(Received for publication, June 29, 1995; and in revised form, January 5, 1996)
From the
Dystrophin-related and -associated proteins are important in the formation and maintenance of the mammalian neuromuscular junction. We have characterized mouse cDNA clones encoding isoforms of the dystrophin-homologous 87-kDa postsynaptic protein, dystrobrevin. In Torpedo, the 87-kDa protein is multiply phosphorylated and closely associated with proteins in the postsynaptic cytoskeleton, including the acetylcholine receptor. In contrast to Torpedo, where only a single transcript is seen, the mouse expresses several mRNAs encoding different isoforms. A 6.0-kilobase transcript in brain encodes a 78-kDa protein (dystrobrevin-1) that is very similar to the Torpedo sequence. A second transcript encodes a 59-kDa protein (dystrobrevin-2) that has a different C terminus, lacking the putative tyrosine kinase substrate domain. In skeletal and cardiac muscle, transcripts of 1.7 and 3.3/3.5 kilobases predominate and encode additional isoforms. Alternative splicing within the coding region and differential usage of untranslated regions produce additional variation. Multiple dystrobrevin-immunoreactive proteins copurify with syntrophin from mouse tissues. In skeletal muscle, dystrobrevin immunoreactivity is restricted to the neuromuscular junction and sarcolemma. The occurrence of many dystrobrevin isoforms is significant because alternative splicing and phosphorylation often have profound effects upon the biological activity of synaptic proteins.
Chemical synapses in the peripheral and central nervous systems
mediate intercellular communication, controlling and coordinating an
overwhelming range of cellular processes. Despite their importance,
comparatively little is known about the structure and function of
synapses in the brain. The majority of our knowledge of synapse
structure originates from the study of the specific contact that occurs
between a motor neuron and a muscle fiber: the neuromuscular junction
(NMJ). ()The NMJ consists of a presynaptic nerve terminal
separated from the postsynaptic muscle cell by the synaptic cleft
containing the basal lamina. The morphology and molecular architecture
of the NMJ are critical for efficient synaptic transmission.
The formation of dense clusters of acetylcholine receptors (AChRs) at the point of contact between the motor neuron and muscle fiber is an early event in postsynaptic differentiation. Of the various factors that influence AChR clustering, agrin, an extracellular matrix protein secreted by the motor neuron, appears to play a pivotal role(2) . In addition to AChR clustering, agrin also induces the redistribution of many other postsynaptic (3, 4) and cytoskeletal (5) proteins. These localized cellular changes lead to the formation of microclusters of AChRs that are subsequently developing into the macroclusters characteristic of the mature synapse.
Central to the role of agrin
in synaptogenesis is the cell-surface agrin receptor.
-Dystroglycan (156-kDa dystrophin-associated glycoprotein), a
component of the dystrophin-associated glycoprotein complex (DGC), has
recently been shown to be an agrin receptor in skeletal
muscle(6, 7, 8, 9) .
Extra-junctionally,
-dystroglycan binds to the laminin
-chain (merosin) in the extracellular matrix and to
components of the DGC that span the sarcolemma(10) . One of
these transmembrane proteins,
-dystroglycan (43-kDa
dystrophin-associated glycoprotein), binds to the cysteine-rich domain
of dystrophin(11) . Since the N terminus of dystrophin binds to
actin (12, 13) , the dystroglycans effectively link
the cytoskeleton of the muscle fiber to the extracellular matrix.
The immobilization of AChR clusters is thought to be accomplished by
proteins in the junctional cytoskeleton(14) . Although
dystrophin is present in the cytoskeleton of the
NMJ(15, 16, 17) , utrophin, an autosomally
encoded homologue of dystrophin(18) , precisely colocalizes
with the AChR clusters at the crests of the junctional
folds(19, 20, 21) . Utrophin, like
dystrophin, also binds to components of the DGC(22) . An
attractive hypothesis is that agrin binding to -dystroglycan at
the NMJ causes a local accumulation and reorganization of cytoskeletal
proteins and, in particular, utrophin, eventually resulting in the
immobilization of AChR clusters by a cytoskeletal scaffold. This
hypothesis is supported by evidence showing that utrophin is present in
spontaneous clusters of AChRs (23) and following agrin
induction (7) .
