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β2–syntrophin: Localization at the neuromuscular junction in skeletal muscle
Abstract
The syntrophins are a multigene family of proteins which bind C-terminal domains of dystrophin, utrophin and homologs thereof. We report here that antibodies specific for one isoform, beta 2-syntrophin, labeled only the neuromuscular junction (NMJ) in rat skeletal muscle. Anti-alpha 1-syntrophin antibodies gave strong labeling of the sarcolemma and NMJ in normal rat and mouse muscle, and similar but much weaker labeling in dystrophin-minus mdx muscle. beta 2-Syntrophin therefore appears to be specific to the NMJ in normal muscle, as is utrophin, and may be involved in acetylcholine receptor clustering. alpha 1-Syntrophin appears to be associated mainly with dystrophin, as expected, but a small portion must be associated with another protein, possibly homologs of the electric tissue 87K protein.
... Within the human promoter there are several non conserved binding sites for the ubiquitous transcription factor S p l but no TATA box or CAAT motif . Both the human and the mouse promoters contain an E-box, a recognition sequence for the muscle specific transcription factors of the MyoD family (Davis et al., 1989;W right et al., 1989), and an N-box, a motif that occurs in promoter regions of genes expressed at muscle-nerve synapses (NMJ), for example the acetylcholine receptor ô subunit (Koike et al., 1995) and p2-syntrophin (Peters et al., 1994). 0.9 Kb of 5' flanking sequence, which includes both the E box and N box, is sufficient to drive transcription of a reporter gene in transfected cell lines. ...
... All the data in the literature indicate that 395kDa utrophin at the NMJ is important for the maintenance of AChR clusters (Froehner et al., 1991;Phillips et al., 1993;Peters et al., 1994). Utrophin levels are reduced in inherited and acquired AChR deficiencies . ...
... Since G-utrophin contains the cysteine rich and carboxyterminal domains involved in interaction with the DAPs it could form part of a specialised post-synaptic membrane complex. This is likely to involve p2syntrophin since its pattern of expression overlaps that of G-utrophin (Peters et al., 1994, Gorecki et al., 1997 and perhaps p-dystrobrevin which associates with dystrophin in neurones (a-dystrobrevin-1 does not occur in neurones, Blake et al., 1999). p-dystroglycan is not likely to be part of the G-utrophin complex since its expression does not overlap that of G-utrophin (Schofield et al., 1995b). ...
Utrophin is the autosomal homologue of dystrophin. The aim of this project was to characterise the short utrophin forms at the mRNA and protein levels and to look for other utrophin transcripts. Mice knocked-out for full-length utrophin (UKOex6) were used as an experimental system. These mice lack full- length utrophin but it was expected that they would express all the short forms. In order to examine the distribution of utrophin protein in UKOex6 mice, polyclonal antibodies were raised against different regions of the protein. Western blotting and immunofluorescence studies, using these antibodies, confirmed that full-length utrophin is absent in UKOex6 tissues and identified two novel short forms Up120 and Up109. These studies also showed that while Up140 and Up71 appear not to be translated, the other short forms are translated in a tissue specific fashion. G-utrophin is neuronal specific; Up120 is expressed specifically in kidney glomeruli; Up109 was identified in foetal hands/feet and appears to localise to the epidermis. A cDNA for Up109 was isolated by 5' RACE; this contains 460bp of unique sequence encoding 5'UTR and 17 amino acids. Immunological studies confirmed that the full-length form is the major utrophin in skeletal muscle, and demonstrated that this form is also the most abundant In kidney and testis. In the testis, full-length utrophin localises to intertubular tissue and to Leydig cells. Western blot analysis suggested that a novel isoform(s) is expressed in the testis, however, this could not be confirmed by immuno-histochemistry. Weak signal detected at the neuromuscular junction in UKOex6 adult muscle was interpreted as being due to a short isoform, perhaps Up140. In contrast to the apparently limited distribution of short forms as proteins, mRNA in situ hybridisation experiments to control and UKOex6 embryo sections showed that they are transcribed abundantly throughout development. Short transcripts are first detected by 8.5 dpc in the neural tube and appear to comprise the majority of utrophin expression during development. Sites of high expression of full-length utrophin mRNA during development are tongue, choroid plexus and the outflow of the heart. A comparison of the mRNA and protein distributions suggests that the transcription of short utrophin mRNAs is not matched by their translation (this is particularly true for Up140 and Up71). The significance of this important observation and possible roles for the short transcripts are discussed. The developmental studies also revealed three novel sites of utrophin expression - pyloric sphincter, the urethra and the semicircular canals. Utrophin is the autosomal homologue of dystrophin. Transcription from the utrophin gene involves five promoters that regulate the expression of two full-length and three short transcripts. The skeletal muscle isoform has been well studied because it has a potential role in the treatment of Duchenne Muscular Dystrophy. However, there is little information on the short isoforms, particularly their patterns of expression and function. The aim of this project was to characterise the short utrophin isoforms at the mRNA and protein levels and to look for other utrophin transcripts. Mice knocked-out for full-length utrophin (UKOex6) were used as the experimental model. These mice lack full-length utrophin but it was believed that they would express all the short isoforms. In order to examine the distribution of utrophin protein in UKOex6 mice, polyclonal antibodies were raised against different regions of the protein. Western blotting and immunofluorescence to tissue sections confirmed that full- length utrophin is absent in tissues and showed that while Up140 and Up71 appear not to be translated, some short isoforms are translated in a limited fashion. Two of these isoforms, Up120 and Up109, had not been reported previously. Up120 is a kidney specific isoform and Up109 might be a foetal isoform. Up109 cDNA, which contains novel sequence in intron 55, was isolated by 5' RACE. Immunological analysis of a selection of tissues showed that the full-length form is the major utrophin in skeletal muscle, kidney and testis and revealed that in the latter full-length utrophin localises to the intertubular tissue and to the Leydig cells. Surprisingly, weak utrophin signal could be detected at the neuromuscular junction in UKOex6 adult muscle. In contrast to the restricted expression of short isoforms as proteins, mRNA in situ hybridisation experiments using probes to different regions of utrophin mRNA showed that short utrophin isoforms are transcribed abundantly throughout development. Thus, for these isoforms the abundance of mRNA is not matched by that of the protein. The significance of this observation and possible roles for the short transcripts are discussed. (Abstract shortened by UMI.)
... The name PDZ comes from the first three proteins found to contain repeats of this domain (PSD-95, Drosophila discs large protein, and the zona occludens protein 1) (Cho et al., 1992). In skeletal muscle, ␣1-and 1-syntrophin are found on the sarcolemma and are relatively concentrated at the neuromuscular junction (NMJ), whereas 2-syntrophin is concentrated at the NMJ but barely detectable on the sarcolemma (Peters et al., 1994(Peters et al., , 1997. Syntrophins bind directly to the C-terminal domain of dystrophin and the dystrophin-related proteins utrophin and dystrobrevin Ahn and Kunkel, 1995;Dw yer and Froehner, 1995;Yang et al., 1995;Ahn et al., 1996). ...
... Syntrophins bind directly to the C-terminal domain of dystrophin and the dystrophin-related proteins utrophin and dystrobrevin Ahn and Kunkel, 1995;Dw yer and Froehner, 1995;Yang et al., 1995;Ahn et al., 1996). The absence of dystrophin in Duchenne muscular dystrophy (DMD) and the md x mouse leads to a dramatic reduction in sarcolemmal syntrophin, although the syntrophins remain concentrated at the NMJ (Butler et al., 1992;Peters et al. 1994Peters et al. , 1997Yang et al., 1995). ...
... mAb 1351 is directed against an epitope within the PDZ domain of syntrophin (S. H. Gee and S. C. Froehner, unpublished data) and recognizes all known mouse syntrophin isoforms (Peters et al., 1994(Peters et al., , 1997. mAb 1808 against dystrophin has been described previously (Sealock et al., 1991). ...
Syntrophins are cytoplasmic peripheral membrane proteins of the dystrophin-associated protein complex (DAPC). Three syntrophin isoforms, α1, β1, and β2, are encoded by distinct genes. Each contains two pleckstrin homology (PH) domains, a syntrophin-unique (SU) domain, and a PDZ domain. The name PDZ comes from the first three proteins found to contain repeats of this domain (PSD-95, Drosophila discs large protein, and the zona occludens protein 1). PDZ domains in other proteins bind to the C termini of ion channels and neurotransmitter receptors containing the consensus sequence (S/T)XV-COOH and mediate the clustering or synaptic localization of these proteins. Two voltage-gated sodium channels (NaChs), SkM1 and SkM2, of skeletal and cardiac muscle, respectively, have this consensus sequence. Because NaChs are sarcolemmal components like syntrophins, we have investigated possible interactions between these proteins. NaChs copurify with syntrophin and dystrophin from extracts of skeletal and cardiac muscle. Peptides corresponding to the C-terminal 10 amino acids of SkM1 and SkM2 are sufficient to bind detergent-solubilized muscle syntrophins, to inhibit the binding of native NaChs to syntrophin PDZ domain fusion proteins, and to bind specifically to PDZ domains from α1-, β1-, and β2-syntrophin. These peptides also inhibit binding of the syntrophin PDZ domain to the PDZ domain of neuronal nitric oxide synthase, an interaction that is not mediated by C-terminal sequences. Brain NaChs, which lack the (S/T)XV consensus sequence, also copurify with syntrophin and dystrophin, an interaction that does not appear to be mediated by the PDZ domain of syntrophin. Collectively, our data suggest that syntrophins link NaChs to the actin cytoskeleton and the extracellular matrix via dystrophin and the DAPC.
... Immunoblot analysis was performed in liver lysates of wild type mice and mice deficient for SNTA, SNTB2 or both syntrophins (12). Using recently described non-commercial antibodies (11) it was confirmed that SNTA was not expressed in the livers of SNTA−/− and SNTA/B2−/− mice and SNTB2 was only detected in wild type and SNTA−/− mice (Fig. 2). The antibody from Abcam recognized SNTA (Fig. 2). ...
... Although it can not be completely excluded that a truncated SNTA is produced in these animals the molecular weight of such a shorter form would be much smaller than 60 kDa. The SNTA specific antibodies described in the literature (11) and the antibody from Abcam which specifically react with SNTA have been produced using the identical peptide sequence. Two of the antibodies tested and claimed to be specific for SNTA reacted with SNTB2 which is highly homologous to SNTA (1). ...
... Analysis of SNTA in the liver of SNTA−/−, SNTB2−/−, SNTA/B2−/− mice and wild type (WT) mice. Immunoblot analysis of SNTA and SNTB2 using antibodies described by Peters et al. (11) and four commercially available antibodies purchased from Abcam, Thermo Fisher Scientific Pierce, Life Span BioScience and Sigma-Aldrich. ...
Alpha-syntrophin (SNTA) is an adaptor protein which regulates several signaling pathways. To analyze expression of SNTA immunoblot assays have to be performed. Here, the specificity of four commercially available SNTA antibodies has been evaluated in immunoblot experiments using liver tissues of wild type and SNTA deficient mice. While one of the antibodies reacts with SNTA, two antibodies specifically recognize beta 2 syntrophin (SNTB2). The antigen detected by the forth antibody has not been identified but is different from SNTA and SNTB2. Therefore, only one of the four tested antibodies is appropriate to analyze SNTA protein levels by immunoblot.
Copyright © 2015. Published by Elsevier Inc.
... The third subcomplex consists of one or more of the 59-kD peripheral membrane proteins encoded by individual yet homologous genes, known as the syntrophins 0xl, 131, and 132). cd-syntrophin is present uniformly around the sarcolemmal membrane and is thought to be present in most dystrophin complexes (36). pl-syntrophin is present at low levels in skeletal muscle, but its precise localization pattern has not been reported (1). ...
... pl-syntrophin is present at low levels in skeletal muscle, but its precise localization pattern has not been reported (1). 132-syntrophin colocalizes at the neuromuscular junction with utrophin, and is hypothesized to associate in complexes with this dystrophin homologue (36). ...
... al-syntrophin is highly expressed in skeletal muscle and displays an identical localization pattern to dystrophin (36). Both al-syntrophin and 131-syntrophin, the latter expressed predominantly in liver but at moderate levels in skeletal muscle, have been shown to bind the exon 73-74 regions of dystrophin in vitro (3,42,45). ...
