the protease inhibitors aprotinin, leupeptin, and antipain at 0.5 µg/ml each, pepstatin A, 0.05 µg/ml, and 2 mM
PMSF) (28). The particulate fraction was pelleted (10 min at 12,000 g,
model JA-20 rotor; Beckman Instruments, Fullerton, CA), resuspended in
HB (10 vol), and recentrifuged. Washed pellets were solubilized in 1%
Triton X-100/HB (3 vol) and incubated on ice for 15 min. For preparation
of individual syntrophin isoforms (see Fig. 2), the particulate fractions
were dissolved in 1% SDS/HB (3 vol) to disrupt possible complexes of
multiple syntrophins. After incubation for 15 min, excess 1% Triton/HB
(30 vol) was added. In all cases, the detergent-solubilized extracts were
clarified by centrifugation (20 min at 53,000 g, model Ti 60 rotor; Beckman Instruments, Fullerton, CA) and incubated with antibody resins prepared by coupling 2 mg of affinity-purified antibody to 2 ml of Affi-gel 10 (BioRad Labs) according to manufacturer's specifications. After agitation for 2 h at 4°C, resins were collected in a column and washed sequentially with 1% Triton/HB (50 ml), 0.1% Triton/HB (10 ml), and HB (5 ml). Bound proteins were eluted with 0.1 M triethylamine, pH 11.5, precipitated with TCA, washed with 95% ethanol, and resuspended in SDS-PAGE sample buffer.
Fig. 2.
Isoform specificity
of syntrophin antibodies. Antibodies were characterized
by immunoblotting samples
enriched in individual syntrophin isoforms (as indicated
above lanes). 1-,
1-, and
2-syntrophins were solubilized from crude membrane
preparations from skeletal
muscle, liver, and kidney, respectively, in 1% SDS to disrupt potential multi-syntrophin complexes. After
addition of excess Triton,
syntrophins were immunoaffinity purified with the appropriate antibody (Abs
SYN17, SYN37, and SYN28).
Blots were probed with mAb
SYN1351 or biotinylated
polyclonal antibodies as indicated. Labeling with each
polyclonal antibody was
eliminated by preincubation
with the appropriate antigenic peptide (not shown). Positions of
two molecular mass markers (52 and 59 kD) are shown on the
right in the top panel.
[View Larger Version of this Image (41K GIF file)]
-mercaptoethanol, 62.5 mM Tris, pH 6.7, and reprobed as
above. Prestained standards (Sigma Chemical Co.) were used after calibration with unstained molecular weight markers (BioRad Labs).
-bungarotoxin (Tx)
conjugated to BODIPY-fluorescence (Molecular Probes, Eugene, OR;
1:300). Washed sections were fixed with 4% paraformaldehyde and
mounted in glycerol containing n-propyl gallate to reduce photobleaching (20). Antibody specificity was tested by preincubating antibodies with
their antigenic peptide (100 µM) for 30 min before labeling. Adjacent
sections were stained with Mayers haematoxylin-eosin (30). Sections
were viewed on a fluorescence microscope (model Axioskop; Carl Zeiss,
Inc., Thornwood, NY) and photographed (TMax 400 film; Kodak, Rochester, NY).
Results
1-syntrophin cDNA
1-syntrophin was
obtained by screening a mouse
gt11 cDNA library with the
human
1-syntrophin cDNA. Five clones were isolated
containing overlapping sequence that included >70% of
the protein coding sequence and 820 base pairs of 3
untranslated region. The remaining 5
coding sequence was
obtained by rapid amplification of cDNA ends (see Materials and Methods). The resulting cDNA (Fig. 1 A) contains 354 nucleotides 5
of a 1,611-nucleotide open reading
frame. The first methionine codon is in a context favoring
translation initiation (27) and aligns with that described
for the human cDNA (5). The deduced protein contains
537 amino acids and has a predicted mass of 58,088 D and
a pI of 8.3. Its amino acid sequence is 90% identical to human
1-syntrophin while sharing only 48 and 55% identity
with human
1- and
2-syntrophins, respectively, indicating that it is the mouse ortholog of human
1-syntrophin
(5). After the initial methionine, the amino termini of both
mouse and human
1-syntrophin have a stretch of nine
hydrophobic amino acids (alanine and valine). A similar
sequence is present in the human
2-syntrophin (6) sequence but is absent in mouse
2-syntrophin (2) and the
1-syntrophins (1, 6, 54). Comparison of the amino acid
sequences of the three mouse syntrophins shows marked
conservation, particularly within the PDZ and SU domains (Fig. 1 B).