In Torpedo electric organ, two
proteins with molecular masses of 87 and 58 kDa (syntrophin) are
associated with dystrophin and postsynaptic complexes. The 87-kDa
protein is a minor component of the postsynaptic membrane that
copurifies with the AChR in the electric organ membranes(24) .
Monoclonal antibodies (mAbs) raised against this protein label Torpedo electric organ, mammalian end plates, and, to a lesser
extent, the extrasynaptic sarcolemma(24) . The 87-kDa protein
coimmunoprecipitates with dystrophin and
syntrophin(25, 26) . Three mammalian syntrophin genes
have been identified by molecular
cloning(27, 28, 29) . The
-form of syntrophin is highly localized to the
neuromuscular postsynaptic membrane, whereas
-syntrophin is
ostensibly located at the sarcolemma(30) .
Cloning of the Torpedo 87-kDa cDNA revealed moderate but significant sequence similarity to the cysteine-rich and C-terminal domains of the dystrophin protein family(1, 31) . Similar regions in dystrophin, utrophin, and the 87-kDa protein contain the binding sites for the mammalian homologues of syntrophin(32, 33, 34, 35) . In Torpedo, the 87-kDa postsynaptic protein is the product of a single 4.6-kb transcript expressed in the electric organ, in brain, and in skeletal muscle(1) . While the function of the 87-kDa protein is unresolved, a role in the formation and stability of synapses has been proposed(1) . The 87-kDa protein is also a major phosphotyrosine-containing protein in Torpedo electric organ (1) . This is particularly significant because agrin-induced AChR clustering is blocked by inhibitors of tyrosine kinase(14, 36, 37) . In mammalian tissues, multiple 87-kDa cross-reactive proteins copurify with syntrophin from many different rat tissues(26) . The 87-kDa protein may therefore have other functions in addition to its role in synaptogenesis.
To investigate the molecular organization of utrophin- and dystrophin-containing complexes in mouse brain and muscle, we have cloned the murine homologue of the Torpedo 87-kDa postsynaptic protein. In this paper, we show that there are many different murine 87-kDa protein isoforms produced from alternatively spliced transcripts. This is at variance with the situation in Torpedo where only a single transcript and protein have been described(1) . Many of these 87-kDa isoforms are associated with syntrophin in different mouse tissues. In rat skeletal muscle, 87-kDa immunoreactivity is restricted to the NMJ and sarcolemma. Our studies parallel the heterogeneity and localization of the mammalian dystrophin/utrophin family and the syntrophin family(38) , all of which are potential ligands for the 87-kDa isoforms described herein. In deference to the 87-kDa protein being a low molecular mass protein with homology to dystrophin and in agreement with other researchers studying this protein, we have adopted the name dystrobrevin for proteins encoded by the murine 87-kDa gene.
Clone 87e1 was hybridized to a Northern blot of mRNAs from mouse brain and two cell lines. 87e1 hybridized to five transcripts of 6.0, 4.3, 3.5, 3.3, and 1.7 kb in adult brain mRNA (Fig. 1). Additionally, a single 6.0-kb band was detected in the mouse muscle cell line BC3H-1 and monkey kidney COS-7 cells (Fig. 1). No hybridization was observed to embryonic mouse brain RNA. 87e1 was used to screen an adult mouse brain cDNA library constructed from an aliquot of the same mRNA sample that was used in the initial Northern blot. Two cDNA clones, m871 (1.9 kb) and m872 (1.7 kb), were isolated from the first screening. Both clones were entirely sequenced. The majority of m872 and half of m871 were similar to the coding region of the Torpedo nucleotide sequence; however, the sequence of m871 diverged from the m872 sequence at the 3`-end. It was thereby concluded that m871 and m872 were partial cDNAs from separate but related transcripts, consistent with the multiple band pattern seen on Northern blots (Fig. 1). With this in mind, the cDNA library was rescreened with both the m871 and m872 cDNA clones.
Figure 1: Northern blot hybridized with a cDNA encoding part of the human dystrobrevin. 5 µg of mRNA prepared from the following sources was hybridized with 87e1: a mouse BC3H-1 smooth muscle-like cell line, monkey kidney COS-7 cells, embryonic mouse brain (EMB), and adult mouse brain (AMB). The 6.0-kb full-length transcript was present in all samples except embryonic mouse brain mRNA. Additional transcripts of 4.3, 3.5, 3.3, and 1.7 kb (represented by asterisks) were also detected in adult mouse brain mRNA.