Dystrophin plays an important role in skeletal muscle by linking the cytoskeleton and the extracellular matrix. The amino terminus of dystrophin binds to actin and possibly other components of the subsarcolemmal cytoskeleton, while the carboxy terminus associates with a group of integral and peripheral membrane proteins and glycoproteins that are collectively known as the dystrophin-associated protein (DAP) complex. We have generated transgenic/mdx mice expressing "full-length" dystrophin constructs, but with consecutive deletions within the COOH-terminal domains. These mice have enabled analysis of the interaction between dystrophin and members of the DAP complex and the effects that perturbing these associations have on the dystrophic process. Deletions within the cysteine-rich region disrupt the interaction between dystrophin and the DAP complex, leading to a severe dystrophic pathology. These deletions remove the beta-dystroglycan-binding site, which leads to a parallel loss of both beta-dystroglycan and the sarcoglycan complex from the sarcolemma. In contrast, deletion of the alternatively spliced domain and the extreme COOH terminus has no apparent effect on the function of dystrophin when expressed at normal levels. The proteins resulting from these latter two deletions supported formation of a completely normal DAP complex, and their expression was associated with normal muscle morphology in mdx mice. These data indicate that the cysteine-rich domain is critical for functional activity, presumably by mediating a direct interaction with beta-dystroglycan. However, the remainder of the COOH terminus is not required for assembly of the DAP complex.
... 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, ␣ 1syntrophin 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). ...
... 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 ␣ 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). ...
... The Ab SYN37 was prepared against the peptide C-RLGGGSAEPLSSQSFSFHRDR, corresponding to amino acids 220-240 of mouse  1-syntrophin, plus an NH 2 -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). ...
The syntrophins are a multigene family of intracellular dystrophin-associated proteins comprising three isoforms, α1, β1, and β2. 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 β1-syntrophin cDNA, and previously isolated cDNAs for α1- and β2-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, α1- and β1-syntrophin are concentrated at the neuromuscular junction but are also present on the extrasynaptic sarcolemma. β1-syntrophin is restricted to fast-twitch muscle fibers, the first fibers to degenerate in Duchenne muscular dystrophy. β2-syntrophin is largely restricted to the neuromuscular junction.
The sarcolemmal distribution of α1- and β1-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 β1- and β2-syntrophins, while dystrophin complexes contain mostly α1- and β1-syntrophins. Dystrobrevin complexes contain dystrophin and α1- and β1-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.
... This was the case at the membrane of both regenerating and non-regenerating muscle fibers. Interestingly, the only site where both proteins were preserved at wild type levels in mdx muscles was at NMJs, where utrophin is strongly expressed [26,43,44]. α1syntrophin expression in mdx muscle was low in regenerating fibers, where utrophin is also highly expressed, and was low to undetectable in non-regenerating fibers. ...
... PTRF) and Cytokeratin 17 (Table 3). Among these, β2-syntrophin is already known to interact with the DAPC present at the NMJ [40,43]. This interaction is mediated by utrophin, not dystrophin, thus confirming the specificity and sensitivity of our proteomics approach [31]. ...
... Anti-dystrophin (MANDYS1), isotype-matched control (MW8), and anti-β-dystroglycan (MANDAG2) antibodies were produced in-house from hybridoma cell lines (DSHB; University of Iowa) and concentrated using the Amicon ultrafiltration cell (Millipore) to be used for immunoprecipitations. Other antibodies used are: DYS1 (Novocastra) to dystrophin, a polyclonal antibody to the C-terminus of dystrophin preabsorbed for cross-reactivity with utrophin was used to detect Dp71 (a kind gift of Jeff Chamberlain, University of Washington), isoform specific anti-α1-, or β1-syntrophin, and anti-α1-or α2-dystrobrevin antibodies [40,41,43] (a kind gift of Stan Froehner, University of Washington); clone IIH6C4 to αdystroglycan (Millipore); anti-nNOS (#610308) and pan anti-αdystrobrevin (#610766, BD Bioscience); anti-β-sarcoglycan (clone 5B1, Leica Microsystems); anti-utrophin (DRP2, Leica Microsystems); anti-caveolin 3 (BD transduction Labs, clone 26); anti-cavin-1 (ab40840, Abcam) anti-Ca v β2 (ab93606, Abcam), anti-Ca v β2 (ab79264, Abcam), and anti-keratin 17 (ab109725, Abcam). For Figure 1 A, B lower immunoblots, mouse monoclonal anti-dystrophin antibody Dy4/6D3 (DYS1) and anti-β-dystroglycan antibody 43DAG1/8D5 were purchased from Novocastra. ...
In skeletal muscle, α- and β-dystroglycan are an integral part of the dystrophin-associated glycoprotein (DAG) complex, helping to link dystrophin in the sub-membranous cytoskeleton to laminin in the myomatrix. Disruption of this structural link results in severe forms of muscular dystrophy. It is generally assumed that almost all α/β-dystroglycan in skeletal muscle fibers is bound to dystrophin and is engaged in this structural link. The remaining α/β-dystroglycan is thought to interact with utrophin, a homolog of dystrophin that is normally concentrated at the neuromuscular junction. Contrary to expectation, we discovered that a significant fraction of α/β-dystroglycan at the myofiber membrane does not directly associate with dystrophin or utrophin, but is part of a separate protein complex or complexes distinct from the DAG. Using a proteomics-based approach, we uncovered evidence for at least three separate pools of α/β-dystroglycan-containing complexes within myofibers. These pools differ in protein composition and are differentially affected by the loss of dystrophin. Our findings indicate a more complex role for α/β-dystroglycan in muscle than is currently recognized and may help explain differences in pathological mechanisms and disease severity among muscular dystrophies associated with mutations in known members of the DAG complex.
... This was the case at the membrane of both regenerating and non-regenerating muscle fibers. Interestingly, the only site where both proteins were preserved at wild type levels in mdx muscles was at NMJs, where utrophin is strongly expressed [26,43,44] . α1- syntrophin expression in mdx muscle was low in regenerating fibers, where utrophin is also highly expressed, and was low to undetectable in non-regenerating fibers. ...
... PTRF) and Cytokeratin 17 (Table 3). Among these, β2-syntrophin is already known to interact with the DAPC present at the NMJ [40,43]. This interaction is mediated by utrophin, not dystrophin, thus confirming the specificity and sensitivity of our proteomics approach [31]. ...
... Anti-dystrophin (MANDYS1), isotype-matched control (MW8), and anti-β-dystroglycan (MANDAG2) antibodies were produced in-house from hybridoma cell lines (DSHB; University of Iowa) and concentrated using the Amicon ultrafiltration cell (Millipore) to be used for immunoprecipitations. Other antibodies used are: DYS1 (Novocastra) to dystrophin, a polyclonal antibody to the C-terminus of dystrophin preabsorbed for cross-reactivity with utrophin was used to detect Dp71 (a kind gift of Jeff Chamberlain, University of Washington), isoform specific anti-α1-, or β1-syntrophin, and anti-α1-or α2-dystrobrevin antibodies [40,41,43] (a kind gift of Stan Froehner, University of Washington); clone IIH6C4 to αdystroglycan (Millipore); anti-nNOS (#610308) and pan anti-αdystrobrevin (#610766, BD Bioscience); anti-β-sarcoglycan (clone 5B1, Leica Microsystems); anti-utrophin (DRP2, Leica Microsystems); anti-caveolin 3 (BD transduction Labs, clone 26); anti-cavin-1 (ab40840, Abcam) anti-Ca v β2 (ab93606, Abcam), anti-Ca v β2 (ab79264, Abcam), and anti-keratin 17 (ab109725, Abcam). ForFigure 1 A, B lower immunoblots, mouse monoclonal anti-dystrophin antibody Dy4/6D3 (DYS1) and anti-β-dystroglycan antibody 43DAG1/8D5 were purchased from Novocastra. ...
The dystroglycan complex contains the transmembrane protein β-dystroglycan and its interacting extracellular mucin-like protein α-dystroglycan. In skeletal muscle fibers, the dystroglycan complex plays an important structural role by linking the cytoskeletal protein dystrophin to laminin in the extracellular matrix. Mutations that affect any of the proteins involved in this structural axis lead to myofiber degeneration and are associated with muscular dystrophies and congenital myopathies. Because loss of dystrophin in Duchenne muscular dystrophy (DMD) leads to an almost complete loss of dystroglycan complexes at the myofiber membrane, it is generally assumed that the vast majority of dystroglycan complexes within skeletal muscle fibers interact with dystrophin. The residual dystroglycan present in dystrophin-deficient muscle is thought to be preserved by utrophin, a structural homolog of dystrophin that is up-regulated in dystrophic muscles. However, we found that dystroglycan complexes are still present at the myofiber membrane in the absence of both dystrophin and utrophin. Our data show that only a minority of dystroglycan complexes associate with dystrophin in wild type muscle. Furthermore, we provide evidence for at least three separate pools of dystroglycan complexes within myofibers that differ in composition and are differentially affected by loss of dystrophin. Our findings indicate a more complex role of dystroglycan in muscle than currently recognized and may help explain differences in disease pathology and severity among myopathies linked to mutations in DAPC members.
... Syntrophins are intracellular peripheral membrane proteins of 58-60 kD, originally identified at the postsynaptic apparatus in Torpedo electric organ (Froehner et al. 1987) and subsequently shown to be present in many mammalian tissues. The syntrophin complex is composed of ocl-syntrophin, pi-syntrophin and p2-syntrophin encoded by different genes (Peters et al. 1994). cDNA cloning and peptide sequencing revealed these proteins to be homologous (Adams et al. 1994;Yang et al. 1994;Ahn et al. 1994). ...
... The Ctermianl 57 amino acids of syntrophin (termed the syntrophin-unique (SU) domain) are highly conserved amongst the three isoforms (Ahn et al. 1996)., In skeletal muscle, al-syntrophin is localized to the sarcolemma along with dystrophin, whereas p2-syntrophin is restricted to the NMJ in a comparable manner to utrophin (Peters et al. 1994). Recently p2-syntrophin was shown to bind and co-localise with the PDZ domain containing proteins, MAST205 (microtubule-associated serine/threonine kinase-205 kD) in the NMJ and with SAST (syntrophin-associated serine/threonine kinase) in cerebral vasculature, spermatic acrosomes and neuronal processes (Lumeng et al. 1999). ...
Duchenne and Becker muscular dystrophies (DMD/BMD) are caused by mutations in the dystrophin gene. DMD is also associated with a variable degree of mental impairment. Several dystrophin transcripts are expressed in the brain including a novel transcript (P-type dystrophin) expressed specifically in Purkinje cells; its expression is controlled by an alternative promoter. This study shows that the P-type mRNA is also expressed in skeletal and cardiac muscle but not in smooth muscle. Its first exon encodes a specific, short amino terminus that is highly conserved in mammals and to a lesser extent in chicken. The nucleotide sequence of the P-type first exon and putative promoter region is also conserved. In mice, the 5'-end of the P-type transcript was found to be structurally diverse arising from alternative splicing events at the 5'-UTR. This may occur separately or in combination with insertion of a part of intron I resulting in premature termination of translation. There are multiple transcription initiation sites, the predominant one being conserved in human and mouse. Moreover, alternative usage of ATG codons may result in alternative N-termini in rodents or short upstream open reading frames in other species. Several regulatory elements are conserved in different species. The TATA box found in human sequence is not conserved and is outside the region that directed CAT reporter gene expression in differentiated myotubes in culture.
... α-syntrophin is the predominant isoform in skeletal and cardiac muscle and is also expressed at a lower level in the brain. β1-Syntrophin is expressed on the sarcolemma of skeletal muscle fast fibers and β2-syntrophin is expressed primarily at the neuromuscular junction (NMJ) (17)(18)(19). α -Syntrophin is also the dominant syntrophin isoform in cardiac muscle where it forms a complex with, nNOS, PMCA4B and SCN5A (20). ...
... While a number of studies have investigated the roles of individual syntrophin isoforms (4,13,17,21,32,33), functional compensation by upregulation of other isoforms can mask a phenotype. We have shown previously that in the absence of α-syntrophin, β1-syntrophin is upregulated in muscle (21). ...
