Interaction of gamma 1-syntrophin with diacylglycerol kinase-zeta. Regulation of nuclear localization by PDZ interactions

Syntrophins are modular adapter proteins that link ion channels and signaling proteins to dystrophin and its homologues. A yeast two-hybrid screen of a human brain cDNA library using the PDZ domain of gamma 1- syntrophin, a recently identified brain-specific isoform, yielded overlapping clones encoding the C terminus of diacylglycerol kinase-zeta (DGK-zeta), an enzyme that converts diacylglycerol into phosphatidic acid. In biochemical assays, the C terminus of DGK-zeta, which contains a consensus PDZ-binding motif, was found to be necessary and sufficient for association with gamma 1-syntrophin. When coexpressed in HeLa cells, DGK-zeta and gamma 1-syntrophin formed a stable complex that partitioned between the cytoplasm and nucleus. DGK-zeta translocates from the cytosol to the nucleus, a process negatively regulated by protein kinase C phosphorylation. We found that DGK-zeta recruits gamma 1-syntrophin into the nucleus and that the PDZ-binding motif is required. Disrupting the interaction altered the intracellular localization of both proteins; DGK-zeta accumulated in the nucleus, whereas gamma 1-syntrophin remained in the cytoplasm. The level of endogenous syntrophins in the nucleus of HeLa cells also reflected the amount of nuclear DGK-zeta. In the brain, DGK-zeta and gamma 1-syntrophin were colocalized in cell bodies and dendrites of cerebellar Purkinjie neurons and other neuronal cell types, suggesting that their interaction is physiologically relevant. Moreover, coimmunoprecipitation and pull-down experiments from brain extracts and cells suggest that DGK-zeta, gamma 1-syntrophin, and dystrophin form a ternary complex. Collectively, our results suggest that gamma 1-syntrophin participates in regulating the subcellular localization of DGK-zeta to ensure correct termination of diacylglycerol signaling.     

Mammalian alpha 1- and beta 1-syntrophin bind to the alternative splice- prone region of the dystrophin COOH terminus

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.     

A PDZ-containing scaffold related to the dystrophin complex at the basolateral membrane of epithelial cells

Membrane scaffolding complexes are key features of many cell types, serving as specialized links between the extracellular matrix and the actin cytoskeleton. An important scaffold in skeletal muscle is the dystrophin-associated protein complex. One of the proteins bound directly to dystrophin is syntrophin, a modular protein comprised entirely of interaction motifs, including PDZ (protein domain named for PSD-95, discs large, ZO-1) and pleckstrin homology (PH) domains. In skeletal muscle, the syntrophin PDZ domain recruits sodium channels and signaling molecules, such as neuronal nitric oxide synthase, to the dystrophin complex. In epithelia, we identified a variation of the dystrophin complex, in which syntrophin, and the dystrophin homologues, utrophin and dystrobrevin, are restricted to the basolateral membrane. We used exogenously expressed green fluorescent protein (GFP)-tagged fusion proteins to determine which domains of syntrophin are responsible for its polarized localization. GFP-tagged full-length syntrophin targeted to the basolateral membrane, but individual domains remained in the cytoplasm. In contrast, the second PH domain tandemly linked to a highly conserved, COOH-terminal region was sufficient for basolateral membrane targeting and association with utrophin. The results suggest an interaction between syntrophin and utrophin that leaves the PDZ domain of syntrophin available to recruit additional proteins to the epithelial basolateral membrane. The assembly of multiprotein signaling complexes at sites of membrane specialization may be a widespread function of dystrophin-related protein complexes.     

