(Received for publication, April 6, 1995; and in revised form, June 27, 1995)
From the
Aberrant expression of the dystrophin-associated protein complex
is thought to underlie the pathogenesis of Duchenne dystrophy, Becker
muscular dystrophy, and severe childhood autosomal recessive muscular
dystrophy. Recently, our laboratory identified an agrin receptor from Torpedo electric organ postsynaptic membranes. It is a
heteromer of 190- and 50-kDa subunits with similarity to two components
of the dystrophin-associated protein complex of - and
-dystroglycan. We now confirm the relationship between the Torpedo agrin receptor and mammalian dystroglycans and provide
further information about the structure of the
-dystroglycan-
-dystroglycan complex. The sequences of three
peptides from each Torpedo subunit were 69% identical to
mammalian dystroglycans. An antiserum to mammalian
-dystroglycan
recognizes the Torpedo 50-kDa polypeptide. Additionally, like
-dystroglycan, the 190-kDa agrin receptor subunit binds laminin.
Previous studies have indicated that
- and
-dystroglycan
arise by cleavage of a precursor protein. Tryptic peptide mapping of
both subunits and amino-terminal sequencing of Torpedo
-dystroglycan indicate a single cleavage site, corresponding
to serine 654 of the mammalian dystroglycan precursor. Gel
electrophoresis analysis indicates there is at least one intrachain
disulfide bond in
-dystroglycan. These results provide precise
primary structures for
- and
-dystroglycan.
Linkages between the extracellular matrix and the cytoskeleton
play key roles in forming and maintaining specialized membrane domains,
ensuring the structural integrity of the plasma membrane, attaching
cells to the extracellular matrix, and cell
signaling(1, 2) . In skeletal muscle, the
dystrophin-associated protein complex (DAPC) ()is likely to
be an essential arbiter of basal lamina cytoskeletal communication in
both synaptic and nonsynaptic
regions(3, 4, 5, 6) . Perturbations
of this membrane protein complex have been linked to the pathogenesis
of muscular dystrophies (MDs). In Duchenne and Becker MD, mutations in
dystrophin, a cytoskeletal protein, lead to decreased levels of the
DAPC complex in the plasma membrane(4, 7) . Some forms
of autosomal recessive MD are linked to mutations affecting the
expression of adhalin, a transmembrane constituent of the
DAPC(8, 9, 10) . Moreover, the basal lamina
protein merosin, a ligand of the DAPC(11) , is missing in many
cases of congenital MD where the genetic defect is
unknown(12, 13) . Interestingly, these latter two MDs
have a phenotype similar to Duchenne MD, even though the dystrophin
gene is unaffected. Together, these results indicate that the DAPC is a
functionally interdependent protein ensemble. As such it is essential
to characterize the structural and functional features that underlie
its organization.
Two components of the DAPC, - and
-dystroglycan, form a heteromeric membrane-spanning complex that
is likely to constitute a principal linkage between the basal lamina
and the cytoskeleton.
-Dystroglycan is a highly glycosylated
extrinsic peripheral membrane protein that binds the basal lamina
elements agrin, laminin, and
merosin(11, 14, 15, 16, 17, 18) .
-Dystroglycan is a transmembrane protein whose cytoplasmic domain
has been reported to bind to the carboxyl-terminal tail of
dystrophin(19) . Utrophin, an autosomally encoded protein that
has close structural similarity to
dystrophin(20, 21) , may also interact with
-dystroglycan. This association is of general interest in view of
utrophin's expression at neuromuscular junctions and in a wide
variety of nonmuscle tissues(22) .