Ten positively hybridizing clones were isolated in the second screening. Each clone was analyzed by restriction mapping and end sequencing. The largest clones were placed into two groups: those that were similar to m871 (m22 (3.2 kb) and m24 (2.8 kb)) and those that were similar to m872 (m32 (3.0 kb), m21 (2.8 kb), and m11 (2.2 kb)) (Fig. 2). Because clones m24 and m32 appeared to extend most 5`, they were entirely sequenced (Fig. 3, A and B).
Figure 2:
Restriction map of the murine cDNA clones
predicted to encode brain dystrobrevin-1 (A) and brain
dystrobrevin-2 (B). The structure of the Torpedo transcript is shown for reference and is not meant to indicate
regions of homology. hI + hII denotes the location of the
-helices predicted to form the coiled-coils conserved in proteins
with homology to the C terminus of dystrophin (31) , and TYR in the Torpedo 87-kDa sequence denotes the
tyrosine kinase substrate domain(1) . The polyadenylation
consensus sequence, ATTAAA, is shown in parentheses because it
is only homologous to the Torpedo sequence and may not be the
true polyadenylation signal. The dashed line at the end of the
dystrobrevin-2 restriction map indicates that the entire
dystrobrevin-2-encoding transcript(s) has not been cloned. B, BamHI; E, EcoRI; H, HindIII; Sc, SacI; S, SphI; X, XhoI.
Figure 3: A, cDNA sequence and conceptual translation of clone m24, encoding dystrobrevin-1. The sequences of the peptides used to produce Ab 308 and Ab 433 are in boldface. B, cDNA sequence and conceptual translation of clone m32, encoding dystrobrevin-2. C, multiple sequence alignment of dystrobrevin-1 and dystrobrevin-2 with the Torpedo 87-kDa protein. Sequence alignments were prepared using the programs PILEUP and PRETTYPLOT (Genetics Computer Group Version 7.3 software package).
The sequence of m24 revealed a number of interesting
features. First, m24 had no obvious similarity to the majority of the
5`-untranslated region (UTR) of the Torpedo sequence. The
homology between m24 and the Torpedo sequence starts at
nucleotide 135 of the Torpedo sequence, in the region encoding
the second in-frame methionine codon. Translation of the m24 sequence
from this ATG codon predicts a protein of 78 kDa that has 83% identity
to the Torpedo protein sequence. Upstream of this initiation
codon are two in-frame stop codons, indicating that this sequence is
most likely to be the 5`-UTR of m24. Second, computer-assisted
alignments of the m24-encoded protein sequence with the Torpedo sequence showed that although there was considerable identity
between the two sequences, there was one sizable gap in the m24
sequence, encompassing amino acids 370-400 of the Torpedo sequence (Fig. 3C). Third, the similarity to the
cysteine-rich and C-terminal domains of dystrophin and utrophin was
retained and included the repeating heptad of leucine residues
predicted to form the -helices of coiled-coils implicated in
protein-protein interactions (1, 31) .
The sequence
of m24 was identical to the m871 sequence throughout the region of
overlap. The sequences of m24 and m871 3` of the TGA stop codon
overlapped with the 5`-end of m22 (Fig. 2A). The other
end of m22 showed significant similarity to the 3`-UTR of the Torpedo cDNA including the proposed polyadenylation site (data
not shown). The m22 cDNA is therefore part of the 3`-UTR of the same
transcript from which m24 and m871 are derived. Because the m24 and m22
cDNAs are 2.8 and 3.2 kb, respectively, and they overlap by 50
base pairs, it was concluded that they are derived from the 6.0-kb
brain transcript (Fig. 1). This was confirmed by Northern
blotting (see below) and by RT-PCR (data not shown). We have named the
78-kDa protein product of the 6.0-kb transcript
``dystrobrevin-1'' to distinguish it from the other proteins
described in this study.