Syntrophins are a family of modular adaptor proteins that are part of the dystrophin protein complex, where they recruit and anchor a variety of signaling proteins. Previously we generated mice lacking α- and/or β2-syntrophin but showed that in the absence of one isoform, other syntrophin isoforms can partially compensate. Therefore, in the current study, we generated mice that lacked α, β1, and β2-syntrophins (tKO mice) and assessed skeletal and cardiac muscle function. The tKO mice showed a profound reduction in voluntary wheel running activity at both 6 and 12 months of age. Function of the tibialis anterior was assessed in situ and we found that the specific force of tKO muscle was decreased by 20-25% compared to wild type mice. This decrease was accompanied by a shift in fiber type composition from fast 2B to more oxidative fast 2A fibers. Using echocardiography to measure cardiac function, it was revealed that tKO hearts had left ventricular cardiac dysfunction and were hypertrophic, with a thicker left ventricular posterior wall. Interestingly, we also found that membrane localized dystrophin expression was lower in both skeletal and cardiac muscles of tKO mice. Since dystrophin mRNA levels were not different in tKO, this finding suggests that syntrophins may regulate dystrophin trafficking to, or stabilization at, the sarcolemma. These results show that the loss of all three major muscle syntrophins has a profound effect on exercise performance, and skeletal and cardiac muscle dysfunction contributes to this deficiency.
... mAb124, a rat monoclonal antibody against the AChR  subunit, was a kind gift from Dr. J. Lindstrom (University of Pennsylvania, Philadelphia, PA). The mouse monoclonal antiserum mAb 88B, reactive with the AChR ␥ and ␦ subunits, and mAb SYN1351 , a mouse monoclonal antibody reactive with all syntrophin isoforms (␣1, 1, and 2) (Peters et al., 1994), were generously provided by Dr. S. C. Froehner (University of North C arolina, Chapel Hill, NC). In C2 myotube extracts, syntrophin isoforms were detected as three major protein bands between 55 and 65 kDa (see Fig. 2). ...
... No evidence was found for binding of syntrophin, the third member of the complex tested. Using a monoclonal antibody, mAb SYN1351, that recognized all three major syntrophin isoforms, ␣1, 1, and 2 (Peters et al., 1994), in C2 myotube extracts, no immunopositive protein was detected bound to toxin beads (Fig. 2 D). The failure to detect association with syntrophin indicates that the binding of -dystroglycan and utrophin is specific and does not reflect nonselective protein aggregation. ...
At the neuromuscular junction, aggregates of acetylcholine receptors (AChRs) are anchored in the muscle membrane by association with rapsyn and other postsynaptic proteins. We have investigated the interactions between the AChR and these proteins in cultured C2 myotubes before and after treatment with agrin, a nerve-derived protein that induces AChRs to cluster. When AChRs were isolated from detergent extracts of untreated C2 myotubes, they were associated with rapsyn and, to a lesser degree, with utrophin, β-dystroglycan, MuSK, and src-related kinases, but not with syntrophin. Treatment with agrin increased the association of AChRs with MuSK, a receptor tyrosine kinase that forms part of the agrin receptor complex, without affecting other interactions. Analysis of rapsyn-deficient myotubes, which do not form protein clusters in response to agrin, revealed that rapsyn is required for association of the AChR with utrophin and β-dystroglycan, and for the agrin-induced increase in association with MuSK, but not for constitutive interactions with MuSK and src-related kinases. In rapsyn −/− myotubes, agrin caused normal tyrosine phosphorylation of AChR-associated and total MuSK, whereas phosphorylation of the AChR β subunit, both constitutive and agrin-induced, was strongly reduced. These results show first that aneural myotubes contain preassembled AChR protein complexes that may function in the assembly of the postsynaptic apparatus, and second that rapsyn, in addition to its role in AChR phosphorylation, mediates selected protein interactions with the AChR and serves as a link between the AChR and the dystrophin/utrophin glycoprotein complex.
... Among them, or-and fl-A1 were recently shown to be the mammalian homologues of a Torpedo 58-kD postsynaptic protein, syntrophin, which has been suggested to be involved in clustering acetylcholine receptors (Froehner 1984(Froehner , 1987Adams et al., 1993;Alan et al., 1994;Yang et al., 1994). In agreement with other laboratories, we renamed a-and fl-A1 as t~l-and fll-syntrophin, respectively (Peters et al., 1994). In the previous paper, we have shown that A0 and fll-syntrophin also bind to the dystrophin COOH-terminal region directly, and their binding sites are located within the distal half of the COOH-terminal domain . ...
... Recent works on cDNA cloning established the heterogeneity of the mammalian syntrophin (Adams et al., 1993;Ahn et al., 1994;Yang et al., 1994): there are three forms of syntrophin (cd-,/31-, and/~2-syntrophin), which originate from different genes (their nomenclature was given in Peters et al. [1994]). In the present work, we have shown that or-and/31syntrophin bind to very close but discrete sites on dystrophin. ...
The carboxy-terminal region of dystrophin has been suggested to be crucially important for its function to prevent muscle degeneration. We have previously shown that this region is the locus that interacts with the sarcolemmal glycoprotein complex, which mediates membrane anchoring of dystrophin, as well as with the cytoplasmic peripheral membrane protein, A0 and beta 1-syntrophin (Suzuki, A., M. Yoshida, K. Hayashi, Y. Mizuno, Y. Hagiwara, and E. Ozawa. 1994. Eur. J. Biochem. 220:283-292). In this work, by using the overlay assay technique developed previously, we further analyzed the dystrophin-syntrophin/A0 interaction. Two forms of mammalian syntrophin, alpha 1- and beta 1-syntrophin, were found to bind to very close but discrete regions on the dystrophin molecule. Their binding sites are located at the vicinity of the glycoprotein-binding site, and correspond to the amino acid residues encoded by exons 73-74 which are alternatively spliced out in some isoforms. This suggests that the function of syntrophin is tightly linked to the functional diversity among dystrophin isoforms. Pathologically, it is important that the binding site for alpha 1-syntrophin, which is predominantly expressed in skeletal muscle, coincides with the region whose deletion was suggested to result in a severe phenotype. In addition, A0, a minor component of dystrophin-associated proteins with a molecular mass of 94 kD which is immunochemically related to syntrophin, binds to the same site as beta 1-syntrophin. Finally, based on our accumulated evidence, we propose a revised model of the domain organization of dystrophin from the view point of protein-protein interactions.
... Indeed, syntrophins have been shown to co-purify in vitro with the SkM1 αsubunit [17,40]. In normal skeletal muscle, the three syntrophin isoforms are highly clustered at the NMJ [23] and β2 appears restricted to this site [33]. In extrajunctional regions, β1-syntrophin is detected exclusively on the sarcolemma of fast-twitch fibres while the α1 isoform is found in all fibre types along the sarcolemma [34]. ...
... Since the SkM1-gene message level appeared unchanged in mdx muscle (our present results) and the translational efficiency was also unaffected by mdx pathology [27], the observed reduction of SkM1 content in all fibres implies that the anchorage mechanism is defective. The dystrophin-associated α1and β1-syntrophins link directly to SkM1 [17,40]; moreover, their number are strongly reduced in dystrophindeficient muscle [33,34]. The loss of syntrophins in mdx muscle could lead to an alteration of SkM1 sarcolemmal anchorage. ...
The membrane cytoskeleton is increasingly considered as both an anchor and a functional modulator for ion channels. The cytoskeletal disruptions that occur in the absence of dystrophin led us to investigate the voltage-gated sodium channel (SkM1) content in the extensor digitorum longus (EDL) muscle of the dystrophin-deficient mdx mouse. Levels of SkM1 mRNA were determined by semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR). A C-terminal portion of the mouse-specific SkM1 α-subunit cDNA (mScn4a) was identified first. SkM1 mRNA levels were as abundant in mdx as in normal muscle, thus suggesting that the transcriptional rate of SkM1 remains unchanged in mdx muscle. However, SkM1 density in the extrajunctional sarcolemma was shown to be significantly reduced in mdx muscle, using confocal immunofluorescence image analysis. This decrease was found to be associated with a reduction in the number of SkM1-rich fast-twitch IIb fibres in mdx muscle. In addition, lowered SkM1 sarco-lemmal labelling was found in all mdx fibres regardless of their metabolic type. These results suggest the existence of a perturbation of SkM1 anchorage to the plasma membrane. Such an alteration is likely to be related to the 50% decrease in mdx muscle of the dystrophin-associated syntrophins, which are presumed to be involved in SkM1 anchorage. However, the moderate reduction in SkM1 density (–12.7%) observed in mdx muscle argues in favour of a non-exclusive role of syntrophins in SkM1 anchorage and suggests that other membrane-associated proteins are probably also involved.
... Despite abnormally high background again, likely due to nonspecifie secondary antibody, the labelling ofmoieties recognized by mAb 1B2 was apparent, existing in proximity to endogenous AChR clusters indicated by a-BTX staining (Figure 13). Interestingly, this result supports previous studies showing that syntrophins are concentrated at NMJs (Peters et al., 1994). An additional feature which was noted, was the staining of what seemed to be costameres in the muscle fibres from the tissue sections. ...
... Efforts made to e1ucidate which, if any, isoform of syntrophin was being recognized by mAb 1B2 resulted in the hypothesis that it may be an a l-syntrophin for several reasons: 1) Fusion protein of al-syntrophin domains PHlab, PDZ and SU were all recognized by 1B2 when analysed using western blot, with the controls of absence of primary antibody and unre1ated antibody showing no staining. 2) On rat muscle sections, staining was similar to what has been shown by others studying syntrophin in muscle (Kramarcyand Sealock, 2000; Peters et al., 1994) that is, specifie labelling of the sarcolemma at NMJs which were identified using a-BTX fluorescent labelling of AChRs. In addition, two groups (Adams et al., 1993; Ahn et al., 1994) have estab1ished that pl syntrophin is ubiquitously expressed in many tissues, but also co-expressed in the same region of the sarcolemma as utrophin, therefore it is possible that 1B2 could be recognizing pl syntrophin. ...
The cytoskeletal component of the muscle membrane, dystrophin and its associated proteins (DAPs), are essential for the maintenance of muscle integrity, since the absence of these molecules results in a variety of muscular dystrophies. The purpose of this work was to create and characterize monoclonal antibodies (mAbs) designed to recognize components of the DAP complex (DAPC), in order to provide tools for the study of its structure and function. The first mAb generated, 1137, was raised against a 33 amino acid sequence of the core protein at the c-terminus of alpha-dystroglycan (alpha DG), a cell surface member of the DAPC linked to dystrophin via its co-transcript, the transmembrane protein, beta-dystroglycan. 1B7 was used to perform a comparative study in denervated rat muscle tissue in parallel with IIH6, a mAb which recognizes a different, more glycosylated form of alpha DG. The second and third mAbs were raised against a complex of proteins purified by succinylated Wheat Germ Agglutinin (sWGA) following extraction from rabbit skeletal muscle. (Abstract shortened by UMI.)
... α1-syntrophin, in addition to being the most predominant in muscle tissue, is located throughout the sarcolemma, whereas β-syntrophin is more strongly enriched at NMJ (Peters et al., 1994; for review, see Bhat et al., 2019). ...