The dystroglycan complex
has also been implicated in cell signaling. Agrin is an extracellular
matrix molecule that induces the clustering of acetylcholine receptors
and other postsynaptic molecules on muscle cell surfaces (reviewed in (23) ). Our search for the agrin receptor in postsynaptic
membranes of Torpedo electroplax led to the discovery of a
heteromeric complex of two membrane glycoproteins that based on amino
acid sequence of two internal peptides share structural similarity with
the dystroglycans(16) . Agrin binds to this complex at
subnanomolar concentrations in a calcium-dependent manner. Further, the
complex spans the plasmalemma and is selectively concentrated in
postsynaptic membranes(16, 24) . Other investigators
have used antibodies to -dystroglycan to show that it binds agrin (15, 17, 18) . Interestingly, agrin induces
the redistribution of several other DAPC components, including utrophin
and adhalin (reviewed in (6) ). Together, these results suggest
that the
-dystroglycan-
-dystroglycan complex participates in
agrin's signaling pathway. They also suggest that agrin may
direct the spatial organization of the DAPC complex on the muscle
surface.
Knowledge of the detailed structure and interactions of the
dystroglycan complex will be essential for understanding the
DAPC's role in muscular dystrophy pathogenesis. Moreover, these
results are important for understanding the role of dystroglycans in
postsynaptic differentiation. In this study we extend our examination
of the relationship between Torpedo agrin receptor subunits
and mammalian dystroglycans to three levels: peptide sequence, ligand
binding, and antigenicity. In addition we have localized the cleavage
site of the dystroglycan precursor in Torpedo, and present
evidence suggesting that a similar site is used in mammalian
dystroglycan. These results provide an unambiguous primary structure
for - and
-dystroglycan.
The ligand overlay
assay was performed essentially as described(16) . Agrin was
purified from Torpedo electric organ(24) . Laminin
(Upstate Biotechnologies Inc.) and anti-agrin antibody MAb-5B1 were
labeled with I using Iodogen (Pierce). All overlay
procedures were at 4 °C. Blots were incubated overnight in block
solution (without Tween 20) followed by 100 ng/ml
I-laminin or 0.2 units/µl Torpedo agrin in
the presence of 1 mM calcium for 3.5 h. Agrin overlays were
incubated in a second layer containing 1 µg/ml
I-MAb-5B1. Agrin- or laminin-binding polypeptides were
revealed by autoradiography.
Figure 1:
Binding of anti-dystroglycan
antibodies, agrin, and laminin to purified Torpedo agrin
receptor. Agrin receptor subunits from Torpedo electric organ (lanes 1-4) or membrane proteins from rat muscle (lane 5) were separated by SDS-PAGE and either silver-stained (lane 1) or blotted to nitrocellulose (lanes
2-5). Blots probed with 0.2 units/µl Torpedo agrin followed by I-labeled anti-agrin antibody 5B1 (lane 2); 100 ng/ml
I-laminin (lane 3);
or an antiserum, 12031C, raised against a peptide in the predicted
cytoplasmic domain of
-dystroglycan (lanes 4 and 5). The
minor bands in lane 5 are nonspecific because they are
detected with nonimmune serum. The bars to the right of the lanes indicate migration of prestained protein
standards (Amersham Corp.): 220, 97.4, 66, 46, and 30
kDa.
Figure 2:
Peptide
microsequencing of Torpedo agrin receptor 190- and 50-kDa
subunits. A, amino acid sequences obtained from purified 190-
(-dystroglycan) and 50-kDa (
-dystroglycan) agrin receptor
subunits (top lines, each pair) aligned with predicted amino
acid sequences from human dystroglycan (bottom
lines)(28) . Sequence 1
was obtained by
amino-terminal sequencing of purified 50-kDa subunit; all other
sequences were obtained by internal microsequencing. Assignments of
amino acid similarity are as defined in the BLASTA
algorithm(34) . B, schematic of the alignment of the Torpedo agrin receptor subunit sequences with the predicted
full-length human dystroglycan precursor polypeptide. The cleavage site
deduced in this study is shown. TM, the predicted
transmembrane domain(28) . Sequences 2
and 3
have been published
previously(16) .