In vivo requirement of the alpha-syntrophin PDZ domain for the sarcolemmal localization of nNOS and aquaporin-4

alpha-Syntrophin is a scaffolding adapter protein expressed primarily on the sarcolemma of skeletal muscle. The COOH-terminal half of alpha-syntrophin binds to dystrophin and related proteins, leaving the PSD-95, discs-large, ZO-1 (PDZ) domain free to recruit other proteins to the dystrophin complex. We investigated the function of the PDZ domain of alpha-syntrophin in vivo by generating transgenic mouse lines expressing full-length alpha-syntrophin or a mutated alpha-syntrophin lacking the PDZ domain (Delta PDZ). The Delta PDZ alpha-syntrophin displaced endogenous alpha- and beta 1-syntrophin from the sarcolemma and resulted in sarcolemma containing little or no syntrophin PDZ domain. As a consequence, neuronal nitric oxide synthase (nNOS) and aquaporin-4 were absent from the sarcolemma. However, the sarcolemmal expression and distribution of muscle sodium channels, which bind the alpha-syntrophin PDZ domain in vitro, were not altered. Both transgenic mouse lines were bred with an alpha-syntrophin-null mouse which lacks sarcolemmal nNOS and aquaporin-4. The full-length alpha-syntrophin, not the Delta PDZ form, reestablished nNOS and aquaporin-4 at the sarcolemma of these mice. Genetic crosses with the mdx mouse showed that neither transgenic syntrophin could associate with the sarcolemma in the absence of dystrophin. Together, these data show that the sarcolemmal localization of nNOS and aquaporin-4 in vivo depends on the presence of a dystrophin-bound alpha-syntrophin PDZ domain.     

Mouse alpha1-syntrophin binding to Grb2: further evidence of a role for syntrophin in cell signaling

Syntrophins have been proposed to serve as adapter proteins. Syntrophins are found in the dystrophin glycoprotein complex (DGC); defects in the constituents of this complex are linked to various muscular dystrophies. Blot overlay experiments demonstrate that alpha-dystroglycan, beta-dystroglycan, and syntrophins all bind Grb2, the growth factor receptor bound adapter protein. Mouse alpha1-syntrophin sequences were produced as chimeric fusion proteins in bacteria and found to also bind Grb2 in a Ca2+-independent manner. This binding was localized to the proline rich sequences adjacent to and overlapping with the N-terminal pleckstrin homology domain (PH1). Grb2 bound syntrophin with an apparent KD of 563 +/- 15 nM. Grb2-C-SH3 domain bound syntrophin with slightly higher affinity than Grb2-N-SH3 domain. Crk-L, an SH2/SH3 protein of similar domain structure but different specificity, does not bind these syntrophin sequences.     

The carboxyterminus of the ATP-binding cassette transporter A1 interacts with a beta2-syntrophin/utrophin complex

Recent work identified ABCA1 as the major regulator of plasma HDL-cholesterol responsible for the removal of excess choline-phospholipids and cholesterol from peripheral cells and tissues. ABCA1 function may depend on the association with heteromeric proteins and to identify these candidates a human liver yeast two-hybrid library was screened with the carboxyterminal 144 amino acids of ABCA1. Beta2-syntrophin was found to interact with ABCA1 and the C-terminal five amino acids of ABCA1 proned to represent a perfect tail for binding to syntrophin PDZ domains. Immunoprecipitation further confirmed the association of ABCA1 and beta2-syntrophin and in addition utrophin, known to couple beta2-syntrophin and its PDZ ligands to the F-actin cytoskeleton, was identified as a constituent of this complex. ABCA1 in the plasmamembrane of human macrophages was found to be partially associated with Lubrol rafts and effluxed choline-phospholipids involve these microdomains. Beta2-syntrophin does not colocalize in these rafts indicating that beta2-syntrophin may participate in the retaining of ABCA1 in cytoplasmic vesicles and for the targeting of ABCA1 to plasmamembrane microdomains when ABCA1 is released from beta2-syntrophin.     