Two-pore domain (K2P) potassium channels belong to a large family of ion channels implicated in determining and maintaining the resting cell membrane potential. K2P channels are proteins extensively conserved throughout evolution, being present in almost all animal cells. In the nematode Caenorhabditis elegans, 47 genes code K2P channels sub-units, but only three of them have been characterized and reported in the literature. By tagging a certain number of them with fluorescent proteins (CRISPR/Cas9), we have found that nine channels are co-expressed in body wall muscle, showing a highly specific sub-cellular distribution. The most fascinating distribution was the one of TWK-28, which exhibits a polarized comet-like pattern that occupy only the anterior tip of each body wall muscle cell. In order to elucidate the cellular mechanisms underlying this particular distribution, we performed a genetic screen on the novel TWK-28 gain-of-function strain. We revealed that genes belonging to Dystrophin-Associated Protein Complex (DAPC) are involved in determining the amount of this channel at the muscle cell surface. DAPC is composed of at least 10 intra and extracellular proteins and plays a key role in physically connecting the extracellular matrix to the actin cytoskeleton. Interestingly, when tagging multiple components of DAPC with fluorescent proteins by CRISPR/Cas9 gene editing, we found that most of the dystrophin-associated proteins, such as syntrophin/STN-1, dystrobrevin/DYB-1 or even sarcoglycans (SGCA-1 and SGCB-1), show a particularly asymmetric distribution in muscle. We also revealed the, to date excluded, presence of dystroglycan/DGN-1 in body wall muscle of C. elegans. Finally, the asymmetric distribution of TWK-28 along the antero-posterior axis on a cellular and tissue scale, suggests that the Planar Cell Polarity pathways might be implicated. By gene candidate approach of the WNT pathway, we showed that proteins such as Disheveled, ROR/CAM-1 or WNT ligand/EGL-20 can modify the localization of TWK-28 by driving it into a new posterior sub-complex in the muscle cells
... Although, syntrophin pairs of SNTA with either SNTB1 or SNTB2 are most common (Peters et al., 1997), experiments observing SNTB1 and SNTB2 pull down along with dystrophin have also been reported (Camp et al., 2015) and correlates with our data. SNTB2 is usually expressed at low levels and generally localises to the neuromuscular junction however it is also weakly detectable at the sarcolemma (Kramarcy & Sealock, 2000;Peters, Kramarcy, Sealock, & Froehner, 1994). SNTA levels have previously been found to decrease as a result of dystrophin deficiency while SNTB1 and SNTB2 levels increase. ...
The aim of this project was to systematically identify new interaction partners of the dystrophin protein within differentiated human skeletal muscle cells in order to uncover new roles in which dystrophin is involved, and to better understand how the global interactome is affected by the absence of dystrophin. hTERT/cdk4 immortalized myogenic human cell lines represent an important tool for skeletal muscle research however, disruption of the cell cycle has the potential to affect many other cellular processes to which it also linked. A transcriptome-wide analysis of healthy and diseased lines comparing immortalized lines with their parent primary populations in both differentiated and undifferentiated states testing their myogenic character by comparison with non-myogenic cells found that immortalization has no measurable effect on the myogenic cascade or on any other cellular processes, and that it was protective against the senescence. In this context the human muscle cell lines are a good in vitro model to study the dystrophin interactome. We investigated dystrophin’s interactors using the high-sensitivity proteomics ‘QUICK’ approach. We identified 18 new physical interactors of dystrophin which displayed a high proportion of vesicle transport related proteins and adhesion proteins, strengthening the link between dystrophin and these roles. The proteins determined through previously published data together with the newly identified interactors were incorporated into a web-based data exploration tool: sys-myo.rhcloud.com/dystrophin-interactome, intended to provide an easily accessible and informative view of dystrophins interactions in skeletal muscle.
... Dès 1987, date de sa découverte [1], et durant environ 2 décennies la dystrophine voyait presque chaque année autour d'elle apparaître de nouveaux partenaires aussi bien ancrés dans la membrane musculaire que distribués dans le cytoplasme environnant et formant ainsi un large complexe macromoléculaire [2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21]. On va ainsi identifier une protéine apparentée à la dystrophine comme la dystrobrévine avec 2 versions différentes (α et β) et 7 à 8 isoformes respectivement. ...
https://doi.org/10.1051/myolog/201715017
... SNTA antibody was kindly provided by Prof. Adams and was described recently. The antibody recognizes a peptide sequence in the PH1b domain of SNTA and was raised in rabbits [24,25]. Antibodies for detection of ACC, pACC, Akt, pAkt, AMPK, pAMPK, ATGL, β-actin, caveolin-1, Cox IV, ERK1/2, pERK1/2, FABP4, FAS, GAPDH, HSL, pHSL, PARP1, perilipin, PPARγ, Rab5 and SCD1 were from New England Biolabs GmbH (Frankfurt am Main, Germany). ...
The scaffold protein alpha-syntrophin (SNTA) regulates lipolysis indicating a role in lipid homeostasis. Adipocytes are the main lipid storage cells in the body, and here, the function of SNTA has been analyzed in 3T3-L1 cells. SNTA is expressed in preadipocytes and is induced early during adipogenesis. Knock-down of SNTA in preadipocytes increases their proliferation. Proteins which are induced during adipogenesis like adiponectin and caveolin-1, and the inflammatory cytokine IL-6 are at normal levels in the mature cells differentiated from preadipocytes with low SNTA. This suggests that SNTA does neither affect differentiation nor inflammation. Expression of proteins with a role in cholesterol and triglyceride homeostasis is unchanged. Consequently, basal and epinephrine induced lipolysis as well as insulin stimulated phosphorylation of Akt and ERK1/2 are normal. Importantly, adipocytes with low SNTA form smaller lipid droplets and store less triglycerides. Stearoyl-CoA reductase and MnSOD are reduced upon SNTA knock-down but do not contribute to lower lipid levels. Oleate uptake is even increased in cells with SNTA knock-down. In summary, current data show that SNTA is involved in the expansion of lipid droplets independent of adipogenesis. Enhanced preadipocyte proliferation and capacity to store surplus fatty acids may protect adipocytes with low SNTA from lipotoxicity in obesity.
... Syntrophins are a family of five dystrophin-binding adapter proteins (α1, β1, β2, γ1 and γ2) with a similar domain structure, containing a pleckstrin homology (PH), a PDZ protein interaction and a C-terminal domain unique to syntrophin (Adams et al., 1995b). In the mouse skeletal muscle, α1and β1-syntrophins are found throughout the sarcolemma and are highly concentrated at the NMJ Kramarcy and Sealock, 2000); β2syntrophin is found almost exclusively at the NMJ (Peters et al., 1994Kramarcy and Sealock, 2000) and γ2-syntrophin is present at the subsynaptic sarcoplasmic reticulum (Alessi et al., 2006). Of these, α-syn is the only isoform closely associated with nAChRs at the crests of the NMJ . ...
The effectiveness of synaptic transmission at most mammalian synapses depends largely on the maintenance of a high density of postsynaptic receptors. In a mature synapse, this density is highly dynamic and can be regulated by several factors including synaptic activity, post-translational modifications of receptors, and scaffold proteins. In my thesis work, I focused on the regulation of AChR clustering, which is the hallmark of a neuromuscular junction, a well characterized cholinergic synapse between the motor neuron and the skeletal muscle. Among several pathways, I first focused on the role of alpha-syntrophin (syn), a member of the dystrophin glycoprotein complex (DGC), in the development and modulation of nAChR dynamics of the mouse NMJ. Using syn knock-out mice, I showed that syn is not required for synapse formation, but it is essential for synapse maturation. Particularly, I demonstrated that during the maturation of synapses, the integrity of the postsynaptic apparatus is altered, the turnover rate of AChRs increases significantly, and the number/density of AChRs is impaired. The synaptic alterations observed in this mouse mutant were explained by the loss of tyrosine phosphorylated alpha-dystrobrevin (dbn). Interestingly, when GFP-dbn1 was electroporated into sternomastoid muscles of syn mutant, most of synaptic abnormalities were partially restored. In the second part of my thesis work, I investigated the role of serine/threonine kinases, particularly PKC and PKA on the regulation of nAChR trafficking. We found that PKC accelerates nAChR removal and inhibits recycling at the NMJ, while PKA has the opposite effect. Finally, I begin to address the role of the Wnt/beta-catenin pathway in the adult NMJ, and we show that beta-catenin interacts with the DGC in mature synapses, via rapsyn. Taken together, these results provide new insights into the cellular and molecular underlying signaling of the regulation of nAChR trafficking and dynamics.
... L'α-syntrophine est la forme prédominante dans les fibres musculaires. Elle est présente sous la membrane plasmique de toutes les fibres musculaires alors que la β1-syntrophine est préférentiellement exprimée dans les fibres rapides et la β2-syntrophine se trouve au niveau de la jonction neuromusculaire Peters et al., 1994). Les syntrophines se lient à la dystrophine et à la dystrobrévine, dans leurs parties C terminales . ...
Duchenne muscular dystrophy is a muscular degenerative disease caused by the absence of dystrophin. Dystrophin function and the causes of muscle degeneration in its absence are still not known.
I combined studies in the Caenorhabditis elegans and murine animal models of this disease to elucidate the mechanisms of muscle degeneration.
We demonstrated that the calcium-dependant potassium channel, SLO-1, and the syntrophin homologue, STN-1, are functionally linked to the C. elegans homologue of dystrophin, DYS-1. We ran a genome-wide screen in search of suppressor genes of muscular degeneration. We showed that the protein degradation pathways and several kinases are involved in muscular degeneration in C. elegans. In parallel, I participated to the search of molecules reducing muscle degeneration in C. elegans, and then in mdx mice. We confirmed the beneficial effect of the activation of the serotonergic pathway on the muscular degeneration of mdx mice.
... skeletal muscle of wild-type, mdx, and various transgenic mdx mice (Fig. 2) that express mutant forms of dystrophin (8,9,(19)(20)(21)35). As previously reported, otl-syntrophin was absent from extrajunctional sarcolemma of mdx mouse, b u t remained at neuromuscular endplates (31,36). n N O S was absent from both junctional and extrajunctional sarcolemma of mdx mouse. ...
Becker muscular dystrophy is an X-linked disease due to mutations of the dystrophin gene. We now show that neuronal-type nitric oxide synthase (nNOS), an identified enzyme in the dystrophin complex, is uniquely absent from skeletal muscle plasma membrane in many human Becker patients and in mouse models of dystrophinopathy. An NH2-terminal domain of nNOS directly interacts with alpha 1-syntrophin but not with other proteins in the dystrophin complex analyzed. However, nNOS does not associate with alpha 1-syntrophin on the sarcolemma in transgenic mdx mice expressing truncated dystrophin proteins. This suggests a ternary interaction of nNOS, alpha 1-syntrophin, and the central domain of dystrophin in vivo, a conclusion supported by developmental studies in muscle. These data indicate that proper assembly of the dystrophin complex is dependent upon the structure of the central rodlike domain and have implications for the design of dystrophin-containing vectors for gene therapy.
... We asked whether or not RNAs encoding other components of the postsynaptic apparatus are regulated similarly. First, we determined the synaptic enrichment of transcripts encoding basal lamina, plasma membrane, and cytoskeletal proteins concentrated in the postsynaptic apparatus (Peters et al., 1994; Miner and Sanes, 1994; Altiok et al., 1995; Moscoso et al., 1995a,b; Zhu et al., 1995; Patton et al., 1997; Sanes and Lichtman, 1999). In addition to those transcripts listed in Tables I and II, RNAs encoding AChR, AChR, rapsyn, ColQ, erbB3, utrophin, N-CAM, laminins 5, and collagen 3(IV) were enriched in the synaptic samples (Table III). ...
In both neurons and muscle fibers, specific mRNAs are concentrated beneath and locally translated at synaptic sites. At the
skeletal neuromuscular junction, all synaptic RNAs identified to date encode synaptic components. Using microarrays, we compared
RNAs in synapse-rich and -free regions of muscles, thereby identifying transcripts that are enriched near synapses and that
encode soluble membrane and nuclear proteins. One gene product, LL5β, binds to both phosphoinositides and a cytoskeletal protein,
filamin, one form of which is concentrated at synaptic sites. LL5β is itself associated with the cytoplasmic face of the postsynaptic
membrane; its highest levels border regions of highest acetylcholine receptor (AChR) density, which suggests a role in “corraling”
AChRs. Consistent with this idea, perturbing LL5β expression in myotubes inhibits AChR aggregation. Thus, a strategy designed
to identify novel synaptic components led to identification of a protein required for assembly of the postsynaptic apparatus.
... Anti-dystrophin (MANDYS1), isotype-matched control (MW8), and anti-b-dystroglycan (MANDAG2) antibodies were produced in-house from hybridoma cell lines (DSHB; University of Iowa) and concentrated using the Amicon ultra-filtration cell (Millipore). Antibodies to DAPC members are: isoform specific anti-a1-, b1- or b2-syntrophin, and anti-a1-or a2-dystrobrevin antibodies [27,30,46]; pan anti-syntrophin (ab11425, Abcam); Manex1011B to dystrophin and MANDAG2 to b-dystroglycan (DSHB); clone IIH6C4 to a-dystroglycan (Upstate); anti-nNOS (#610308) and anti-a-dystrobrevin (#610766, BD Bioscience); anti-b-sarcoglycan (clone 5B1, Leica Microsystems). ...
