ABSTRACT
The dystrophin glycoprotein complex (DGC) is a large multisubunit complex located throughout the sarcolemma of striated muscle fibers. This complex is critical for maintaining the structural integrity of muscle fibers during muscle contraction and also provides a scaffold for signaling molecules. Defects in some components of the DGC, such as dystrophin and sarcoglycans, disrupt the complex and lead to muscular dystrophies. Alpha-dystrobrevin is a dystrophin-related component of the DGC that is localized to the cytoplasmic side of the sarcolemma. In skeletal muscle, alpha-dystrobrevin is also highly concentrated at the neuromuscular junction, a highly specialized region of the sarcolemma responsible for receiving motor nerve signals necessary for muscle contraction. Current evidence suggests that alpha-dystrobrevin plays an important role in signaling at the sarcolemma and in the maturation and maintenance of the postsynaptic apparatus at the neuromuscular junction. In this review, we summarize the currently known cellular and molecular properties of alpha-dystrobrevin in skeletal muscle and discuss its potential functions at both the sarcolemma and neuromuscular junction.
Subject(s)
Cytoskeletal Proteins/metabolism , Dystrophin-Associated Proteins , Membrane Proteins/metabolism , Muscle, Skeletal/metabolism , Alternative Splicing , Animals , Cloning, Molecular , Cytoskeletal Proteins/deficiency , Cytoskeletal Proteins/genetics , DNA, Complementary/genetics , Gene Expression , Gene Expression Regulation, Developmental , Membrane Proteins/deficiency , Membrane Proteins/genetics , Mice , Mice, Knockout , Muscle, Skeletal/cytology , Muscle, Skeletal/ultrastructure , Muscular Dystrophies/metabolism , Muscular Dystrophies/pathology , Neuromuscular Junction/metabolism , Organ Specificity , Protein Binding , Receptors, Cholinergic/metabolism , Sarcolemma/metabolism , Subcellular Fractions/metabolismABSTRACT
Muscle nicotinic acetylcholine receptors (AChRs) are immobilized at the neuromuscular junction in high-density clusters by rapsyn, a 43-kDa protein located at the cytoplasmic face of the postsynaptic membrane. When expressed in nonmuscle cells, rapsyn induces the aggregation of both assembled and unassembled AChR subunits. Here, we investigated the mechanism of rapsyn-induced clustering of the AChR alpha subunit by testing a series of alpha subunit mutants for colocalization with rapsyn patches in transfected QT6 cells. Partial or total deletion of the large intracellular domain of the alpha subunit dramatically reduced its ability to colocalize with rapsyn patches. Furthermore, insertion of the alpha subunit large intracellular domain into a potassium channel resulted in a significant increase in the channel's colocalization with rapsyn patches. We conclude that the large intracellular domain of the alpha subunit plays an important role in mediating rapsyn-induced coclustering of the AChR alpha subunit.
Subject(s)
Muscle Proteins/metabolism , Potassium Channels, Voltage-Gated , Receptors, Nicotinic , Animals , Antibodies, Monoclonal , Cell Line , DNA Primers , Fibroblasts/chemistry , Fibroblasts/cytology , Fibroblasts/metabolism , Fluorescent Antibody Technique , Gene Deletion , Kv1.2 Potassium Channel , Mutagenesis/physiology , Plasmids , Potassium Channels/analysis , Potassium Channels/genetics , Potassium Channels/immunology , Protein Structure, Tertiary , Quail , Receptors, Nicotinic/chemistry , Receptors, Nicotinic/genetics , Receptors, Nicotinic/metabolism , Recombinant Fusion Proteins/analysis , Recombinant Fusion Proteins/immunology , Recombinant Fusion Proteins/metabolism , TransfectionABSTRACT
Alpha-dystrobrevin is a dystrophin-related protein expressed primarily in skeletal muscle, heart, lung and brain. In skeletal muscle, alpha-dystrobrevin is a component of the dystrophin-associated glycoprotein complex and is localized to the sarcolemma, presumably through interactions with dystrophin and utrophin. Alternative splicing of the alpha-dystrobrevin gene generates multiple isoforms which have been grouped into three major classes: alpha-DB1, alpha-DB2, and alpha-DB3. Various isoforms have been shown to interact with a variety of proteins; however, the physiological function of the alpha-dystrobrevins remains unknown. In the present study, we have cloned a novel alpha-dystrobrevin cDNA encoding a protein (referred to as alpha-DB2b) with a unique 11 amino acid C-terminal tail. Using RT PCR with primers specific to the new isoform, we have characterized its expression in skeletal muscle, heart, and brain, and in differentiating C2C12 muscle cells. We show that alpha-DB2b is expressed in skeletal muscle, heart and brain, and that exons 12 and 13 are alternatively spliced in alpha-DB2b to generate at least three splice variants. The major alpha-DB2b splice variant expressed in adult skeletal muscle and heart contains exons 12 and 13, while in adult brain, two alpha-DB2b splice variants are expressed at similar levels. This is consistent with the preferential expression of exons 12 and 13 in other alpha-dystrobrevin isoforms in skeletal muscle and heart. Similarly, in alpha-DB1 the first 21 nucleotides of exon 18 are preferentially expressed in skeletal muscle and heart relative to brain. We also show that the expression of alternatively spliced alpha-DB2b is developmentally regulated in muscle; during differentiation of C2C12 cells, alpha-DB2b expression switches from an isoform lacking exons 12 and 13 to one containing them. We demonstrate similar developmental upregulation of exons 12, 13, and 18 in alpha-DB1 and of exons 12 and 13 in alpha-DB2a. Finally, we show that alpha-DB2b protein is expressed in adult skeletal muscle, suggesting that it has a functional role in adult muscle. Together, these data suggest that alternatively spliced variants of the new alpha-dystrobrevin isoform, alpha-DB2b, are differentially expressed in various tissues and developmentally regulated during muscle cell differentiation in a fashion similar to that previously described for alpha-dystrobrevin isoforms.