The extreme 5`-end of m32 had no homology to the Torpedo sequence or to the m24 sequence. In common with m24, the homology to the Torpedo sequence started at nucleotide 135. Upstream of this sequence are stop codons in every reading frame, indicating that this sequence is not translatable and therefore is the 5`-UTR. Conceptual translation of the m32 sequence yielded a long open reading frame encoding a 59-kDa protein. There are some significant differences between the sequences of m32 and m24 and the Torpedo sequence. The m32 sequence diverges from m24 at amino acid 504 of the m32 sequence (Fig. 3C). A TAA stop codon occurs after 30 nucleotides, resulting in a protein with a different C terminus (Fig. 3B). m32 therefore encodes a protein devoid of the tyrosine kinase substrate domain described by Wagner et al.(1) . In common with the 5`-UTRs, m32 and m24 also have different 3`-UTRs. Comparison of the m24 and m32 sequences revealed two other minor differences. m32 has an insertion of 9 nucleotides that code for the tripeptide DTW. This sequence is not present in m24 or in the Torpedo sequence (Fig. 3C). Additionally, there is a single base difference between the two sequences, resulting in a neutral amino acid difference of Leu-251 in m32 and Val-251 in m24. Valine is found at this position in the Torpedo sequence (Fig. 3C). Like m24, the m32 sequence contains a deletion of amino acids 370-400 of the Torpedo sequence (Fig. 3C). This observation is investigated below.
The sequences of m32 and m872 3` of the stop codon are identical.
The 3`-ends of m32, m21, and m11 also overlap, suggesting a common
3`-UTR in all the clones. In contrast, the extreme 5`-end of m21,
within the predicted 5`-UTR, differs from the sequence of m32, although
the remainder of the 5`-UTR is identical in these clones. This result
indicates that the 5`-UTRs of some of the dystrobrevin transcripts may
be encoded by more than one exon. Because clones m32 and m21 are both
3.0 kb and their 5`-UTRs are different, the corresponding
transcript sizes (4.3, 3.5, or 3.3 kb) cannot be directly deduced. We
have named the 59-kDa protein encoded by the m32 cDNA clone
``dystrobrevin-2.''
In addition to the sequence differences in the 5`-UTRs of clones m24, m32, and m21 described above, there are three variable regions within the protein coding sequence. An extra 3 amino acids (DTW) occur at position 335 of the m32 peptide sequence that are not present in the m24 sequence. We have designated this site ``variable region (vr) 1.''
A stretch of charged residues (EEELKQGTR) corresponding to amino acids 495-503 of the m24 sequence (Fig. 3A) is replaced by the sequence TQG encoded by m871 (Fig. 4A). We have designated this region ``vr2.'' vr2 covers the point where the predicted C termini of the brain dystrobrevin-1 and dystrobrevin-2 sequences diverge and is immediately adjacent to the coiled-coil region conserved in the dystrophin-related protein family(31) . The Torpedo sequence is also similar to the 9 amino acids at vr2 (Fig. 3C).
Figure 4:
Alternatively spliced dystrobrevin
isoforms. A, the predicted amino acid sequence of the splice
variants at the vr2 site (C). The sequence encoded by the cDNA
clone m871 was compared with the dystrobrevin-1 (encoded by m24),
dystrobrevin-2 (encoded by m32), and Torpedo 87-kDa protein
sequences. The amino acid number of the m871 sequence is not shown
because m871 does not contain a complete open reading frame. The asterisk denotes the stop codon of the dystrobrevin-2
transcript. B, the predicted amino acid sequence of the
tissue-specific splice variants at the vr3 site. The sequences of
dystrobrevin-1 (brain) and the Torpedo 87-kDa protein were
compared with the deduced amino acid sequence of RT-PCR products cloned
from skeletal muscle first strand cDNA. The skeletal muscle sequence is
identical to that obtained from heart cDNA. The sequences of
dystrobrevin-1 and dystrobrevin-2 at vr1 are also identical. C, diagrammatic representation of the dystrobrevin isoforms in
brain and muscle. hI and hII denote the location of
the -helices predicted to form the coiled-coils conserved in
proteins with homology to the C terminus of dystrophin(31) .
vr1, -2, and -3 are the sites of alternative splicing described in the
text. The locations of the peptides used to produce Ab 308 and Ab 433
and the syntrophin-binding site are indicated. The N-terminal regions
of dystrobrevin-3 and dystrobrevin-4 are represented by broken
boxes; this reflects the fact that the N-terminal encoding
sequences have not been determined.