Mutations affecting the expression of dystrophin result in progressive loss of skeletal muscle function and cardiomyopathy leading to early mortality. Interestingly, clinical studies revealed no correlation in disease severity or age of onset between cardiac and skeletal muscles, suggesting that dystrophin may play overlapping yet different roles in these two striated muscles. Since dystrophin serves as a structural and signaling scaffold, functional differences likely arise from tissue-specific protein interactions. To test this, we optimized a proteomics-based approach to purify, identify and compare the interactome of dystrophin between cardiac and skeletal muscles from as little as 50 mg of starting material. We found selective tissue-specific differences in the protein associations of cardiac and skeletal muscle full length dystrophin to syntrophins and dystrobrevins that couple dystrophin to signaling pathways. Importantly, we identified novel cardiac-specific interactions of dystrophin with proteins known to regulate cardiac contraction and to be involved in cardiac disease. Our approach overcomes a major challenge in the muscular dystrophy field of rapidly and consistently identifying bona fide dystrophin-interacting proteins in tissues. In addition, our findings support the existence of cardiac-specific functions of dystrophin and may guide studies into early triggers of cardiac disease in Duchenne and Becker muscular dystrophies.
... Antibodies used for immunoblotting were: anti-beta dystroglycan (43DAG or MANDAG2), anti-dystrophin (Dys1 or MANEX1011b) from Novo Castra and DSHB, antiutrophin from DSHB, anti alpha dystroglycan (clone IIH6C4) from Upstate Biotechnology and anti alpha dystrobrevin (610766) from BD Bioscience. Antibodies specific to alpha-and beta-syntrophins were a kind gift of Dr. Stanley Froehner (University of Washington) and have been previously characterized by his group [38][39][40] . Antibodies to Interleukin 15 receptor alpha were from Bioss or R & D systems, antibodies to Spire2 were from Abgent or Proteintech, and antibody to alpha B crystallin was from Enzo Life Sciences. ...
Dystroglycan is a major cell surface glycoprotein receptor for the extracellular matrix in skeletal muscle. Defects in dystroglycan glycosylation cause muscular dystrophy and alterations in dystroglycan glycosylation can impact extracellular matrix binding. Here we describe an immunoprecipitation technique that allows isolation of beta dystroglycan with members of the dystrophin-associated protein complex (DAPC) from detergent-solubilized skeletal muscle. Immunoprecipitation, coupled with shotgun proteomics, has allowed us to identify new dystroglycan-associated proteins and define changed associations that occur within the DAPC in dystrophic skeletal muscles. In addition, we describe changes that result from overexpression of Galgt2, a normally synaptic muscle glycosyltransferase that can modify alpha dystroglycan and inhibit the development of muscular dystrophy when it is overexpressed. These studies identify new dystroglycan-associated proteins that may participate in dystroglycan's roles, both positive and negative, in muscular dystrophy.
... In mature skeletal muscle, a-syntrophin associates with dystrophin to provide scaffolding that localizes to the sarcolemma various channels, kinases and signaling proteins910111314151617. The a-syntrophin scaffolding has been extensively studied in mature skeletal muscle both at the neuromuscular junction and on the sarcolemma [41]. However, the function of a-syntrophin during early development of skeletal muscle has not been previously characterized. ...
α-Syntrophin is a scaffolding protein linking signaling proteins to the sarcolemmal dystrophin complex in mature muscle. However, α-syntrophin is also expressed in differentiating myoblasts during the early stages of muscle differentiation. In this study, we examined the relationship between the expression of α-syntrophin and myogenin, a key muscle regulatory factor.
The absence of α-syntrophin leads to reduced and delayed myogenin expression. This conclusion is based on experiments using muscle cells isolated from α-syntrophin null mice, muscle regeneration studies in α-syntrophin null mice, experiments in Sol8 cells (a cell line that expresses only low levels of α-syntrophin) and siRNA studies in differentiating C2 cells. In primary cultured myocytes isolated from α-syntrophin null mice, the level of myogenin was less than 50% that from wild type myocytes (p<0.005) 40 h after differentiation induction. In regenerating muscle, the expression of myogenin in the α-syntrophin null muscle was reduced to approximately 25% that of wild type muscle (p<0.005). Conversely, myogenin expression is enhanced in primary cultures of myoblasts isolated from a transgenic mouse over-expressing α-syntrophin and in Sol8 cells transfected with a vector to over-express α-syntrophin. Moreover, we find that myogenin mRNA is reduced in the absence of α-syntrophin and increased by α-syntrophin over-expression. Immunofluorescence microscopy shows that α-syntrophin is localized to the nuclei of differentiating myoblasts. Finally, immunoprecipitation experiments demonstrate that α-syntrophin associates with Mixed-Lineage Leukemia 5, a regulator of myogenin expression.
We conclude that α-syntrophin plays an important role in regulating myogenesis by modulating myogenin expression.
... Among five syntrophin isoforms, α1-syntrophin is present at its highest levels in skeletal muscle [59,60] where it is located close to the inner surface of muscle plasma membrane together with β1-syntrophin. In contrast, β2-syntrophin is mainly concentrated at the neuromuscular junction [61]. AQP4 molecule turned out to be associated with α1-syntrophin as described below, although the association is not always the case. ...
Freeze-fracture electron microscopy enabled us to observe the molecular architecture of the biological membranes. We were studying the myofiber plasma membranes of health and disease by using this technique and were interested in the special assembly called orthogonal arrays (OAs). OAs were present in normal myofiber plasma membranes and were especially numerous in fast twitch type 2 myofibers; while OAs were lost from sarcolemmal plasma membranes of severely affected muscles with dystrophinopathy and dysferlinopathy but not with caveolinopathy. In the mid nineties of the last century, the OAs turned out to be a water channel named aquaporin 4 (AQP4). Since this discovery, several groups of investigators have been studying AQP4 expression in diseased muscles. This review summarizes the papers which describe the expression of OAs, AQP4, and other AQPs at the sarcolemma of healthy and diseased muscle and discusses the possible role of AQPs, especially that of AQP4, in normal and pathological skeletal muscles.
... 64 In addition, the skeletal muscle γ 2 -syntrophin was found at high levels only at the postsynaptic membrane of the neuromuscular junctions. 65,66 Alsoγ 2 -syntrophin has been detected in skeletal muscle, where it has been reported to localize to the sarcolemma 55 and to be able to bind the C-terminus of Nav1.5 through its PDZ domain, regulating the gating properties of the sodium channel. 67 In addition to syntrophin, other scaffolding proteins such as caveolin-3 (CAV3), which is present in the caveolae, flask-shaped plasma membrane microdomains, are involved in signal transduction and vesicle trafficking in myocytes, modulating cardiac remodeling during heart failure. ...
The heart is a force-generating organ that responds to self-generated electrical stimuli from specialized cardiomyocytes. This function is modulated by sympathetic and parasympathetic activity. In order to contract and accommodate the repetitive morphological changes induced by the cardiac cycle, cardiomyocytes depend on their highly evolved and specialized cytoskeletal apparatus. Defects in components of the cytoskeleton affect the ability of the cell to compensate at both functional and structural levels in the long term. In addition to structural remodeling, the myocardium becomes increasingly susceptible to altered electrical activity, leading to arrhythmogenesis. The development of arrhythmias secondary to structural remodeling defects has been noted, although the detailed molecular mechanisms are still elusive. Here, the author reviews the current knowledge of the molecular and functional relationships between the cytoskeleton and ion channels, and discusses the future impact of new data on molecular cardiology research and clinical practice.
... Samples were quantified by the Coomassie Plus Protein Assay Reagent (Pierce Chemical Co.) and equivalent protein loading was verified by SDS-PAGE. KCl-washed microsomes were analyzed by Western blot using antibodies against  2- syntrophin (Peters et al., 1994), pan syntrophin (Froehner et al., 1987), nNOS (Transduction Laboratories),  -dystroglycan, ␣ -sarcoglycan (Novocastra Laboratories), and other proteins described above. ...
Dystrophin is a multidomain protein that links the actin cytoskeleton to laminin in the extracellular matrix through the dystrophin associated protein (DAP) complex. The COOH-terminal domain of dystrophin binds to two components of the DAP complex, syntrophin and dystrobrevin. To understand the role of syntrophin and dystrobrevin, we previously generated a series of transgenic mouse lines expressing dystrophins with deletions throughout the COOH-terminal domain. Each of these mice had normal muscle function and displayed normal localization of syntrophin and dystrobrevin. Since syntrophin and dystrobrevin bind to each other as well as to dystrophin, we have now generated a transgenic mouse deleted for the entire dystrophin COOH-terminal domain. Unexpectedly, this truncated dystrophin supported normal muscle function and assembly of the DAP complex. These results demonstrate that syntrophin and dystrobrevin functionally associate with the DAP complex in the absence of a direct link to dystrophin. We also observed that the DAP complexes in these different transgenic mouse strains were not identical. Instead, the DAP complexes contained varying ratios of syntrophin and dystrobrevin isoforms. These results suggest that alternative splicing of the dystrophin gene, which naturally generates COOH-terminal deletions in dystrophin, may function to regulate the isoform composition of the DAP complex.
Visceral adiposity is strongly associated with liver steatosis, which predisposes to the development of non-alcoholic steatohepatitis (NASH). Mice with loss of the molecular adapter protein beta-2 syntrophin (SNTB2) have greatly reduced intra-abdominal fat mass. Hepatic expression of proteins with a role in fatty acid metabolism such as fatty acid synthase was nevertheless normal. This was also the case for proteins regulating cholesterol synthesis and uptake. Yet, a slight induction of hepatic cholesterol was noticed in the mutant mice. When mice were fed a methionine choline deficient (MCD) diet to induce NASH, liver cholesteryl ester content was induced in the wild type but not the mutant mice. Serum cholesterol of the mice fed a MCD diet declined and this was significant for the SNTB2 null mice. Though the mutant mice lost less fat mass than the wild type animals, hepatic triglyceride levels were similar between the groups. Proteins involved in fatty acid or cholesterol metabolism such as fatty acid synthase, apolipoprotein E and low-density lipoprotein receptor did not differ between the genotypes. Hepatic oxidative stress and liver inflammation of mutant and wild type mice were comparable. Mutant mice had lower hepatic levels of secondary bile acids and higher cholesterol storage in epididymal fat, and this may partly prevent hepatic cholesterol deposition. In summary, the current study shows that SNTB2 null mice have low intra-abdominal fat mass and do not accumulate hepatic cholesteryl esters when fed a MCD diet. Nevertheless, the SNTB2 null mice develop a similar NASH pathology as wild type mice suggesting a minor role of intra-abdominal fat and liver cholesteryl esters in this model.
The acetylcholine receptor (AChR) is highly concentrated at the neuromuscular junction (NMJ), ensuring efficient signal transmission from motoneurons to muscle fibers. This requires the agrin-LRP4-MuSK signaling as well as rapsyn, a peripheral, intracellular protein that is enriched at the NMJ. Mutations of rapsyn have been associated with NMJ diseases including congenital myasthenia syndromes. Rapsyn is a prototype of synaptic adaptor proteins that is thought to bind and anchor neurotransmitter receptors to the postsynaptic membrane. In accord, it interacts with the AChR and a plethora of proteins that associate or regulate the cytoskeleton. Rapsyn also interacts with signaling molecules. Recent studies show that it possesses E3 ligase activity that is required for NMJ formation, revealing a novel function of this classic adaptor protein. Identifying rapsyn as a signaling molecule provides a handle in studies of mechanisms of NMJ formation, maintenance, aging and disorders.