1-,
1-, or
2-syntrophin (skeletal
muscle, liver, and kidney, respectively). To ensure that each
preparation contained only a single syntrophin, samples
were first treated with SDS to disrupt possible multimeric
complexes and were then diluted with Triton X-100 before
immunopurification. As expected from previous results
(38), immunoblotting with the anti-
1-syntrophin antibody, SYN17, labeled a single ~60-kD protein in the
1-syntrophin preparation but did not recognize proteins in
either the
1- or
2-syntrophin preparations (Fig. 2). Likewise, the anti-
1-syntrophin antibody, SYN37, strongly labeled proteins in the
1-syntrophin preparation without
recognizing proteins in either the
1- or
2-syntrophin
preparations. Finally, the
2-syntrophin antibodies, SYN24
and SYN28, recognized only
2-syntrophin. In contrast,
mAb SYN1351, which recognizes an epitope in the PDZ
domain (M.E. Adams, S.H. Gee, and S.C. Froehner, unpublished results), strongly labeled each syntrophin isoform (Fig. 2). Thus, we conclude that mAb SYN1351 is
pan specific, while each of the syntrophin peptide antibodies is isoform-specific in immunoblotting and in immunoaffinity purification.
1- and anti-
2-syntrophin antibodies (Fig. 2).
These proteins were also recognized by mAb SYN1351,
thus confirming their identities as syntrophins. The tissue
distribution patterns for these smaller syntrophin-related
proteins are distinct from each other and from the corresponding full-length isoforms (Fig. 3). Although it is possible that the smaller proteins are proteolytic fragments, a
more likely possibility is that they are generated by posttranslational modification or by alternative splicing of the
1- and
2-syntrophin mRNAs. Northern blot analysis
identified multiple transcripts for
1- and
2-syntrophins,
while only a single transcript for
1-syntrophin was found
(1, 5, 6). Although the basis of these multiple transcripts is
unknown, it is certainly possible that they encode modified
forms of
1- and
2-syntrophins.
Fig. 3.
Tissues express multiple syntrophin isoforms. Syntrophins were isolated from Triton X-100-solubilized tissue extracts
with the pan-specific syntrophin antibody SYN1351. Sample
loadings were adjusted to contain approximately equal amounts
of total syntrophin, as judged by immunoblotting with SYN1351
(pan anti-syn). Individual syntrophin isoforms were identified by
blotting with Abs SYN17 (anti-1 syn), SYN37 (anti-
1 syn), or
SYN24 (anti-
2 syn). Positions of two molecular mass markers
(52 and 59 kD) are shown on the right in the top panel.
[View Larger Version of this Image (63K GIF file)]
1-syntrophin, while liver expresses the highest levels of
1-syntrophin,
and testis expresses predominantly
2-syntrophin. Almost
every tissue expresses two or three syntrophin isoforms,
but there did not appear to be any bias for particular pairs
of syntrophins to be expressed together.
1-syntrophin is the predominant isoform
(see Fig. 3 and results in references 1, 5, 6). Since the distributions of dystrophin, utrophin, and dystrobrevin are
known in this tissue, a comparison of their localizations
with the syntrophin isoforms can be used to corroborate
the biochemical studies on isolated complexes. Furthermore, previous studies have shown that two syntrophins,
1 and
2, have distinct distributions in muscle, an observation that supports the idea of differential association of
syntrophins and dystrophin family members (38). Finally,
Duchenne muscular dystrophy has its most profound effects on skeletal muscle. Since the absence of dystrophin
results in a loss of dystrophin-associated proteins, including syntrophins, understanding syntrophin's interactions in
this tissue may be especially important in deciphering the molecular causes of this disease.
1-syntrophin was strong on sarcolemma with
particular enrichment at NMJs (Fig. 4). A similar labeling
pattern was identified for
1-syntrophin, although the staining intensity of individual fibers varied. Slow-twitch fibers
(type I) displayed little or no
1-syntrophin labeling, while
a subset of fast-twitch fibers (type II) showed intense labeling (Fig. 5 A). Among fast fibers, the most glycolytic
ones (type IIB) showed stronger labeling than oxidative fibers (type IIA and D) (Fig. 5 B). Although this was the
typical staining pattern, exceptions could be found. For example, in predominantly fast muscles, staining for
1-syntrophin in slow fibers was weak but clearly detectable (not
shown). In contrast, differential staining of fiber types was
not seen for
1-syntrophin (Fig. 5 A) or dystrophin (22).