Molecular heterogeneity of the dystrophin-associated protein complex in the mouse kidney nephron: differential alterations in the absence of utrophin and dystrophin

The dystrophin-associated protein complex (DPC) consisting of syntrophin, dystrobrevin, and dystroglycan isoforms is associated either with dystrophin or its homolog utrophin. It is present not only in muscle cells, but also in numerous tissues, including kidney, liver, and brain. Using high-resolution immunofluorescence imaging and Western blotting, we have investigated the effects of utrophin and dystrophin gene deletion on the formation and membrane anchoring of the DPC in kidney epithelial cells, which co-express utrophin and low levels of the C-terminal dystrophin isoform Dp71. We show that multiple, molecularly distinct DPCs co-exist in the nephron; these DPCs have a segment-specific distribution and are only partially associated with utrophin in the basal membrane of tubular epithelial cells. In utrophin-deficient mice, a selective reduction of 2-syntrophin has been observed in medullary tubular segments, whereas 1-syntrophin and 1-syntrophin are retained, concomintant with an upregulation of -dystroglycan, -dystrobrevin, and Dp71. These findings suggest that 2-syntrophin is dependent on utrophin for association with the DPC, and that loss of utrophin is partially compensated by Dp71, allowing the preservation of the DPC in kidney epithelial cells. This hypothesis is confirmed by the almost complete loss of all DPC proteins examined in mice lacking full-length utrophin and all C-terminal dystrophin isoforms (utrophin^0/0/mdx^3Cv). The DPC thus critically depends on these proteins for assembly and/or membrane localization in kidney epithelial cells. This project was supported by the Swiss National Science Foundation (no. 31-63901.00 to J.M.F.).     

The phosphoinositol 3,4-bisphosphate-binding protein TAPP1 interacts with syntrophins and regulates actin cytoskeletal organization

Syntrophins are scaffold proteins of the dystrophin glycoprotein complex (DGC), which target ion channels, receptors, and signaling proteins to specialized subcellular domains. A yeast two-hybrid screen of a human brain cDNA library with the PSD-95, Discs-large, ZO-1 (PDZ) domain of gamma1-syntrophin yielded overlapping clones encoding the C terminus of TAPP1, a pleckstrin homology (PH) domain-containing adapter protein that interacts specifically with phosphatidylinositol 3,4-bisphosphate (PI(3,4)P(2)). In biochemical assays, the C terminus of TAPP1 bound specifically to the PDZ domains of gamma1-, alpha1-, and beta2-syntrophin and was required for syntrophin binding and for the correct subcellular localization of TAPP1. TAPP1 is recruited to the plasma membrane of cells stimulated with platelet-derived growth factor (PDGF), a motogen that produces PI(3,4)P(2). Cell migration in response to PDGF stimulation is characterized by a rapid reorganization of the actin cytoskeleton, which gives rise to plasma membrane specializations including peripheral and dorsal circular ruffles. Both TAPP1 and syntrophins were localized to PDGF-induced circular membrane ruffles in NIH-3T3 cells. Ectopic expression of TAPP1 potently blocked PDGF-induced formation of dorsal circular ruffles, but did not affect peripheral ruffling. Interestingly, coexpression of alpha1- or gamma1-syntrophin with TAPP1 prevented the blockade of circular ruffling. In addition to syntrophins, several other proteins of the DGC were enriched in circular ruffles. Collectively, our results suggest syntrophins regulate the localization of TAPP1, which may be important for remodeling the actin cytoskeleton in response to growth factor stimulation.     

Dystroglycan contributes to the formation of multiple dystrophin-like complexes in brain

In muscle, dystrophin anchors a complex of proteins at the cell surface which includes alpha-dystroglycan, beta-dystroglycan, syntrophins and dystrobrevins. Mutations in the dystrophin gene lead to muscular dystrophy and mental retardation. In contrast to muscle, little is known about the localization and the molecular interactions of dystrophin and dystrophin associated proteins (DAPs) in brain. In the present study, we show that alpha-dystroglycan and dystrophin are localized to large neurones in cerebral cortex, hippocampus, cerebellum and spinal cord. Furthermore, we show that dystroglycan is a member of three distinct dystrophin-containing complexes. Two of these complexes contain syntrophin and both dystrophin and syntrophin are enriched in post-synaptic densities. These data suggest that dystrophin and DAPs may have a role in the organization of CNS synapses. Interestingly, the enrichment for syntrophin in post-synaptic densities is not affected in mice mutant for all dystrophin isoforms. Thus in the brain, unlike in muscle, the association of syntrophin with dystrophin is not crucial for the DAP complex which suggests that it may be associated with other proteins.     

