Identification of dystrophin isoforms in Torpedo postsynaptic membranes

Cartaud, Annie; Ludosky, Marie-Aline; Stetzkowski‐Marden, Françoise; Tomé, Fernando; Khurana, T.S.; Cartaud, Jean

doi:10.1016/0248-4900(91)90111-y

Cited by 5 publications

(10 citation statements)

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“…Recent work from several laboratories has shown that mammalian syntrophins are capable of binding directly to different members of the dystrophin family [24][25][26][27] Torpedo ~-syntrophin also bound to an additional member of the dystrophin family, the 87K postsynaptic protein. The 87K protein has 27% amino acid identity to dystrophin scattered throughout the CRCT domain ( [14], see also Fig.…”

Section: Discussionmentioning

confidence: 99%

Direct binding of Torpedo syntrophin to dystrophin and the 87 kDa dystrophin homologue

Dwyer

Froehner

1995

FEBS Letters

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Syntrophin, a 58-kDa membrane-associated protein, is one component of a protein complex associated with dystrophin and other members of the dystrophin family, including the 87-kDa homologue (87K protein). To characterize interactions between syntrophin and 87K protein, we used an in vitro overlay binding assay. We demonstrate that purified Torpedo syntrophin binds directly to dystrophin and 871(. By expressing overlapping regions of the 87K protein as bacterial fusion proteins for binding targets, we show that a 52-amino acid region of 87K (residues 375-426) is sufficient for binding syntrophin.Key words: Syntrophin; Dystrophin; Binding site; Torpedo into two groups based on isoelectric point: acidic (~, pI = 6.7) or basic (ill and f12, pI = 9 +) [19]. In skeletal muscle, c~-syntrophin is found on the sarcolemm a, in a distribution like that of dystrophin and the 87K protein, while fl2-syntrophin is localized to the neuromuscular junction [20].Here, we characterize the binding of Torpedo syntrophin to dystrophin and 87K. Using an in vitro blot overlay assay, we show that Torpedo syntrophin (an ~-syntrophin) binds directly to dystrophin and 87K. Using fusion proteins encoding portions of 87K, we map a binding site of syntrophin to residues 375-426.

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Section: Discussionmentioning

confidence: 99%

Direct binding of Torpedo syntrophin to dystrophin and the 87 kDa dystrophin homologue

Dwyer

Froehner

1995

FEBS Letters

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“…Myoblasts were allowed to fuse into multinucleated myotubes for 3-4 days and were then used for experiments. To examine the effects of nerve-derived trophic factors, 0.1 M rat CGRP (Sigma) or 10 ng/ml purified Torpedo agrin (29) was added directly to the culture medium for 24 -48 h. Additionally, the effects of 1 nM recombinant neural (C-Ag 12,4,8 ) or muscle (C-Ag 12,0,0 ) isoforms of agrin were also examined (30).…”

Section: Methodsmentioning

confidence: 99%

Muscle and Neural Isoforms of Agrin Increase Utrophin Expression in Cultured Myotubes via a Transcriptional Regulatory Mechanism

Gramolini¹,

Burton²,

Tinsley³

et al. 1998

Journal of Biological Chemistry

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Duchenne muscular dystrophy is a prevalent X-linked neuromuscular disease for which there is currently no cure. Recently, it was demonstrated in a transgenic mouse model that utrophin could functionally compensate for the lack of dystrophin and alleviate the muscle pathology (Tinsley, J. M., Potter, A. C., Phelps, S. R., Fisher, R., Trickett, J. I., and Davies, K. E. (1996) Nature 384, 349 -353). In this context, it thus becomes essential to determine the cellular and molecular mechanisms presiding over utrophin expression in attempts to overexpress the endogenous gene product throughout skeletal muscle fibers. In a recent study, we showed that the nerve exerts a profound influence on utrophin gene expression and postulated that nerve-derived trophic factors mediate the local transcriptional activation of the utrophin gene within nuclei located in the postsynaptic sarcoplasm (Gramolini, A. O., Dennis, C. L., Tinsley, J. M., Robertson, G. S., Davies, K. E, Cartaud, J., and Jasmin, B. J. (1997) J. Biol. Chem. 272, 8117-8120). In the present study, we have therefore focused on the effect of agrin on utrophin expression in cultured C2 myotubes. In response to Torpedo-, muscle-, or nerve-derived agrin, we observed a significant 2-fold increase in utrophin mRNAs. By contrast, CGRP treatment failed to affect expression of utrophin transcripts. Western blotting experiments also revealed that the increase in utrophin mRNAs was accompanied by an increase in the levels of utrophin. To determine whether these changes were caused by parallel increases in the transcriptional activity of the utrophin gene, we transfected muscle cells with a 1.3-kilobase pair utrophin promoter-reporter (nlsLacZ) gene construct and treated them with agrin for 24 -48 h. Under these conditions, both muscle-and nerve-derived agrin increased the activity of ␤-galactosidase, indicating that agrin treatment led, directly or indirectly, to the transcriptional activation of the utrophin gene. Furthermore, this increase in transcriptional activity in response to agrin resulted from a greater number of myonuclei expressing the 1.3-kilobase pair utrophin promoter-nlsLacZ construct. Deletion of 800 base pairs 5 from this fragment decreased the basal levels of nlsLacZ expression and abolished the sensitivity of the utrophin promoter to exogenously applied agrin. In addition, site-directed mutagenesis of an N-box motif contained within this 800-base pair fragment demonstrated its essential contribution in this regulatory mechanism. Finally, direct gene transfer studies performed in vivo further revealed the importance of this DNA element for the synapse-specific expression of the utrophin gene along multinucleated muscle fibers. These data show that both muscle and neural isoforms of agrin can regulate expression of the utrophin gene and further indicate that agrin is not only involved in the mechanisms leading to the formation of clusters containing presynthesized synaptic molecules but that it can also participate in the local regulation of genes encoding s...