Fig. 4.
Localization of syntrophin isoforms in skeletal muscle. Regions of mouse gastrocnemius muscle containing NMJs were identified by -bungarotoxin (
-BgTx). Distributions of
1-,
1-, and
2-syntrophins were determined with Abs SYN17, SYN37, and SYN28,
respectively. In each case, the specificity of immunolabeling was confirmed in adjacent serial sections by preincubating antibodies with
the appropriate antigenic peptide. Bar, 50 µm.
[View Larger Version of this Image (77K GIF file)]
Fig. 5.
Fiber-type specificity of 1-syntrophin. (A)
Syntrophins were examined
in sections of mouse hind
limb muscle containing the mixed slow/fast-twitch plantaris (left of arrows) with the
adjacent fast-twitch gastrocnemius (right of arrows). Labeling for
1- and
1-syntrophins (SYN17 and SYN37) is
compared to that of fast fiber
(type II) MHC staining
(mAb NCL-MHCf). Note
that
1-syntrophin labeling is
highly restricted to a subset
of type II myofibers, while
1-syntrophin labeling shows
uniform fiber-type staining. (B)
1-syntrophin labeling
was examined in subtypes of
fast fibers identified by mAb
BF-F3 specific for type IIB
MHC (IIB) and mAb A4.74
specific for IIA, and to a
lesser extent type IID MHC
(IIA, D).
1 and IIA, D show
a single cryosection double
labeled as indicated, while
IIB shows an adjacent serial
section. Note the correspondence between strong labeling for
1-syntrophin (
1) and that of type IIB myosin
(IIB). Bars: (A) 50 µm; (B)
80 µm.
[View Larger Version of this Image (141K GIF file)]
2-syntrophin is concentrated at NMJs (38). This labeling pattern was established
on rat skeletal muscle with Ab SYN24, which reacts only
weakly with mouse
2-syntrophin. We have now prepared
an additional antibody, SYN28, which gives much stronger
labeling of
2-syntrophin at the neuromuscular junction in
mouse and rat skeletal muscle. In both rat and mouse,
faint but specific labeling for
2-syntrophin was occasionally detected on the sarcolemma with Ab SYN28 (Fig. 4).
Thus, although it is likely that small amounts of this isoform are also present on the sarcolemma, these results are
in general agreement with our original finding that
2-syntrophin is concentrated at the NMJ. Thus, each of the
three syntrophins exhibits a differential distribution in
skeletal muscle, either within a single muscle fiber or
across muscle fiber types.
1-
and
1-syntrophins, while utrophin complexes contained
mostly
1- and
2-syntrophins.
Fig. 6.
Dystrophin and utrophin complexes contain distinct pairs
of syntrophin isoforms. (A) Dystrophin and utrophin complexes
were immunoaffinity purified from Triton-solubilized extracts of
mouse skeletal muscle with antibodies DYS3669 and UTR3165,
respectively. Sample loadings were adjusted to contain approximately equal amounts of syntrophin, as judged by immunoblotting (pan-Syn, mAb SYN1351). Duplicate blots were probed with
mAbs Mandys-8 (Dys), and DRP-1 (Utr) or biotinylated polyclonal antibodies SYN17 (1-syn), SYN37 (
1-syn), and SYN28
(
2-syn). (B) Syntrophins were immunoaffinity purified from
skeletal muscle extracts with Abs SYN17, SYN37, and SYN28.
Sample loadings were adjusted to contain similar amounts of total syntrophin, as judged by immunoblotting (pan-Syn, mAb SYN1351). A duplicate blot was probed with mAb Mandys-8
(Dys), stripped, and reprobed with mAb DRP-1 (Utr). Positions
of molecular mass markers (in kD) are shown in some panels.
These results were replicated twice, and representative blots from a
single experiment are shown.