We next asked if the Torpedo 50-kDa
subunit was antigenically related to mammalian -dystroglycan (Fig. 1). A polyclonal antiserum directed against mammalian
-dystroglycan recognized the 50-kDa Torpedo subunit. In
addition, the electrophoretic mobility of the Torpedo polypeptide was virtually identical to that of rat muscle
-dystroglycan.
We then examined the ligand binding properties
of the agrin receptor using a blot overlay method. Both laminin and
agrin bound to the polydisperse 190-kDa subunit (Fig. 1, lanes 2 and 3). In agreement with previous
results(15) , the binding of both ligands was calcium-dependent
and inhibited by heparin (data not shown). We also asked if heparin
inhibited the binding of either agrin or laminin to intact postsynaptic
membranes. Solid phase radioassay showed that heparin (10 µg/ml)
reduced agrin and laminin binding by 64 ± 21.2% and 75.5
± 13.2%, respectively (p < 0.01, n =
3). Because their ligand binding properties, antigenicity, and sequence
are similar to the mammalian dystroglycans, the 190- and 50-kDa agrin
receptor subunits are likely to represent Torpedo - and
-dystroglycan, and we will hence refer to them as such.
The apparent molecular mass of Torpedo -dystroglycan (190 kDa) is substantially greater than that
reported for its counterparts in mammalian brain (120 kDa) or muscle
(156 kDa)(14, 27) . Analysis of all three forms in our
gel system confirmed these differences (data not shown), raising the
possibility that the Torpedo 190-kDa polypeptide might
represent the full-length dystroglycan precursor (see below). However,
- and
-dystroglycan share no common peptides, as judged by
tryptic peptide mapping (Fig. 3). Additionally, an antiserum
directed against the predicted cytoplasmic domain of the dystroglycan
precursor fails to bind the Torpedo 190-kDa glycoprotein (Fig. 1). Taken together, these data suggest that the 190 kDa
glycoprotein represents fully proteolytically processed
-dystroglycan. Moreover, a similar result was obtained with crude Torpedo postsynaptic membranes or with electric organ that had
been lysed directly with boiling SDS-PAGE sample buffer (data not
shown), indicating that cleavage is not an artifact of purification.
Figure 3: Tryptic peptide maps of Torpedo agrin receptor 190- and 50-kDa subunits. Purified Torpedo agrin receptor 190- and 50-kDa subunits were separated by SDS-PAGE, electroblotted to polyvinylidene difluoride, and digested in situ with trypsin. The released peptides were resolved by HPLC on a C8 column and detected by absorbance at 210 nm. No peptides common to the 190- and 50-kDa subunits were detected. The asterisk denotes artefactual peak from digest buffer.
Figure 4:
Analysis of disulfide linkages in 190- and
50-kDa polypeptides. A, Torpedo postsynaptic
membranes were incubated for 10 min at 60 °C in sample buffer
either in the presence (+) or absence(-) of 20 mM
dithiothreitol and then separated by SDS-PAGE. 190 () and 50 kDa
(
) were visualized by agrin-binding ligand blot overlay and by
immunoblot with antiserum 12031C, respectively. The mobility of
-dystroglycan is greater under nonreducing conditions, indicating
the presence of intrachain disulfide bonds in this polypeptide. B, schematic model of
-dystroglycan topology, drawn
approximately to scale. The deduced disulfide bond in the extracellular
domain is depicted.
There are only three cysteines in the predicted
amino acid sequence of mammalian -dystroglycan ( Fig. 2and (27) and (28) ). One (Cys
) is in the
intracellular domain, and two (Cys
and Cys
)
are in the extracellular domain. Further, Cys
is
conserved in Torpedo (Fig. 2, sequence
1
). It is thus likely that Cys
and Cys
form a disulfide bond in
-dystroglycan (Fig. 4B). These data also indicate that
-dystroglycan, which is tightly associated with the plasma
membrane(16, 24, 29) , does not do so via
interchain disulfide bonding to
-dystroglycan.