Protein trafficking and anchoring complexes revealed by proteomic analysis of inward rectifier potassium channel (Kir2.x)-associated proteins

Inward rectifier potassium (Kir) channels play important roles in the maintenance and control of cell excitability. Both intracellular trafficking and modulation of Kir channel activity are regulated by protein-protein interactions. We adopted a proteomics approach to identify proteins associated with Kir2 channels via the channel C-terminal PDZ binding motif. Detergent-solubilized rat brain and heart extracts were subjected to affinity chromatography using a Kir2.2 C-terminal matrix to purify channel-interacting proteins. Proteins were identified with multidimensional high pressure liquid chromatography coupled with electrospray ionization tandem mass spectrometry, N-terminal microsequencing, and immunoblotting with specific antibodies. We identified eight members of the MAGUK family of proteins (SAP97, PSD-95, Chapsyn-110, SAP102, CASK, Dlg2, Dlg3, and Pals2), two isoforms of Veli (Veli-1 and Veli-3), Mint1, and actin-binding LIM protein (abLIM) as Kir2.2-associated brain proteins. From heart extract purifications, SAP97, CASK, Veli-3, and Mint1 also were found to associate with Kir2 channels. Furthermore, we demonstrate for the first time that components of the dystrophin-associated protein complex, including alpha1-, beta1-, and beta2-syntrophin, dystrophin, and dystrobrevin, interact with Kir2 channels, as demonstrated by immunoaffinity purification and affinity chromatography from skeletal and cardiac muscle and brain. Affinity pull-down experiments revealed that Kir2.1, Kir2.2, Kir2.3, and Kir4.1 all bind to scaffolding proteins but with different affinities for the dystrophin-associated protein complex and SAP97, CASK, and Veli. Immunofluorescent localization studies demonstrated that Kir2.2 co-localizes with syntrophin, dystrophin, and dystrobrevin at skeletal muscle neuromuscular junctions. These results suggest that Kir2 channels associate with protein complexes that may be important to target and traffic channels to specific subcellular locations, as well as anchor and stabilize channels in the plasma membrane.     

α-Syntrophin is required for the hepatocyte growth factor-induced migration of cultured myoblasts

Syntrophins are adaptor proteins that link intracellular signaling molecules to the dystrophin based scaffold. In this study, we investigated the function of syntrophins in cell migration, one of the early steps in myogenic differentiation and in regeneration of adult muscle. Hepatocyte growth factor (HGF) stimulates migration and lamellipodia formation in cultured C2 myoblasts. In the migrating cells, syntrophin concentrated in the rear-lateral region of the cell, opposite of the lamellipodia, instead of being diffusely present throughout the cytoplasm of non-migrating cells. When the expression of α-syntrophin, the major syntrophin isoform of skeletal muscle, was reduced by transfection with the α-syntrophin-specific siRNA, HGF stimulation of lamellipodia formation was prevented. Likewise, migration of myoblasts from α-syntrophin knockout mice could not be stimulated by HGF. However, HGF-induced migration was restored in myoblasts isolated from a transgenic mouse expressing α-syntrophin only in muscle cells. Treatment of C2 myoblasts with inhibitors of PI3-kinase not only reduced the rate of cell migration, but also impaired the accumulation of syntrophins in the rear-lateral region of the migrating cells. Phosphorylation of Akt was reduced in the α-syntrophin siRNA-treated C2 cells. These results suggest that α-syntrophin is required for HGF-induced migration of myoblasts and for proper PI3-kinase/Akt signaling.     