[View Larger Version of this Image (58K GIF file)]
1-syntrophin preparations
were particularly enriched in dystrophin, while
1- and
2-syntrophin preparations contained smaller amounts of
dystrophin. The same blot was stripped and reprobed for
utrophin. Highest levels of utrophin were found in the
2-syntrophin preparations, while lower levels were detected
in the
1-syntrophin preparations (Fig. 6 B). Only with
much longer exposures was utrophin detected in
1-syntrophin complexes. Together, these results indicate that, in
skeletal muscle, dystrophin associates preferentially with
1- and
1-syntrophin. In contrast, utrophin complexes
contain predominantly
1- and
2-syntrophin.
1- and
1-syntrophins on the sarcolemma. However, in mdx muscle, the absence of dystrophin results in a dramatic reduction of both
1- and
1-syntrophin sarcolemmal staining
(compare Fig. 7, A and B, to Fig. 4). This result provides
further support for the association of
1- and
1-syntrophins with dystrophin.
Fig. 7.
Localization of
syntrophins in mdx mouse
skeletal muscle. (A) The distribution of syntrophin isoforms was examined in regions containing NMJs
(identified with -bungarotoxin; Tx). Labeling in the
perisynaptic regions with antibodies SYN17 (
1), SYN37 (
1), SYN28 (
2), and
UTR3165 (Utr) was characterized. (B) In 8-wk-old mdx
mice, regenerating fibers
with central nuclei were identified by haematoxylin-eosin (H&E) staining. Serial
sections were labeled with
antibodies UTR3165 (Utr),
DYS3669 (Dys), SYN17 (
1), SYN37 (
1), and
SYN28 (
2). Bars: (A;
1,
1, and Utr) 50 µm; (A; B2)
30 µm; (B) 50 µm.
[View Larger Version of this Image (128K GIF file)]
1-syntrophin in mdx muscle was particularly strong at the
NMJ, a site of high utrophin concentration (Fig. 7 A). Staining for
1- and
2-syntrophin was also retained at the
NMJ (Fig. 7 A). Utrophin labeling frequently extended beyond its normal highly restricted distribution on the postsynaptic membrane, spilling over onto the perisynaptic sarcolemma and diminishing in intensity with distance from
the NMJ (Fig. 7 A).
1- and
1-syntrophins, and to a lesser
extent
2-syntrophin, were also found perisynaptically in
mdx muscle (Fig. 7 A).
1-syntrophin and especially
1-syntrophin mirrored that of utrophin.
2-syntrophin
was not detectable under standard labeling conditions.
These fibers are not revertants since antidystrophin labeling was negative (Fig. 7 B). Thus, although syntrophin immunoreactivity is dramatically reduced on the sarcolemma of mdx skeletal muscle, staining for at least two isoforms is retained in regions that express utrophin. When considered with the results of immunoaffinity purification, these
data suggest that utrophin is capable of associating with
each syntrophin isoform.
71-74 transgenic mdx
mouse is particularly useful since it expresses a dystrophin
transgene in skeletal muscle that lacks exons 71-74, which
includes the syntrophin-binding site (for review see reference 18). In mdx muscle,
71-74 dystrophin produced by
the transgene is targeted to the sarcolemma, such that normal levels of membrane-bound dystrophin-associated proteins are restored (39, 40). Surprisingly, the
71-74 dystrophin transgene restores syntrophin immunoreactivity to
the sarcolemma, despite lacking the syntrophin-binding
site (39, 40). Since the
71-74 dystrophin transgene product retains most of the coiled-coil region encoded by exon
75, it might still associate with dystrobrevin, which could in
turn bind syntrophin and target it to the membrane. Indeed, we find dystrobrevin localized on the sarcolemma
along with both
1- and
1-syntrophins (Fig. 8 B). Thus,
1- and
1-syntrophins may be restored to the membrane
in these transgenic mice via association with dystrobrevin
bound to dystrophin.
Fig. 8.
Dystrobrevin associates with 1- and
1-syntrophins in
71-74/mdx skeletal
muscle. (A) Schematic comparing the structure of the cysteine-rich COOH-terminal region of normal dystrophin, of the dystrophin transgene lacking exons 71-74, and the corresponding structure of dystrobrevin. The locations of the
sequences used for preparation of dystrobrevin antipeptide antibodies DB308 and DB433 are indicated by bars.