Computer-assisted alignment of the
predicted protein sequences of m24 and m32 with the Torpedo peptide sequences shows that the region encompassing amino acids
363-400 of the Torpedo sequence is not present in the
two mouse sequences (Fig. 3C). The mouse brain cDNA
clones only have a small additional stretch of nonhomologous sequence
(FITRSM) before the similarity with the Torpedo sequence is
restored after amino acid 400. Oligonucleotides were made that flanked
this region and were used to amplify first strand cDNA prepared from
various mouse tissues. Products of the predicted size were obtained in
brain cDNA; however, the products in skeletal and cardiac muscle were
200 base pairs larger (data not shown). The products from both
tissues were cloned and sequenced. Sequence analysis showed that the
additional sequence, accounting for the increased mobility of the PCR
products in heart and muscle, was very similar to the region encoding
amino acids 364-399 of the Torpedo sequence, but absent
in m24 and m32. The region of similarity between the Torpedo and mouse sequences is not continuous, but is interrupted by two
insertions of 5 and 21 amino acids in the mouse sequence (Fig. 4B). RT-PCR showed that this region, which we
have called ``vr3,'' appears to be specific to skeletal and
cardiac muscle (data not shown).
Since the dystrobrevin-1 and dystrobrevin-2 transcripts differ in their 3`-ends (Fig. 3, A and B) and hybridize to transcripts in heart and skeletal muscle (Fig. 5, A and C), it was important to determine whether vr3 was specific to either transcript. RT-PCR using a forward primer in the common coding sequence and a reverse primer in the 3`-UTR of either the dystrobrevin-1 or dystrobrevin-2 sequence only gave products that included the vr3 sequence. This was confirmed by cloning and sequencing (data not shown). These data indicate that the major dystrobrevin-encoding transcripts in heart and skeletal muscle contain vr3 and are thus different from the transcripts present in brain. We have named the protein that contains the vr3 sequence and is encoded by a transcript with the same 3`-UTR as the dystrobrevin-1 transcript ``dystrobrevin-3.'' Similarly, we have named the protein that contains the vr3 sequence but is encoded by a transcript with the same 3`-UTR as the dystrobrevin-2 transcript ``dystrobrevin-4.'' The organization of dystrobrevin-1, -2, -3, and -4 is summarized in Fig. 4C.
Figure 5: Northern blot analysis of dystrobrevin transcripts in mouse tissues. 25 µg of total RNA was hybridized with the following probes: A, m872 (Fig. 2B); B, 1.1-kb EcoRI fragment of m871 (Fig. 2A); C, 0.9-kb XhoI-EcoRI fragment of m21 (Fig. 2B). Sizes of the RNA markers and the ribosomal RNAs are indicated with arrows.
Northern blots hybridized with clone m872 (Fig. 2), encoding the majority of dystrobrevin-2, gave a complex band pattern (Fig. 5A). The m872 cDNA clone was chosen because it contains coding sequence that is common to both dystrobrevin-1 and dystrobrevin-2. In brain RNA, m872 hybridized strongly to transcripts of 6.0, 4.3, 3.5, 3.3, 1.7, 1.2, and 0.7 kb. The hybridization pattern in the other tissues is less complex. In heart, m872 hybridized to two transcripts of 3.3 and 1.7 kb. In skeletal muscle and lung, m872 hybridized predominantly to three transcripts of 6.0, 3.5 (3.3 kb in muscle), and 1.7 kb. No strongly hybridizing transcripts were detected in RNA from other tissues. The 6.0-kb full-length transcript as well as the 4.3-, 3.5-, 3.3-, and 1.7-kb transcripts are also present in the adult mouse brain mRNA (Fig. 1) used in the cDNA library construction.
To establish the molecular basis of this complex pattern of transcripts, Northern blots were hybridized with cDNA subclones. Using the 1.1-kb EcoRI restriction fragment from m871, composed of sequence unique to the dystrobrevin-1 transcript, the major transcript detected is 6.0 kb (Fig. 5B). This transcript is abundant in brain, but can also be detected at lower levels in muscle and lung and very weakly in kidney. Additionally, a weakly hybridizing 3.3-kb transcript is also detected in brain, lung, and muscle RNAs. This confirms that the brain transcript encoding dystrobrevin-1 is 6.0 kb. m22 gave the same hybridization pattern as the 1.1-kb EcoRI subclone of m871 (data not shown).