Interaction of alpha1-syntrophin with multiple isoforms of heterotrimeric G protein alpha subunits

Syntrophins are components of the dystrophin-glycoprotein complex of the plasma membrane in muscular and neuronal cells, and recruit signaling proteins such as neuronal nitric oxide synthase via their multiple protein-protein interaction motifs. In this study, we found that alpha1-syntrophin binds to various subtypes of guanine nucleotide-binding protein alpha subunits (Galpha). A pull-down analysis using full-length recombinant alpha1-syntrophin and MS analysis showed that alpha1-syntrophin was coprecipitated with several isoforms of Galpha proteins in addition to known binding partners such as dystrobrevin and neuronal nitric oxide synthase. Further analysis using recombinant Galpha isoforms showed that alpha1-syntrophin associates with at least Galphai, Galphao, Galphas and Galphaq subtypes. The region of alpha1-syntrophin required for its interaction with Galphas was determined as the N-terminal half of the first pleckstrin homology domain. In addition, the syntrophin unique domain of alpha1-syntrophin was suggested to contribute to this interaction. In COS-7 cells, downregulation of alpha1-syntrophin by RNAi resulted in enhanced cAMP production and cAMP response element-binding protein phosphorylation induced by isoproterenol treatment. These results suggest that alpha1-syntrophin provides a scaffold for the Galpha family of heterotrimeric G proteins in the brain to regulate the efficiency of signal transduction evoked by G-protein-coupled receptors.     

gamma-Syntrophin scaffolding is spatially and functionally distinct from that of the alpha/beta syntrophins

The syntrophins are a family of scaffolding proteins with multiple protein interaction domains that link signaling proteins to dystrophin family members. Each of the three most characterized syntrophins (alpha, beta1, beta2) contains a PDZ domain that binds a unique set of signaling proteins including kinases, ion and water channels, and neuronal nitric oxide synthase (nNOS). The PDZ domains of the gamma-syntrophins do not bind nNOS. In vitro pull-down assays show that the gamma-syntrophins can bind dystrophin but have unique preferences for the syntrophin binding sites of dystrophin family members. Despite their ability to bind dystrophin in vitro, neither gamma-syntrophin isoform co-localizes with dystrophin in skeletal muscle. Furthermore, gamma-syntrophins do not co-purify with dystrophin isolated from mouse tissue. These data suggest that the interaction of gamma-syntrophin with dystrophin is transient and potentially subject to regulatory mechanisms. gamma1-Syntrophin is highly expressed in brain and is specifically localized in hippocampal pyramidal neurons, Purkinje neurons in cerebellum, and cortical neurons. gamma2-Syntrophin is expressed in many tissues including skeletal muscle where it is found only in the subsynaptic space beneath the neuromuscular junction. In both neurons and muscle, gamma-syntrophin isoforms localize to the endoplasmic reticulum where they may form a scaffold for signaling and trafficking.     

Phosphorylation of dystrophin and alpha-syntrophin by Ca(2+)-calmodulin dependent protein kinase II.

A Ca(2+)-calmodulin dependent protein kinase activity (DGC-PK) was previously shown to associate with skeletal muscle dystrophin glycoprotein complex (DGC) preparations, and phosphorylate dystrophin and a protein with the same electrophoretic mobility as alpha-syntrophin (R. Madhavan, H.W. Jarrett, Biochemistry 33 (1994) 5797-5804). Here, we show that DGC-PK and Ca(2+)-calmodulin dependent protein kinase II (CaM kinase II) phosphorylate a common site (RSDS(3616)) within the dystrophin C terminal domain that fits the consensus CaM kinase II phosphorylation motif (R/KXXS/T). Furthermore, both kinase activities phosphorylate exactly the same three fusion proteins (dystrophin fusions DysS7 and DysS9, and the syntrophin fusion) out of a panel of eight fusion proteins (representing nearly 100% of syntrophin and 80% of dystrophin protein sequences), demonstrating that DGC-PK and CaM kinase II have the same substrate specificity. Complementing these results, anti-CaM kinase II antibodies specifically stained purified DGC immobilized on nitrocellulose membranes. Renaturation of electrophoretically resolved DGC proteins revealed a single protein kinase band (M(r) approximately 60,000) that, like CaM kinase II, underwent Ca(2+)-calmodulin dependent autophosphorylation. Based on these observations, we conclude DGC-PK represents a dystrophin-/syntrophin-phosphorylating skeletal muscle isoform of CaM kinase II. We also show that phosphorylation of the dystrophin C terminal domain sequences inhibits their syntrophin binding in vitro, suggesting a regulatory role for phosphorylation.     