H1, H2, and WW, position of
helix 1 and helix 2 and the WW
domain, respectively. (B) Immunofluorescence labeling for
1-syntrophin (
1, Ab
SYN17),
1-syntrophin (
1, Ab SYN37), and dystrobrevin
labeling (Db, mAb 13H1), with
the corresponding labeling with
-bungarotoxin (Tx) shown to
the right of each image. The labeling for
2-syntrophin (
2,
Ab SYN28) and
-bungarotoxin (Tx) was reduced in size
and intensity compared to control mice. (C) Dystrobrevin
complexes were immunoaffinity purified from Triton extracts of
71-74/mdx skeletal
muscle with antipeptide dystrobrevin antibodies that recognize either a central region (Ab
DB308, left lane) or the linker
between the two coiled-coils (Ab DB433, right lane). Sample
loadings were adjusted for approximately equal amounts of
dystrobrevin immunoreactivity
in each lane (Db, mAb 13H1).
Complexes purified with Ab
DB433 contained dramatically lower amounts of dystrophin,
as expected if this antibody inhibits a coiled-coil interaction
between dystrophin and dystrobrevin; see text (Dys,
Mandys-8). The total amount
of syntrophin (pan-Syn, mAb
SYN1351) purified with dystrobrevins was approximately
the same with either Db antibody. Both
1-syntrophin (
1-syn, Ab SYN17) and
1-syntrophin (
1-syn, Ab SYN37) copurified with dystrobrevin, independent of the presence of dystrophin. Utrophin and
2-syntrophin were detectable only with much longer exposure
times.
[View Larger Versions of these Images (16 + 88K GIF file)]
1- and
1-syntrophins with dystrobrevin, we immunoaffinity purified
dystrobrevin from
71-74 transgenic mdx mouse skeletal
muscle. Using an antibody (DB308) directed against a central region of dystrobrevin, we purified complexes and
found that both
1- and
1-syntrophins and
71-74 dystrophin copurified with dystrobrevin (Fig. 8 C, lanes DB308).
Complexes isolated with a second dystrobrevin antibody, Ab DB433 made against the short linker region between
dystrobrevin's coiled-coils (see Fig. 8 A), also contained
1- and
1-syntrophins. These results suggest that dystrobrevin can associate with either syntrophin isoform in vivo.
Discussion
1-syntrophin, it may instead be the 60-kD form of dystrobrevin. Indeed, the peptide also bound a larger protein
(A0) (50), which has since been identified as full-length
dystrobrevin (58). Thus, the putative second syntrophin-binding site, which encompasses the first coiled-coil of dystrophin, may instead be a site of interaction between dystrophin and dystrobrevin. A second possibility is that two
syntrophins in a dystrophin complex could result from syntrophin dimerization. Syntrophins have been shown to
bind an ~60-kD DAP, leading to the suggestion that syntrophins form dimers (55). However, this binding may also
represent syntrophin association with the ~60-kD dystrobrevin. In fact, syntrophin also bound a larger protein of
the approximate molecular weight of full-length dystrobrevin (55). Finally, multiple syntrophins may also be recruited to dystrophin complexes by association with other
DAPs (31).
-d-glucoside (57). Detailed characterization of this association
will be needed to determine the precise stoichiometry and
binding orientation of the coiled-coil interaction. Despite
this reservation, the evidence for an interaction between
dystrophin and dystrobrevin is quite strong and leads us to
suggest a new model for the dystrophin COOH-terminal
region in which dystrophin and dystrobrevin combine to
recruit two syntrophins per complex (Fig. 9 A).
Fig. 9.
(A) Hypothetical model of the dystrophin complex
containing two syntrophin-binding proteins. Syntrophins bind the
exon 74-encoded region of dystrophin and the homologous region of dystrobrevin (6, 15). In both proteins, the syntrophin-binding site is followed by two heptad repeats of leucines (H1
and H2), separated by a short linker region (7). (B) Hypothetical
examples of syntrophin isoform combinations as adapters linking
membrane proteins or effector enzymes to dystrophin and utrophin. In fast-twitch myofibers, 1- and/or
1-syntrophin bind
nNOS (10). In slow-twitch myofibers,
1-syntrophin is the predominant isoform present on the sarcolemma. Since slow-twitch
fibers lack nNOS, the
1-syntrophin binding partners are unknown. Utrophin complexes contain
1- and
2-syntrophins. The
binding partners for both syntrophins may be of particular relevance to NMJ function. DGC, dystroglycan complex; SGC, sarcoglycan complex.