The 0.9-kb XhoI-EcoRI fragment of m21 covers part of the 3`-UTR of the transcript(s) that encodes dystrobrevin-2. Hybridization of this probe to Northern blots gave a complex band pattern (Fig. 5C). In brain, this probe hybridizes to transcripts of 6.0, 4.3, 3.5, 3.3, 1.7, 1.2, 0.8, and 0.7 kb. In skeletal muscle, heart, and lung, the 0.9-kb XhoI-EcoRI fragment of m21 predominantly hybridizes to 3.5-kb (3.3-kb in muscle) and 1.7-kb transcripts. In muscle and lung, this probe also hybridizes weakly to a 6.0-kb transcript. Additionally, in muscle, a 4.3-kb transcript can also be detected.
This complex pattern is very similar to the pattern of hybridization of m872 to the same Northern blots. m872 also contains part of the dystrobrevin-2 3`-UTR as well as the majority of the coding region. This indicates that the 3`-UTR of the brain dystrobrevin-2 transcript, present in m872, m32, m21, and m11, cross-hybridizes to several different dystrobrevin-encoding transcripts. This hybridization pattern may be due to a repetitive element within the 3`-UTR or may reflect the number of dystrobrevin-encoding transcripts that utilize this UTR or a UTR with a very similar sequence.
Figure 6: Immunoblot analysis of dystrobrevin gene products in mouse tissues. Dystrobrevin isoforms associated with syntrophin were purified from Triton X-100 extracts of tissues using mAb SYN1351. Sample loadings were adjusted so that the amount of syntrophin in each lane was approximately equal (upper panel, anti-syn). Dystrobrevin isoforms were identified with Ab 308 (middle panel) and Ab 433 (lower panel). The sizes of the molecular mass markers (in kilodaltons) are indicated.
Immunoblotting with a second antibody, Ab 433, confirmed the results obtained with Ab 308, but also revealed additional complexity. A high molecular mass isoform, clearly larger than the predicted muscle ``long'' isoform, dystrobrevin-3, was identified by Ab 433 in skeletal muscle (Fig. 6). Ab 308 and other dystrobrevin antibodies failed to detect this band, raising the possibility that this high molecular mass protein may not be a dystrobrevin isoform. In addition, the major band in liver recognized by both antibodies appears to be larger than the corresponding bands in heart and muscle. Interestingly, in the intestine, a tissue with a considerable smooth muscle content, dystrobrevin immunoreactivity was very low in the syntrophin preparation. Weak and diffuse dystrobrevin immunoreactivity is also apparent in the kidney. Although the proteins identified by immunoblotting are largely consistent with the major isoforms predicted by cDNA cloning, there appears to be additional complexity yet to be identified.
Figure 7:
Localization of dystrobrevin in rat
skeletal muscle. Regions of the section containing NMJs were identified
by labeling with -bungarotoxin (A and C).
Dystrobrevin distribution was determined by staining with Ab 433 alone (B). The specificity of this staining was confirmed by
preincubation of Ab 433 with the appropriate peptide (D). Bar = 20 µm.
This paper describes the isolation and characterization of cDNA clones encoding isoforms of the murine homologue of the 87-kDa postsynaptic protein originally identified in the electric organ of Torpedo californica. In contrast to the situation in Torpedo, where there appears to be a single transcript, in the mouse, there are multiple transcripts encoding different protein isoforms. In mouse brain, the ``full-length'' transcript is 6.0 kb and encodes a protein with a predicted molecular mass of 78 kDa. We have called this protein dystrobrevin-1. In addition to the dystrobrevin-1 transcript, several other mRNAs can be detected in brain. One of these transcripts encodes a protein with a predicted molecular mass of 59 kDa; we have called this product dystrobrevin-2. The dystrobrevin-1 transcript is most similar to the Torpedo mRNA because there is significant sequence similarity between the 3`-UTRs as well as in the coding sequence.
The major difference between dystrobrevin-1 and dystrobrevin-2 is that they have different C termini. Dystrobrevin-2 lacks the proposed tyrosine kinase substrate domain (1) and may not be a suitable substrate for tyrosine phosphorylation. However, dystrobrevin-2 may still be phosphorylated on serine and threonine residues in a similar manner to the Torpedo 87-kDa protein(40) . The other major differences between the dystrobrevin-1 and dystrobrevin-2 transcripts occur in the untranslated regions. The cDNA clones m32, m24, and m21 all have different 5`-UTRs. The occurrence of several 5`-UTRs is suggestive of independent regulation. Each transcript may be regulated by a different promoter and may have separate 5`-noncoding regions, but still utilize the same coding regions and initiator methionine. The rat gene for brain-derived neurotrophic factor has a similarly complex organization. Four short 5`-noncoding exons can be spliced to a common coding exon and are each regulated by separate promoters(41) . These promoters confer tissue-specific, axotomy- and neuronal activity-induced expression in transgenic mice(42) . It is therefore possible that several of the dystrobrevin transcripts in the brain are transcribed in a region-specific manner from different promoters.