The receptor tyrosine phosphatase-like protein ICA512 binds the PDZ domains of beta2-syntrophin and nNOS in pancreatic beta-cells

Islet cell autoantigen (ICA) 512 of type I diabetes is a receptor tyrosine phosphatase-like protein associated with the secretory granules of neurons and endocrine cells including insulin-secreting beta-cells of the pancreas. Here we show that in a yeast two-hybrid assay its cytoplasmic domain binds beta2-syntrophin, a modular adapter which in muscle cells interacts with members of the dystrophin family including utrophin, as well as the signaling molecule neuronal nitric oxide synthase (nNOS). The cDNA isolated by two-hybrid screening corresponded to a novel beta2-syntrophin isoform with a predicted molecular mass of 28 kDa. This isoform included the PDZ domain, but not the C-terminal region, which in full-length beta2-syntrophin is responsible for binding dystrophin-related proteins. In vitro binding of the beta2-syntrophin PDZ domain to ICA512 required both ICA512's C-terminal region and an internal polypeptide preceding its tyrosine phosphatase-like domain. Immunomicroscopy and co-immunoprecipitations from insulinoma INS-1 cells confirmed the occurrence of ICA512-beta2-syntrophin complexes in vivo. ICA512 also interacted in vitro with the PDZ domain of nNOS and ICA512-nNOS complexes were co-immunoprecipitated from INS-1 cells. Finally, we show that INS-1 cells, like muscle cells, contain beta2-syntrophin-utrophin oligomers. Thus, we propose that ICA512, through beta2-syntrophin and nNOS, links secretory granules with the actin cytoskeleton and signaling pathways involving nitric oxide.     

Altered alpha1-syntrophin expression in myofibers with Duchenne and Fukuyama muscular dystrophies

Alpha1-syntrophin, a scaffolding adapter and modular protein, is a cytoplasmic component of the dystrophin glycoprotein complex. This study investigated immunohistochemically the expression of alpha1-syntrophin in Duchenne and Fukuyama muscular dystrophies (DMD and FCMD, respectively). Biopsied muscles of five DMD, five FCMD, five normal controls and five disease controls (three myotonic and two facioscapulohumeral dystrophies) were analyzed. Immunoblot analysis showed that anti-alpha1-syntrophin antibody had a decreased reaction in both DMD and FCMD muscle extracts. Biopsied muscle sections and their serial sections were immunostained with rabbit anti-alpha1-syntrophin and rabbit anti-muscle-specific beta-spectrin antibodies, respectively. Immunoreactive patterns of sarcolemma were classified into (i) a continuously positive immunostaining pattern, (ii) a partially positive immunostaining pattern, (iii) a negative immunostaining pattern and (iv) a faint but entire surface positive immunostaining pattern. The group mean percentages of alpha1-syntrophin and beta-spectrin immunonegative myofibers in the DMD group were 39.3% and 10.8%, respectively, while those in the FCMD group were 45.5% and 10.4%, respectively. These values were statistically significant compared with those of disease control and normal control muscles. Thus we found that dystrophin-deficient DMD muscles contained significant numbers of alpha1-syntrophin-positive fibers and significant numbers of alpha1-syntrophin-negative fibers were present in dystrophin-positive muscles of severe muscular dystrophy such as FCMD. Alpha-dystrobrevin immunoreactivity was tested in DMD muscles and appreciable amounts of alpha-dystrobrevin that binds to syntrophin were found in DMD muscle membranes.     

Dystrophin/α1-syntrophin scaffold regulated PLC/PKC-dependent store-operated calcium entry in myotubes