Syntrophins are a family of 59 kDa peripheral membrane‐associated adapter proteins, containing multiple protein‐protein and protein‐lipid interaction domains. The syntrophin family consists of five isoforms that exhibit specific tissue distribution, distinct sub‐cellular localization and unique expression patterns implying their diverse functional roles. These syntrophin isoforms form multiple functional protein complexes and ensure proper localization of signalling proteins and their binding partners to specific membrane domains and provide appropriate spatiotemporal regulation of signalling pathways. Syntrophins consist of two PH domains, a PDZ domain and a conserved SU domain. The PH1 domain is split by the PDZ domain. The PH2 and the SU domain are involved in the interaction between syntrophin and the dystrophin‐glycoprotein complex (DGC). Syntrophins recruit various signalling proteins to DGC and link extracellular matrix to internal signalling apparatus via DGC. The different domains of the syntrophin isoforms are responsible for modulation of cytoskeleton. Syntrophins associate with cytoskeletal proteins and lead to various cellular responses by modulating the cytoskeleton. Syntrophins are involved in many physiological processes which involve cytoskeletal reorganization like insulin secretion, blood pressure regulation, myogenesis, cell migration, formation and retraction of focal adhesions. Syntrophins have been implicated in various pathologies like Alzheimer’s disease, muscular dystrophy, cancer. Their role in cytoskeletal organization and modulation makes them perfect candidates for further studies in various cancers and other ailments that involve cytoskeletal modulation. The role of syntrophins in cytoskeletal organization and modulation has not yet been comprehensively reviewed till now. This review focuses on syntrophins and highlights their role in cytoskeletal organization, modulation and dynamics via its involvement in different cell signalling networks.
Dystrophin protein in association with several other cellular proteins and glycoproteins leads to the formation of a large multifaceted protein complex at the cell membrane referred to as dystrophin glycoprotein complex (DGC), that serves distinct functions in cell signalling and maintaining the membrane stability as well as integrity. In accordance with this, several findings suggest exquisite role of DGC in signalling pathways associated with cell development and/or maintenance of homeostasis. In the present review, we summarize the established facts about the various components of this complex with emphasis on recent insights into specific contribution of the DGC in cell signalling at the membrane. We have also discussed the recent advances made in exploring the molecular associations of DGC components within the cells and the functional implications of these interactions. Our review would help to comprehend the composition, role and functioning of DGC and may lead to a deeper understanding of its role in several human diseases. This article is protected by copyright. All rights reserved
The shape and organization of the plasma membrane are influenced not only by the lipid bilayer and its integral membrane proteins, but also by structures lying either just inside or just outside of the cell that provide a scaffolding for the membrane. Structures that interact closely with the extracellular face of the membrane may act as an “exoskeleton.” Structures on the cytoplasmic face of the plasma membrane are generally considered to be part of the “cytoskeleton.” A subset of the cytoskeleton that associates primarily or exclusively with the plasma membrane has been termed the “membrane skeleton.” This chapter considers the biochemistry and morphology of the exoskeleton and of the membrane-associated cytoskeleton, how these two macromolecular complexes interact with the plasma membrane, and finally, how they interact with each other through receptors located in the plasma membrane. A major emphasis is placed on the role of these complexes in forming distinct membrane domains, enriched in particular integral membrane proteins with specific biological functions.
The syntrophin family of dystrophin-associated proteins consists of three isoforms, alpha 1, beta 1, and beta 2, each encoded by a distinct gene. We have cloned and characterized the mouse alpha 1- and beta 2-syntrophin genes. The mouse alpha 1-syntrophin gene (>24 kilobases) is comprised of eight exons. The mouse beta 2-syntrophin gene (>33 kilobases) contains seven exons, all of which have homologues at the corresponding position in the alpha 1-syntrophin gene. Primer extension analysis reveals two transcription initiation sites in the alpha 1-syntrophin gene and a single site in the beta 2-syntrophin gene. The sequence immediately 5' of the transcription start sites of both genes lacks a TATA box but is GC-rich and has multiple putative SP1 binding sites. The alpha 1-syntrophin gene is located on human chromosome 20 and mouse chromosome 2, while the beta 2-syntrophin gene is on human chromosome 16 and mouse chromosome 8. Analysis of the amino acid sequence of the syntrophins reveals the presence of four conserved domains. The carboxyl-terminal 56 amino acids are highly conserved and constitute a syntrophin unique domain. Two pleckstrin homology domains are Located at the amino-terminal end of the protein. The first pleckstrin homology domain is interrupted by a domain homologous to repeated sequences originally found in the Drosophila discs-large protein.
This chapter discusses the regulation of membrane-protein organization at the neuromuscular junction. Agrin is an extracellular-matrix protein released by the motor neuron at the time of synapse formation. It resides in the synaptic cleft of the neuromuscular junction (NMJ), where it forms part of the stable basal lamina. In this location, agrin induces the formation of Ach receptor (AchR) clusters under the nerve-muscle synapse. The underlying molecular mechanisms of agrin's actions are likely to involve tethering AChR to the cytoskeleton. Several lines of evidence support this hypothesis. First, agrin induces cluster formation by redistributing AChR already present on the muscle-cell membrane and has no effect on AchR-subunit synthesis. Second, AChR clusters are more resistant to detergent extraction than unclustered receptors. Third, many spectrin-like molecules, including syntrophin, utrophin, p-spectrin, the 87-kDa protein, and rapsyn, are specifically co-localized with AChRs at the NMJ. These molecules are likely to serve as a link between AChRs and the actin cytoskeleton. Agrin binds to α-dystroglycan (DG), a glycoprotein complex (GC) member. The GC is linked to the cytoskeleton by binding dystrophin or utrophin, spectrin-like proteins known to bind F-actin. These data provide a model in which agrin, by binding to a-DG, traps the AChRs as they diffuse into the agrin-receptorcytoskeleton complex.
The extracellular matrix is a well organized structure with profound effects on the development and the integrity of adherent tissues. Agrin is a component of many matrices, such as the basement membrane of kidney, blood capillaries and the muscle fiber basal lamina, where it is highly concentrated at the neuromuscular junction. During synapse formation agrin is believed to promote differentiation of the postsynaptic muscle fibers and the presynaptic motor neuron. This complex process is, at least in part, based on specific interactions of agrin with other matrix molecules and with membrane-associated or integral membrane proteins of the abutting cells. This review summarizes studies concerning the integration of agrin with other molecules and highlights possible functions of agrin in the synaptic basal lamina and in other matrices.
alpha-Syntrophin, a member of the dystrophin-associated protein complex, is required for proper localization of the water channel aquaporin-4 at the blood-brain barrier. Mice lacking alpha-syntrophin have reduced levels of aquaporin-4 in perivascular astroglial endfeet. Consequently, they exhibit reduced edema and infarct volume in brain trauma models and reduced K+ clearance from the neuropil, leading to increased seizure susceptibility. We have used the alpha-syntrophin null mice to investigate whether alpha-syntrophin is required for proper localization of other components of the dystrophin complex at the blood-brain barrier. We find that alpha-syntrophin is required for the full recruitment of gamma2-syntrophin and alpha-dystrobrevin-2 to glial endfeet in adult cerebellum. In contrast, the localization of beta1- and beta2-syntrophin and alpha-dystrobrevin-1 at the blood-brain barrier is not dependent on the presence of alpha-syntrophin. The localization patterns of alpha-dystrobrevin-1 and -2 in wild type cerebellum are strikingly different; while alpha-dystrobrevin-1 is present in glial endfeet throughout the cerebellum, alpha-dystrobrevin-2 is restricted to glial endfeet in the granular layer alone. Finally, we show that the enrichment of dystrophin in glial endfeet depends on the presence of alpha-syntrophin. This finding is the first demonstration that dystrophin localization is dependent on syntrophin. Since the localization of gamma2-syntrophin, alpha-dystrobrevin-2, and dystrophin is contingent on alpha-syntrophin, we conclude that alpha-syntrophin is a central organizer of the astrocyte dystrophin complex, an important molecular scaffold for localization of aquaporin-4 at the blood-brain barrier.
alpha 1-Syntrophin, a member of dystrophin-associated proteins, is expressed at the sar colemma and at perivascular astrocytes, and participates in protein-protein interactions through its PDZ domain. Aquaporin-4 (AQP4) is the predominant water channel protein in the brain, and also expressed at the sarcolemma of fast-twitch muscle fibers. AQP4 is concentrated in orthogonal array particles (OAPs), and its expression has been reported to be decreased at the sarcolemma of dystrophin-deficient mdx mice. We examined whether alpha 1-syntrophin targets AQP4 at the sarcolemma. Immunohistochemistry showed that AQP4 is absent at the sarcolemma in alpha 1-syntrophin knockout mice and that its expression is also lost from the perivascular astrocyte endfeet. On the other hand, expression of AQP4 is not decreased at the sarcolemma of the knockout mice in the neonatal stage. Moreover, AQP4 is expressed in lung, stomach, and kidney of wild-type and alpha 1-syntrophin null mice. Our results show that alpha 1-syntrophin is a key molecule to localize AQP4 to the sarcolemma of mature fast myofibers and astrocyte endfeet, but AQP4 is targeted to the plasma membrane by different molecules in lung, stomach, and kidney.
Synaptic retraction occurs when the presynaptic nerve terminal withdraws from the postsynaptic structure. This process occurs both during development where it is especially prevalent during the process of synapse elimination, and throughout life as synapses change shape and size during synaptic remodeling. While much is known about the cellular mechanisms that control the formation of the synapse, relatively little is known about the cellular mechanisms that function during the retraction of a synapse. The smallest unit of the synapse must be the molecules that together form that cellular structure. Thus, to understand the formation of the synapse one must understand how those molecules come to be located at the synapse, while to understand synaptic retraction one must know how or if those molecules are removed from the synapse. In this article I will consider changes in the distribution of acetylcholinesterase, acetylcholine receptors, agrin, presynaptic molecules, synapse specific carbohydrates and other synapse specific molecules during synaptic retraction. By determining how these molecules are controlled during synapse formation and retraction, the basic mechanisms that control synapse formation and elimination may be determined.
The mature neuromuscular junction (NMJ) is the best characterized cholinergic synapse. The maintenance of a high number and density of nicotinic acetylcholine receptors (nAChRs) at the postsynaptic membrane adjacent to the nerve terminal are crucial for NMJ function. This density is maintained by several factors, ranging from synaptic activity to postsynaptic scaffold proteins. Decreases in postsynaptic nAChR density are related to myasthenic syndromes in the peripheral NMJ, but are also associated in central synapses with neurodegenerative diseases such as Alzheimer's. In this review, we focus particularly on our increasing knowledge about the molecular dynamics of nAChR at the peripheral cholinergic NMJ and their regulation by the postsynaptic proteins of the dystrophin glycoprotein complex (DGC).
Syntrophins are a family of cytoplasmic membrane-associated adaptor proteins, characterized by the presence of a unique domain organization comprised of a C-terminal syntrophin unique (SU) domain and an N-terminal pleckstrin homology (PH) domain that is split by insertion of a PDZ domain. Syntrophins have been recognized as an important component of many signaling events, and they seem to function more like the cell's own personal 'Santa Claus' that serves to 'gift' various signaling complexes with precise proteins that they 'wish for', and at the same time care enough for the spatial, temporal control of these signaling events, maintaining overall smooth functioning and general happiness of the cell. Syntrophins not only associate various ion channels and signaling proteins to the dystrophin-associated protein complex (DAPC), via a direct interaction with dystrophin protein but also serve as a link between the extracellular matrix and the intracellular downstream targets and cell cytoskeleton by interacting with F-actin. They play an important role in regulating the postsynaptic signal transduction, sarcolemmal localization of nNOS, EphA4 signaling at the neuromuscular junction, and G-protein mediated signaling. In our previous work, we reported a differential expression pattern of alpha-1-syntrophin (SNTA1) protein in esophageal and breast carcinomas. Implicated in several other pathologies, like cardiac dys-functioning, muscular dystrophies, diabetes, etc., these proteins provide a lot of scope for further studies. The present review focuses on the role of syntrophins in membrane targeting and regulation of cellular proteins, while highlighting their relevance in possible development and/or progression of pathologies including cancer which we have recently demonstrated.
The membrane cytoskeleton is increasingly considered as both an anchor and a functional modulator for ion channels. The cytoskeletal disruptions that occur in the absence of dystrophin led us to investigate the voltage-gated sodium channel (SkM1) content in the extensor digitorum longus (EDL) muscle of the dystrophin-deficient mdx mouse. Levels of SkM1 mRNA were determined by semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR). A C-terminal portion of the mouse-specific SkM1 alpha-subunit cDNA (mScn4a) was identified first. SkM1 mRNA levels were as abundant in mdx as in normal muscle, thus suggesting that the transcriptional rate of SkM1 remains unchanged in mdx muscle. However, SkMI density in the extrajunctional sarcolemma was shown to be significantly reduced in mdx muscle, using confocal immunofluorescence image analysis. This decrease was found to be associated with a reduction in the number of SkM1-rich fast-twitch IIb fibres in mdx muscle. In addition, lowered SkM1 sarcolemmal labelling was found in all mdx fibres regardless of their metabolic type. These results suggest the existence of a perturbation of SkM1 anchorage to the plasma membrane. Such an alteration is likely to be related to the 50% decrease in mdx muscle of the dystrophin-associated syntrophins, which are presumed to be involved in SkM1 anchorage. However, the moderate reduction in SkM1 density (-12.7%) observed in mdx muscle argues in favour of a non-exclusive role of syntrophins in SkM1 anchorage and suggests that other membrane-associated proteins are probably also involved.