Mammalian dystroglycan isolated from muscle plasma membranes
is a detergent-soluble heteromer comprised of - and
-subunits (30) . We assessed the relationship between the 190-kDa
50-kDa agrin receptor complex from Torpedo electric organ and
mammalian dystroglycan at three levels: ligand binding, sequence
similarity, and antigenicity. First, like mammalian
-dystroglycan,
the 190-kDa subunit requires calcium to bind laminin and agrin, and
this binding is inhibited by heparin. Second, the peptides that we
sequenced from the Torpedo subunits (118 total amino acids)
are 69% identical and 85% similar to mammalian dystroglycan. Third,
antibodies raised against mammalian
-dystroglycan recognize the Torpedo 50-kDa polypeptide. Finally, our previous findings
that the 190-kDa
50-kDa subunits form a stable complex in n-octyl-
-D-glucopyranoside (16) are in
agreement with those obtained for mammalian dystroglycan(30) .
Taken together, these results indicate that the Torpedo agrin
receptor 190- and 50-kDa subunits are homologues of
- and
-dystroglycan in mammals.
In this study, we have deduced the
location of a cleavage site in the dystroglycan precursor that yields
- and
-dystroglycan. Alignment of the predicted mammalian
sequence with the amino-terminal sequence of Torpedo
-dystroglycan and the internal sequences from
-dystroglycan indicates that cleavage occurs between Gly
and Ser
(Fig. 2). Moreover, this locus is
likely to be the major if not the only cleavage site: 1) a single
amino-terminal sequence was obtained from
-dystroglycan; 2) all
internal peptide sequences for the
- and
-dystroglycan
subunits were located on the amino- and carboxyl-terminal sides,
respectively, of this site; and 3) the tryptic peptide maps of
-
and
-dystroglycan were distinct and nonoverlapping. Cleavage is
also efficient; we find no evidence for intact dystroglycan precursor
in Torpedo postsynaptic membranes (Fig. 1).
Based
upon a Gly cleavage site, the predicted molecular masses
of the mammalian
- and
-dystroglycan polypeptides are 72- and
27-kDa, respectively(27, 28) . Interestingly, as
judged by SDS-PAGE, the apparent molecular mass of
-dystroglycan
ranges from 120 to 190 kDa, whereas that of
-dystroglycan is
approximately 43 kDa (50 kDa in the gel system used here). Both
subunits must therefore undergo significant post-translational
modification. Some of these modifications include asparagine-linked
glycosylation and sialylation(14, 16, 29) .
However, because only a fraction of the carbohydrates are sensitive to
any of the glycosidases or glycosaminoglycanases surveyed to date,
additional, perhaps novel, carbohydrate structures are probably
present. Such structures are likely to be key to the function of
-dystroglycan. For example, variant glycosaminoglycan-deficient
muscle cells show greatly attenuated agrin-induced acetylcholine
receptor clustering(31, 32, 33) , and the
-dystroglycan they produce shows reduced agrin
binding(15) . Elucidation of the carbohydrate structure of
dystroglycan is thus an important area for future investigation.
The
ability of the dystroglycan complex to function as a receptor is likely
to depend upon interaction between its subunits. -Dystroglycan is
tightly associated with the plasma membrane, probably via a strong,
albeit noncovalent, interaction with
-dystroglycan.
-Dystroglycan spans the plasma membrane and is thus well
positioned to mediate signal transduction events initiated by ligand
binding to
-dystroglycan. The localization of the cleavage site
provides a starting point for delimiting the domains of
-dystroglycan that are required for association with
-dystroglycan. These domains may also be important for the
interaction of
- and/or
-dystroglycan with other members of
the DAPC, such as adhalin. It is noteworthy that
-dystroglycan is
also found in soluble pools(14) , raising the possibility that
the association between
- and
-dystroglycan may be regulated.
Such regulation would have important implications for whether or not
binding of extracellular matrix components to
-dystroglycan leads
to signaling events.