In skeletal muscles from patient suffering of Duchenne Muscular Dystrophy and from mdx mice, the absence of the cytoskeleton protein dystrophin has been shown to be essential for maintaining a normal calcium influx. We showed that a TRPC store-dependent cation influx is increased by loss of dystrophin or a scaffolding protein α1-syntrophin, however the mechanisms of this calcium mishandling are incompletely understood. First of all, we confirmed that TRPC1 but also STIM1 and Orai1 are supporting the store-operated cation entry which is enhanced in dystrophin-deficient myotubes. Next, we demonstrated that inhibition of PLC or PKC in dystrophin-deficient myotubes restores elevated cation entry to normal levels similarly to enforced minidystrophin expression. In addition, silencing α1-syntrophin also increased cation influx in a PLC/PKC dependent pathway. We also showed that α1-syntrophin and PLCβ are part of a same protein complex reinforcing the idea of their inter-relation in calcium influx regulation. This elevated cation entry was decreased to normal levels by chelating intracellular free calcium with BAPTA-AM. Double treatments with BAPTA-AM and PLC or PKC inhibitors suggested that the elevation of cation influx by PLC/PKC pathway is dependent on cytosolic calcium. All these results demonstrate an involvement in dystrophin-deficient myotubes of a specific calcium/PKC/PLC pathway in elevation of store-operated cation influx supported by the STIM1/Orai1/TRPC1 proteins, which is normally regulated by the α1-syntrophin/dystrophin scaffold.     

Absence of alpha-syntrophin leads to structurally aberrant neuromuscular synapses deficient in utrophin

The syntrophins are a family of structurally related proteins that contain multiple protein interaction motifs. Syntrophins associate directly with dystrophin, the product of the Duchenne muscular dystrophy locus, and its homologues. We have generated alpha-syntrophin null mice by targeted gene disruption to test the function of this association. The alpha-Syn(-/)- mice show no evidence of myopathy, despite reduced levels of alpha-dystrobrevin-2. Neuronal nitric oxide synthase, a component of the dystrophin protein complex, is absent from the sarcolemma of the alpha-Syn(-/)- mice, even where other syntrophin isoforms are present. alpha-Syn(-/)- neuromuscular junctions have undetectable levels of postsynaptic utrophin and reduced levels of acetylcholine receptor and acetylcholinesterase. The mutant junctions have shallow nerve gutters, abnormal distributions of acetylcholine receptors, and postjunctional folds that are generally less organized and have fewer openings to the synaptic cleft than controls. Thus, alpha-syntrophin has an important role in synapse formation and in the organization of utrophin, acetylcholine receptor, and acetylcholinesterase at the neuromuscular synapse.     

The dystrophin / utrophin homologues in Drosophila and in sea urchin

The gene which is defective in Duchenne muscular dystrophy (DMD) is the largest known gene containing at least 79 introns, some of which are extremely large. The product of the gene in muscle, dystrophin, is a 427 kDa protein. The same gene encodes at least two additional non-muscle full length dystrophin isoforms transcribed from different promoters located in the 5'-end region of the gene, and four smaller proteins transcribed from internal promoters located further downstream, and lack important domains of dystrophin. Several other genes, encoding evolutionarily related proteins, have been identified. To study the evolution of the DMD gene and the significance of its various products, we have searched for genes encoding dystrophin-like proteins in sea urchin and in Drosophila. We previously reported on the characterization of a sea urchin gene encoding a protein which is an evolutionary homologue of Dp116, one of the small products of the mammalian DMD gene, and on the partial sequencing of a large product of the same gene. Here we describe the full-length product which shows strong structural similarity and sequence identity to human dystrophin and utrophin. We also describe a Drosophila gene closely related to the human dystrophin gene. Like the human gene, the Drosophila gene encodes at least three isoforms of full length dystrophin-like proteins (dmDLP1, dmDLP2 and dmDLP3,), regulated by different promoters located at the 5' end of the gene, and a smaller product regulated by an internal promoter (dmDp186). As in mammals, dmDp186 and the dmDLPs share the same C-terminal and cysteine-rich domains which are very similar to the corresponding domains in human dystrophin and utrophin. In addition, dmDp186 contains four of the spectrin-like repeats of the dmDLPs and a unique N-terminal region of 512 amino acids encoded by a single exon. The full length products and the small product have distinct patterns of expression. Thus, the complex structure of the dystrophin gene, encoding several large dystrophin-like isoforms and smaller truncated products with different patterns of expression, existed before the divergence between the protostomes and deuterostomes. The conservation of this gene structure in such distantly related organisms, points to important distinct functions of the multiple products.