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“…Al/59-DAP and its Torpedo homologue, 58K (10), have recently been shown by two separate groups to coimmunoprecipitate with dystrophin (11,12). The Torpedo 58K and two homologues in mouse have recently been cloned and named syntrophins (13).…”

Section: Introductionmentioning

confidence: 99%

Cloning of human basic A1, a distinct 59-kDa dystrophin-associated protein encoded on chromosome 8q23-24.

Ahn

Yoshida

Anderson

et al. 1994

Proc. Natl. Acad. Sci. U.S.A.

128

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Duchenne and Becker muscular dystrophies are caused by defects of dystrophin, which forms a part of the membrane cytoskeleton of sp d cells such as muscle. It has been previously shown that the dystrophin-asocited protein Al (59-kDa DAP) is actually a heterogeneous group of phosphorylated proteins consisting of an acidic (a-Al) and a distinct basic ((-Al) component. Partial peptide sequence of the Al complex purified from rabbit muscle permitted the design of oligonucleotide probes that were used to isolate a cDNA for one human isoform ofAl. This cDNA encodes a basic Al isoform that is distinct from the recently described syntrophins in Torpedo and mouse and is expressed in many tissues with at least five distinctmRNA speciesof5.9, 4.8, 4.3, 3.1, and 1.5 kb. A comparison of our human cDNA sequence with the GenBank expressed sequence tag (EST) data base has identified a relative from human skeletal muscle, EST25263, which is probably a human homologue of the published mouse syntrophin 2. We have mapped the human basic component of Al and EST25263 genes to chromosomes 8q23-24 and 16, respectively.Dystrophin is the protein product of a large X chromosomeencoded gene that, when defective, can lead to Duchenne and Becker muscular dystrophies (1). When dystrophin is purified from skeletal muscle membranes by absorption onto wheat germ agglutinin-Sepharose, it is found in association with a large oligomeric complex of membrane glycoproteins, the dystrophin-associated glycoprotein (DAG) complex (DAGC) (2). The dystrophin-associated proteins (DAPs) named Al (62 kDa), A2 (52 kDa), A3 (43 kDa), A4 (36 kDa), and A5 (24 kDa), respectively, correspond to the 59-kDa DAP (59-DAP), 50-kDa DAG, 43-kDa DAG, 35-kDa DAG, and 25-kDa DAP reported elsewhere (3, 4). In addition, AO (94 kDa) is distinct from the 156-kDa DAG; the latter stains weakly with Coomp-;sie brilliant blue *1F These proteins, together with dystrcy'in, a-* thought tv participate in the actin-based membrane cytoskeleton of the muscle cell to maintain its stability. The 156-kDa DAG, dystroglycan, biochemically interacts with the extracellular component laminin, functionally implicating dystrophin and the DAGC as a link between the extracellular matrix and the membrane cytoskeleton (5).The site of interaction between dystrophin and the DAGC has been mapped to within the cysteine-rich domain and the first half of the C-terminal domain of dystrophin (6, 7), suggesting that the DAGC may also interact with the C-terminal dystrophin protein of 71 kDa (Dp7l) (8) and the autosomal near-relative of dystrophin, dystrophin-related protein (DRP or utrophin) (9). Al/59-DAP and its Torpedo homologue, 58K (10), have recently been shown by two separate groups to coimmunoprecipitate with dystrophin (11,12). The Torpedo 58K and two homologues in mouse have recently been cloned and named syntrophins (13).A separate study has shown that purified Al/59-DAP consists ofan acidic (a-Al) and a basic ((3-Al) Al population, which can be discriminated by a monoclonal antibody (14 cDNA C...

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Identification of dystrophin isoforms in Torpedo postsynaptic membranes

Cited by 5 publications

References 0 publications

Direct binding of Torpedo syntrophin to dystrophin and the 87 kDa dystrophin homologue

Direct binding of Torpedo syntrophin to dystrophin and the 87 kDa dystrophin homologue

Muscle and Neural Isoforms of Agrin Increase Utrophin Expression in Cultured Myotubes via a Transcriptional Regulatory Mechanism

Cloning of human basic A1, a distinct 59-kDa dystrophin-associated protein encoded on chromosome 8q23-24.

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