[View Larger Version of this Image (19K GIF file)]
1- and
1-syntrophins with dystrophin could represent either
1/
1 heterodimers or similar amounts of
1/
1 and
1/
1 homodimers with dystrophin. Indeed, these two possibilities are
not mutually exclusive and may both occur in a single
membrane region or may be differentially regulated at particular membrane regions. Distinguishing between these
two possibilities in native complexes may prove technically difficult. Perhaps identification of the mechanisms that determine the syntrophin pairs may help to address
these possibilities.
1-syntrophin is enriched
in fast-twitch type IIB muscle fibers raises new issues with
regard to the importance of syntrophins in DMD. Normally, dystrophin is expressed in all myofiber types (23),
yet DMD preferentially affects human fast type IIB fibers (52). Although the mechanisms underlying this selective
pathology remain to be determined, an intriguing possibility is that among the syntrophins, the function of
1-syntrophin is particularly important in maintaining sarcolemmal integrity.
1-syntrophin
may be particularly relevant to understanding the molecular origin of the DMD myopathy. One possibility is that
1-syntrophin mediates the targeting of the neuronal form
of nitric oxide synthase (nNOS) to the membrane. Like
1-syntrophin, nNOS is expressed preferentially in fast-twitch myofibers and is the only other protein known to correlate so closely with the fiber-type specific onset of
DMD (26, 52). In normal muscle, nNOS is found on the
sarcolemma in association with the dystrophin complex
(9). This association is mediated, at least in part, by PDZ-
PDZ heterodimerization between nNOS and syntrophin
(10). Thus far, this nNOS interaction has been studied
only in vitro with
1-syntrophin. It is possible that in vivo
1-syntrophin alone or paired with
1-syntrophin may be
essential for targeting of nNOS to the sarcolemma, and could thus explain the fiber-type differences seen for this
enzyme.
2-syntrophin is highly restricted to the synapse.
1-
and
1-syntrophins are found on the entire sarcolemma
with particularly high concentrations at synapses, a distribution similar to dystrophin. In contrast, the distribution
of
2-syntrophin more closely resembles that of utrophin.
Using immunoaffinity purification from whole skeletal muscle, we find that dystrophin preparations are enriched
in
1- and
1-syntrophins, while utrophin complexes contain
1- and
2-syntrophin. Thus, while it is tempting to
speculate that these preferential syntrophin-dystrophin/
utrophin pairings hold within the postsynaptic apparatus,
two considerations make this conclusion premature. First,
we find that all three syntrophins remain concentrated at
the NMJ in mdx mice, possibly in association with utrophin. Second, the utrophin complexes isolated from skeletal muscle by biochemical means are not derived exclusively from the postsynaptic membrane, but come in large
part from nonmuscle cells. Thus, it remains to be seen
which syntrophin or combination of syntrophins colocalize
with utrophin at the AChR-rich crests of the postsynaptic folds, or with dystrophin in the troughs, the site of high sodium channel density. Future studies will address this issue
and the identification of synapse-specific binding partners
for
2-syntrophin since it is unique among all the DAPs in
its restriction to the postsynaptic apparatus.
Received for publication 8 April 1997 and in revised form 29 May 1997.
1. Abbreviations used in this paper: DAPs, dystrophin-associated proteins; DMD, Duchenne muscular dystrophy; DRP, dystrophin-related protein; HB, homogenization buffer; MAGUK, membrane-associated guanylate kinase; MHC, myosin heavy chain; NMJ, neuromuscular junction; nNOS, neuronal nitric oxide synthase; PDZ, protein domain originally identified in postsynaptic density-95, discs large, ZO-1; PH, pleckstrin homology; SU, syntrophin-unique.We thank our colleagues in the Froehner and Sealock laboratories for
helpful discussions and comments on the manuscript. We are indebted to
A.H. Ahn and L.M. Kunkel for providing human 1-syntrophin cDNA
and results before publication, to J.S. Chamberlain for supplying the
71-
74/mdx transgenic mice, to Katherine North for assistance with skeletal
muscle fiber-typing, to J.B. Cohen for providing mAB 13H1, to R. Sealock and N.R. Kramarcy for providing Ab DYS3669, and to Kirk McNaughton and Curtis Conner for extensive assistance with cryosectioning.
This work was supported by National Institutes of Health Grants NS33145 (to S.C. Froehner and R. Sealock) and NS14871 (to S.C. Froehner) and a Muscular Dystrophy Association (MDA) Grant (to S.C. Froehner). M.E. Adams was an MDA postdoctoral fellow.
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