The cellular distribution of utrophin, the autosomal homologue of dystrophin, was investigated in developing and adult rat and mouse brain by in situ hybridization and immunohistochemistry. Digoxigenin-labeled cRNA probes complementary to N-terminal, rod-domain, and C-terminal encoding sequences of utrophin were used to differentiate between full-length and short C-terminal isoforms. Largely overlapping distribution patterns were seen for the three probes in neurons of cerebral cortex, accessory olfactory bulb, and several sensory and motor brainstem nuclei as well as in blood vessels, pia mater, and choroid plexus. The C-terminal probe was detected in addition in the main olfactory bulb, striatum, thalamic reticular nucleus, and hypothalamus, suggesting a selective expression of G-utrophin in these neurons. Western blot analysis with isoform-specific antisera confirmed the expression of both full-length and G-utrophin in brain. Immunohistochemically, only full-length utrophin was detected in neurons, in close association with the plasma membrane. In addition, intense staining was seen in blood vessels, meninges, and choroid plexus, selectively localized in the basolateral membrane of immunopositive epithelial cells. The expression pattern of utrophin was already established at early postnatal stages and did not change thereafter. Double-labeling analysis revealed that utrophin and dystrophin are differentially expressed on the cellular and subcellular levels in juvenile and adult brain. Likewise, in mice lacking full-length dystrophin isoforms (mdx mice), no change in utrophin expression and distribution could be detected in brain, although utrophin was markedly up-regulated in muscle cells. These results suggest that utrophin and dystrophin are independently regulated and have distinct functional roles in CNS neurons. J. Comp. Neurol. 422:594–611, 2000. © 2000 Wiley-Liss, Inc.
1. The post-synaptic membranes of neurons and muscle cells are characterized by clusters of transmitter receptors, the number and type of which help to determine synaptic efficacy. Here I briefly review what is known of the mechanism of clustering of nicotinic acetylcholine receptors (AChR) at neuromuscular synapses.
2. The extracellular protein agrin is thought to be secreted by the motor nerve terminal and trigger localized clustering of AChR in the post-synaptic membrane of the skeletal muscle cell.
3. Binding of agrin to its receptor, α-dystroglycan, is followed by rearrangements of the muscle membrane cytoskeleton with localized replacement of dystrophin by utrophin. It remains unclear how these changes relate to the clustering of AChR.
4. In separate studies, RAPsyn/43k protein, a protein associated with the inner face of the post-synaptic membrane was shown to be able to cluster AChR and link them to the cytoskeleton when both proteins were co-transfected into fibroblasts.
5. Mutational studies on RAPsyn identified putative binding domains for AChR and for the cytoskeleton within the RAPsyn primary structure. Targeted disruption of the RAPsyn gene in mice prevented post-synaptic AChR clustering and led to neonatal lethality. Thus RAPsyn might be the final link in the pathway that leads to AChR immobilization in the post-synaptic membrane.
6. The recent observation that active forms of agrin are not restricted to cholinergic regions of the brain suggests that analogous pathways may exist for clustering other receptor types.
Recent advances in the molecular, biochemical, and anatomical aspects of postsynaptic membrane components at the neuromuscular junction (NMJ) are briefly reviewed focussing on assembly, architecture, and function of the multi-subunit dystrophin-protein complex (DPC) and its associated nitric oxide (NO)-signaling complex. Elucidation of unique structural binding motifs of NO-synthases (NOS), and microscopical codistribution of neuronal NOS (nNOS), the major isoform of NOS expressed at the NMJ, with known synaptic proteins, i.e., family members of the DPC, nicotinic acetylcholine receptor (AChR), NMDA-receptor, type-1 sodium and Shaker K+-channel proteins, and linker proteins (e.g., PSD-95, 43K-rapsyn), suggests targeting and assembly of the NO-signaling pathway at postsynaptic membrane components. NO mediates agrin-induced AChR-aggregation and downstream signal transduction in C2 skeletal myotubes while administration of L-arginine, the limiting substrate for NO-biosynthesis, enhances aggregation of synapse-specific components such as utrophin. At the NMJ, NO appears to be a mediator of (1) early synaptic protein clustering, (2) synaptic receptor activity and transmitter release, or (3) downstream signaling for transcriptional control. Multidisciplinary data obtained from cellular and molecular studies and from immunolocalization investigations have led us to propose a working model for step-by-step binding of nNOS, e.g., to subunit domains of targeted and/or preexisting membrane components. Formation of NOS-membrane complexes appears to be governed by agrin-signaling as well as by NO-signaling, supporting the idea that parallel signaling pathways may account for the spatiotemporally defined postsynaptic assembly thereby linking the NOS/NO-signaling cascade to early membrane aggregations and at the right places nearby preexisting targets (e.g., juxtaposition of NO source and target) in synapse formation. Microsc. Res. Tech. 55:171–180, 2001. © 2001 Wiley-Liss, Inc.
Utrophin is an autosomally-encoded homologue of dystrophin, the protein product of the Duchenne muscular dystrophy (DMD) gene. Although, Utrophin is very similar in sequence to dystrophin and possesses many of the protein-binding properties ascribed to dystrophin, both proteins are expressed in an apparently reciprocal manner and may be coordinately regulated. In normal skeletal muscle, Utrophin is found at the neuromuscular junction (NMJ) whereas dystrophin predominates at the sarcolemma. However, during development, and in some myopathies including DMD, utrophin is also found at the sarcolemma. This re-distribution is often associated with a significant increase in the levels of utrophin. At the NMJ utrophin co-localizes with the acetylcholine receptors (AChR) and may play a role in the stabilization of the synaptic cytoskeleton. Because utrophin and dystrophin are so similar, utrophin may be able to replace dystrophin in dystrophin deficient muscle. This review compares the structure and function of utrophin to dystrophin and discusses the rationale behind the use of utrophin as a potential therapeutic agent.
Mutations in the dystrophin gene (DMD) and in genes encoding several dystrophin-associated proteins result in Duchenne and other forms of muscular dystrophy. alpha-Dystroglycan (Dg) binds to laminins in the basement membrane surrounding each myofibre and docks with beta-Dg, a transmembrane protein, which in turn interacts with dystrophin or utrophin in the subplasmalemmal cytoskeleton. alpha- and beta-Dgs are thought to form the functional core of a larger complex of proteins extending from the basement membrane to the intracellular cytoskeleton, which serves as a superstructure necessary for sarcolemmal integrity. Dgs have also been implicated in the formation of synaptic densities of acetylcholine receptors (AChRs) on skeletal muscle. Here we report that chimaeric mice generated with ES cells targeted for both Dg alleles have skeletal muscles essentially devoid of Dgs and develop a progressive muscle pathology with changes emblematic of muscular dystrophies in humans. In addition, many neuromuscular junctions are disrupted in these mice. The ultrastructure of basement membranes and the deposition of laminin within them, however, appears unaffected in Dg-deficient muscles. We conclude that Dgs are necessary for myofibre survival and synapse differentiation or stability, but not for the formation of the muscle basement membrane, and that Dgs may have more than a purely structural function in maintaining muscle integrity.
At the neuromuscular junction (NMJ), the dystrophin protein complex provides a scaffold that functions to stabilize acetylcholine receptor (AChR) clusters. Syntrophin, a key component of that scaffold, is a multidomain adapter protein that links a variety of signaling proteins and ion channels to the dystrophin protein complex. Without syntrophin, utrophin and neuronal nitric oxide synthase mu (nNOSmu) fail to localize to the NMJ and the AChRs are distributed abnormally. Here we investigate the contribution of syntrophin domains to AChR distribution and to localization of utrophin and nNOSmu at the NMJ. Transgenic mice expressing alpha-syntrophin lacking portions of the first pleckstrin homology (PH) domain (DeltaPH1a or DeltaPH1b) or the entire PDZ domain (DeltaPDZ) were bred onto the alpha-syntrophin null background. As expected the DeltaPDZ transgene did not restore the NMJ localization of nNOS. The DeltaPH1a transgene did restore postsynaptic nNOS but surprisingly did not restore sarcolemmal nNOS (although sarcolemmal aquaporin-4 was restored). Mice lacking the alpha-syntrophin PDZ domain or either half of the PH1 domain were able to restore utrophin to the NMJ but did not correct the aberrant AChR distribution of the alpha-syntrophin knock-out mice. However, mice expressing both the transgenic DeltaPDZ and the transgenic DeltaPH1a constructs did restore normal AChR distribution, demonstrating that both domains are required but need not be confined within the same protein to function. We conclude that the PH1 and PDZ domains of alpha-syntrophin work in concert to facilitate the localization of AChRs and nNOS at the NMJ.
Purpose. To determine whether differences in gene expression occur between areas of focal retinal ganglion cell (RGC) loss and of relative RGC preservation in the DBA/2 mouse retina and whether they can provide insight into the pathophysiology of glaucoma. Methods. Areas of focal RGC loss (judged by lack of Fluorogold labeling; Fluorochrome, Denver, CO), adjacent areas with relative RGC preservation in DBA/2 retina, and Fluorogold-labeled retina from DBA/2(-pe) (pearl) mice were dissected and used for microarray analysis. RT-PCR and immunoblot analysis were used to confirm differential gene expression. Bioinformatic analysis was used to identify gene networks affected in the glaucomatous retina. Results. Microarray analysis identified 372 and 115 gene chip IDs as up- and downregulated, respectively, by 0.5-fold in areas of RGC loss. Differentially expressed genes included those coding for cytoskeletal proteins, enzymes, transport proteins, extracellular matrix (ECM) proteins, and immune response proteins. Several genes were confirmed by RT-PCR. For at least two genes, differential protein expression was verified. Bioinformatics analysis identified multiple affected functional gene networks. Pearl mice appeared to have significantly different gene expression, even when compared with relatively preserved areas of the DBA/2 retina. Conclusions. Regional gene expression changes occur in areas of focal RGC loss in the DBA/2 retina. The genes involved code for proteins with diverse cellular functions. Further investigation is needed to determine the cellular localization of the expression of these genes during the development of spontaneous glaucoma in the DBA/2 mouse and to determine whether some of these gene expression changes are causative or protective of RGC loss.
The 58K protein is a peripheral membrane protein enriched in the acetylcholine receptor (AChR)-rich postsynaptic membrane of Torpedo electric organ. Because of its coexistence with AChRs in the postsynaptic membrane in both electrocytes and skeletal muscle, it is thought to be involved in the formation and maintenance of AChR clusters. Using an mAb against the 58K protein of Torpedo electric organ, we have identified a single protein band in SDS-PAGE analysis of Xenopus myotomal muscle with an apparent molecular mass of 48 kD. With this antibody, the distribution of this protein was examined in the myotomal muscle fibers with immunofluorescence techniques. We found that the 48K protein is concentrated at the myotendinous junctions (MTJs) of these muscle fibers. The MTJ is also enriched in talin and vinculin. By double labeling muscle fibers with antibodies against talin and the 48K protein, these two proteins were found to colocalize at the membrane invaginations of the MTJ. In cultured myotomal muscle cells, the 48K protein and talin are also colocalized at sites of membrane-myofibril interaction. The 48K protein is, however, not found at focal adhesion sites in nonmuscle cells, which are enriched in talin. These data suggest that the 48K protein is specifically involved in the interaction of myofibrillar actin filaments with the plasma membrane at the MTJ. In addition to the MTJ localization, 48K protein is also present at AChR clusters both in vivo and in vitro. Thus, this protein is shared by both the MTJ and the neuromuscular junction.