The 6.0-kb transcript is most abundant in adult brain,
but is also expressed at lower levels in skeletal muscle and lung. In
muscle, heart, and lung, two transcripts of 3.3/3.5 and 1.7 kb
predominate. The major dystrobrevin-encoding isoforms in muscle and
heart differ from their brain counterparts and include an additional 57
amino acids (vr3) preceding the coiled-coil domain (Fig. 4C). The vr3 sequence is only present in skeletal
and cardiac muscle and thus represents a significant difference between
the products in brain and muscle. In addition, the vr3 sequence is also
present in muscle transcripts that are predicted to encode proteins
with different C termini, i.e. with C termini identical to
those of dystrobrevin-1 and dystrobrevin-2. We have called these muscle
isoforms dystrobrevin-3 and dystrobrevin-4 (Fig. 4C).
An important difference between dystrobrevin expression in mouse and Torpedo is that dystrobrevin is not expressed in Torpedo heart(1) . In mouse heart, the 3.5- and 1.7-kb
dystrobrevin transcripts are expressed at levels comparable to those in
brain and skeletal muscle. This finding is consistent with our
hypothesis that the dystrobrevins are ligands for dystrophin/utrophin
and syntrophin since these proteins and their transcripts are also
abundant in mouse heart (43) . ()
In addition to the differences within the noncoding regions and the major tissue-specific splice variant, vr3, alternative splicing at two other sites, vr1 and vr2 (Fig. 4C), occurs. vr1 is an insertion of 3 amino acids, whereas the vr2 splice is a replacement of 3 amino acids with a stretch of 9 amino acids. While the function of this splice variation is unknown, it is noteworthy that an insertion of 8 amino acids into the coding sequence of neuronally secreted agrin is associated with a 1000-fold stimulation in AChR clustering activity (44, 45) .
Several findings have shown that the dystrobrevins are components of one or more protein complexes. Our data demonstrate that multiple isoforms of the mammalian dystrobrevin are associated with syntrophin (Fig. 6). Because mAb 1351 detects all three forms of syntrophin, it is not known whether one form of syntrophin is preferentially associated with the dystrobrevins. It is a distinct possibility that there are other dystrobrevin isoforms that are not associated with syntrophin, presumably because they lack the syntrophin-binding domain and would therefore not be present in the affinity-purified syntrophin complex. In addition to syntrophin, dystrophin and utrophin are also present in dystrobrevin-containing protein complexes(25, 26) . In this study, we have not established whether the dystrobrevins bind directly to dystrophin and utrophin or bind via syntrophin. The assembly of complexes containing dystrophin/utrophin, syntrophin, and the dystrobrevins is currently being investigated.
Finally, a recent paper by Yoshida et al.(46) describes the expression cloning of a partial cDNA for A0, a 94-kDa protein that copurifies with dystrophin and other components of the DGC including the syntrophin triplet(47, 48) . The derived sequence has high similarity to the Torpedo 87-kDa protein sequence and is therefore likely to be the rabbit orthologue of the Torpedo gene. Interestingly, the mAb used to screen the library detects a 62-kDa protein in addition to the 94-kDa A0 protein on immunoblots of purified DGC from skeletal muscle. This observation supports our description of two predominant dystrobrevin isoforms, both of which we have cloned. Both forms of A0 are components of the DGC(46) , and both are associated with syntrophin (this study).
In conclusion, we have described several isoforms of murine dystrobrevin. Multiple dystrobrevin isoforms are present in syntrophin preparations from different tissues. In muscle, anti-dystrobrevin antibodies label the NMJ and sarcolemma, in common with other components of the DGC. We predict that the dystrobrevins will be general ligands for both dystrophin/utrophin and syntrophin at the NMJ and sarcolemma, but may have additional roles in nonmuscle tissues.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) X95226 [GenBank]and X95227[GenBank].