Duchenne and Becker muscular dystrophies are caused by defects of the dystrophin gene. Expression of this large X-linked gene is under elaborate transcriptional and splicing control. At least five independent promoters specify the transcription of their respective alternative first exons in a cell-specific and developmentally controlled manner. Three promoters express full-length dystrophin, while two promoters near the C terminus express the last domains in a mutually exclusive manner. Six exons of the C terminus are alternatively spliced, giving rise to several alternative forms. Genetic, biochemical and anatomical studies of dystrophin suggest that a number of distinct functions are subserved by its great structural diversity. Extensive studies of dystrophin may lead to an understanding of the cause and perhaps a rational treatment for muscular dystrophy.
Duchenne and Becker muscular dystrophies are caused by defects of dystrophin, which forms a part of the membrane cytoskeleton of specialized cells such as muscle. It has been previously shown that the dystrophin-associated protein A1 (59-kDa DAP) is actually a heterogeneous group of phosphorylated proteins consisting of an acidic (alpha-A1) and a distinct basic (beta-A1) component. Partial peptide sequence of the A1 complex purified from rabbit muscle permitted the design of oligonucleotide probes that were used to isolate a cDNA for one human isoform of A1. This cDNA encodes a basic A1 isoform that is distinct from the recently described syntrophins in Torpedo and mouse and is expressed in many tissues with at least five distinct mRNA species of 5.9, 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 A1 and EST25263 genes to chromosomes 8q23-24 and 16, respectively.
The primary sequence of two components of the dystrophin-glycoprotein complex has been established by complementary, DNA cloning. The transmembrane 43K and extracellular 156K dystrophin-associated glycoproteins (DAGs) are encoded by a single messenger RNA and the extracellular 156K DAG binds laminin. Thus, the 156K DAG is a new laminin-binding glycoprotein which may provide a linkage between the sarcolemma and extracellular matrix. These results support the hypothesis that the dramatic reduction in the 156K DAG in Duchenne muscular dystrophy leads to a loss of a linkage between the sarcolemma and extracellular matrix and that this may render muscle fibres more susceptible to necrosis.
Two high-affinity mAbs were prepared against Torpedo dystrophin, an electric organ protein that is closely similar to human dystrophin, the gene product of the Duchenne muscular dystrophy locus. The antibodies were used to localize dystrophin relative to acetylcholine receptors (AChR) in electric organ and in skeletal muscle, and to show identity between Torpedo dystrophin and the previously described 270/300-kD Torpedo postsynaptic protein. Dystrophin was found in both AChR-rich and AChR-poor regions of the innervated face of the electroplaque. Immunogold experiments showed that AChR and dystrophin were closely intermingled in the AChR domains. In contrast, dystrophin appeared to be absent from many or all AChR-rich domains of the rat neuromuscular junction and of AChR clusters in cultured muscle (Xenopus laevis). It was present, however, in the immediately surrounding membrane (deep regions of the junctional folds, membrane domains interdigitating with and surrounding AChR domains within clusters). These results suggest that dystrophin may have a role in organization of AChR in electric tissue. Dystrophin is not, however, an obligatory component of AChR domains in muscle and, at the neuromuscular junction, its roles may be more related to organization of the junctional folds.
Dystrophin-related protein (DRP or 'utrophin') is localized in normal adult muscle primarily at the neuromuscular junction. In the absence of dystrophin in Duchenne muscular dystrophy (DMD) patients, DRP is also present in the sarcolemma. DRP is expressed in fetal and regenerating muscle and may play a similar role to dystrophin in early development, although it remains to be determined whether DRP can functionally replace dystrophin in adult tissue. Previously we described a 3.5-kilobase complementary DNA clone that exhibits 80 per cent homology to the C-terminal domain of dystrophin. This sequence identifies a 13-kilobase transcript that maps to human chromosome 6 (refs 2, 11). Antibodies raised against the gene product identify a polypeptide with a relative molecular mass of about 400K in all tissues examined. To investigate the relationship between DRP and dystrophin in more detail, we have cloned and sequenced the whole DRP cDNA. Homology between DRP and dystrophin extends over their entire length, suggesting that they derive from a common ancestral gene. Comparative analysis of primary sequences highlights regions of functional importance, including those that may mediate the localization of DRP and dystrophin in the muscle cell.
Dystrophin, a protein product of the Duchenne muscular dystrophy gene, is thought to associate with the muscle membrane by way of a glycoprotein complex which was co-purified with dystrophin. Here, we firstly demonstrate direct biochemical evidence for association of the carboxy-terminal region of dystrophin with the glycoprotein complex. The binding site is found to lie further inward than previously expected and confined to the cysteine-rich domain and the first half of the carboxy-terminal domain. Since this portion corresponds well to the region that, when missing, results in severe phenotypes, our finding may provide a molecular basis of the disease.
Dystrophin-related protein (DRP) is an autosomal gene product with high homology to dystrophin. We have used highly specific antibodies to the unique C-terminal peptide sequences of DRP and dystrophin to examine the subcellular localization and biochemical properties of DRP in adult skeletal muscle. DRP is enriched in isolated sarcolemma from control and mdx mouse muscle, but is much less abundant than dystrophin. Immunofluorescence microscopy localized DRP almost exclusively to the neuromuscular junction region in rabbit and mouse skeletal muscle, as well as mdx mouse muscle and denervated mouse muscle. DRP is also present in normal size and abundance and localizes to the neuromuscular junction region in muscle from the dystrophic mouse model dy/dy. Thus, DRP is a junction-specific membrane cytoskeletal protein that may play an important role in the organization of the postsynaptic membrane of the neuromuscular junction.
The stoichiometry, cellular location, glycosylation, and hydrophobic properties of the components in the dystrophin-glycoprotein complex were examined. The 156, 59, 50, 43, and 35 kd dystrophin-associated proteins each possess unique antigenic determinants, enrich quantitatively with dystrophin, and were localized to the skeletal muscle sarcolemma. The 156, 50, 43, and 35 kd dystrophin-associated proteins contained Asn-linked oligosaccharides. The 156 kd dystrophin-associated glycoprotein contained terminally sialylated Ser/Thr-linked oligosaccharides. Dystrophin, the 156 kd, and the 59 kd dystrophin-associated proteins were found to be peripheral membrane proteins, while the 50 kd, 43 kd, and 35 kd dystrophin-associated glycoproteins and the 25 kd dystrophin-associated protein were confirmed as integral membrane proteins. These results demonstrate that dystrophin and its 59 kd associated protein are cytoskeletal elements that are tightly linked to a 156 kd extracellular glycoprotein by way of a complex of transmembrane proteins.
Monoclonal antibodies against dystrophin and the postsynaptic 58 kDa protein from Torpedo electric organ were used to localize homologs of these proteins in cultured skeletal muscle (Xenopus laevis). The Xenopus homolog is an Mr 48,000 protein and, like dystrophin, is a sarcolemmal protein. Both proteins localized precisely to talin-positive sites, hence with each other, on the substrate-apposed sarcolemma. Therefore, the first sites of appearance of dystrophin on cultured muscle cells are focal adhesions, i.e. specific sites of cytoskeleton/extracellular matrix interaction. These data also add to evidence that dystrophin and the 58 kDa act together.
The complete sequence of the human Duchenne muscular dystrophy (DMD) cDNA has been determined. The 3685 encoded amino acids of the protein product, dystrophin, can be separated into four domains. The 240 amino acid N-terminal domain has been shown to be conserved with the actin-binding domain of alpha-actinin. A large second domain is predicted to be rod-shaped and formed by the succession of 25 triple-helical segments similar to the repeat domains of spectrin. The repeat segment is followed by a cysteine-rich segment that is similar in part to the entire COOH domain of the Dictyostelium alpha-actinin, while the 420 amino acid C-terminal domain of dystrophin does not show any similarity to previously reported proteins. The functional significance of some of the domains is addressed relative to the phenotypic characteristics of some Becker muscular dystrophy patients. Dystrophin shares many features with the cytoskeletal protein spectrin and alpha-actinin and is a large structural protein that is likely to adopt a rod shape about 150 nm in length.
The protein product of the human Duchenne muscular dystrophy locus (DMD) and its mouse homolog (mDMD) have been identified by using polyclonal antibodies directed against fusion proteins containing two distinct regions of the mDMD cDNA. The DMD protein is shown to be approximately 400 kd and to represent approximately 0.002% of total striated muscle protein. This protein is also detected in smooth muscle (stomach). Muscle tissue isolated from both DMD-affected boys and mdx mice contained no detectable DMD protein, suggesting that these genetic disorders are homologous. Since mdx mice present no obvious clinical abnormalities, the identification of the mdx mouse as an animal model for DMD has important implications with regard to the etiology of the lethal DMD phenotype. We have named the protein dystrophin because of its identification via the isolation of the Duchenne muscular dystrophy locus.
Syntrophin, a 58 kd extrinsic membrane protein, is concentrated at postsynaptic sites at the neuromuscular junction and may be involved in clustering acetylcholine receptors. In muscle and nonmuscle tissues, syntrophin is associated with dystrophin, utrophin, and two homologs of the dystrophin carboxy-terminal region. We have isolated three cDNAs encoding Torpedo and mouse syntrophins. The Torpedo cDNA encodes a full-length protein, and on Northern blots recognizes a 3.5 kb mRNA. The two mouse syntrophin cDNAs are products of separate genes but encode proteins that share 50% identity. Syntrophin-1 mRNA (2.2 kb) is expressed at highest levels in skeletal muscle. Syntrophin-2 mRNAs (2.2, 5.0, and 10 kb) are expressed in all mouse tissues examined. These patterns of expression suggest that syntrophin-1 and syntrophin-2 may associate with different members of the dystrophin family.
Direct interaction between the C-terminal portion of dystrophin and dystrophin-associated proteins was investigated. The binding of dystrophin to each protein was reconstituted by overlaying bacterially expressed dystrophin fusion proteins onto the blot membranes to which dystrophin-associated proteins were transferred after separation by SDS/PAGE with the following results. (a) Among the components of the glycoprotein complex which links dystrophin to the sarcolemma, a 43-kDa dystrophin-associated glycoprotein binds directly to dystrophin. Although at least one of the binding sites of this protein resides within the cysteine-rich domain of dystrophin, a contribution of additional amino acid residues within the first half of the C-terminal domain was also suggested for more secure binding. (b) Two other proteins also directly bind to dystrophin. Their binding sites are suggested to reside within the last half of the C-terminal domain which is alternatively spliced depending on the tissue type.
Previously, based on the enzyme digestion experiments, we showed that the binding site for the glycoprotein complex on dystrophin is present within the cysteine-rich domain and the first half of the C-terminal domain [Suzuki, A., Yoshida, M., Yamamoto, H. & Ozawa, E. (1992) FEBS Lett. 308, 154–160]. Here, we have extended this work and found that the region which is involved in interaction with the complex is widely extended to the entire length of this part of the molecule. On the basis of the present results, we propose a model of molecular architecture at the binding site for the complex on dystrophin.
Postsynaptic peripheral membrane proteins at the neuromuscular junction have been proposed to participate in the immobilization of the nicotinic acetylcholine receptor at the synapse. An 87 kd cytoplasmic peripheral membrane protein has been demonstrated to colocalize with the nicotinic acetylcholine receptor in the Torpedo electric organ and at the mammalian neuromuscular junction. We have cloned the cDNA encoding the 87K protein from Torpedo electric organ, and the predicted protein sequence is homologous to the C-terminal domains of dystrophin, the protein product of the Duchenne muscular dystrophy gene. The 87K protein displays a restricted pattern of expression detected only in electric organ, brain, and skeletal muscle. Analysis of the in vitro and in vivo phosphorylation of the 87K protein indicates that it is multiply phosphorylated on serine, threonine, and tyrosine residues. The 87K protein is in a complex with other proteins associated with the postsynaptic membrane, including dystrophin and a 58 kd protein. These results suggest that the 87K protein is involved in the formation and stability of synapses and is regulated by protein phosphorylation.
An X chromosome-linked mouse mutant (gene symbol, mdx) has been found that has elevated plasma levels of muscle creatine kinase and pyruvate kinase and exhibits histological lesions characteristic of muscular dystrophy. The mutants show mild clinical symptoms and are viable and fertile. Linkage analysis with four X chromosome loci indicates that mdx maps in the Hq Bpa region of the mouse X chromosome. This gives a gene order of mdx-Tfm-Pgk-1-Ags, the same as for the equivalent genes on the human X chromosome.