Dystrophin-related and -associated proteins are important in the formation and maintenance of the mammalian neuromuscular junction. We have characterized mouse cDNA clones encoding isoforms of the dystrophin-homologous 87-kDa postsynaptic protein, dystrobrevin. In Torpedo, the 87-kDa protein is multiply phosphorylated and closely associated with proteins in the postsynaptic cytoskeleton, including the acetylcholine receptor. In contrast to Torpedo, where only a single transcript is seen, the mouse expresses several mRNAs encoding different isoforms. A 6.0-kilobase transcript in brain encodes a 78-kDa protein (dystrobrevin-2) that has a different C terminus, lacking the putative tyrosine kinase substrate domain. In skeletal and cardiac muscle, transcripts of 1.7 and 3.3/3.5 kilobases predominate and encode additional isoforms. Alternative splicing within the coding region and differential usage of untranslated regions produce additional variation. Multiple dystrobrevin-immunoreactive proteins copurify with syntrophin from mouse tissues. In skeletal muscle, dystrobrevin immunoreactivity is restricted to the neuromuscular junction and sarcolemma. The occurrence of many dystrobrevin isoforms is significant because alternative splicing and phosphorylation often have profound effects upon the biological activity of synaptic proteins.

The importance of dystrophin and its associated proteins in normal muscle function is now well established. Many of these proteins are expressed in nonmuscle tissues, particularly the brain. Here we describe the characterization of ␤-dystrobrevin, a dystrophin-related protein that is abundantly expressed in brain and other tissues, but is not found in muscle. ␤-dystrobrevin is encoded by a 2.5-kb alternatively spliced transcript that is found throughout the brain. In common with dystrophin, ␤-dystrobrevin is found in neurons of the cortex and hippocampal formation but is not found in the brain microvasculature. In the brain, ␤-dystrobrevin coimmunoprecipitates with the dystrophin isoforms Dp71 and Dp140. These data provide evidence that the composition of the dystrophinassociated protein complex in the brain differs from that in muscle. This finding may be relevant to the cognitive dysfunction affecting many patients with Duchenne muscular dystrophy.

Dystrophin coordinates the assembly of a complex of structural and signaling proteins that are required for normal muscle function. A key component of the dystrophin protein complex is ␣-dystrobrevin, a dystrophinassociated protein whose absence results in neuromuscular junction defects and muscular dystrophy. To gain further insights into the role of ␣-dystrobrevin in skeletal muscle, we used the yeast two-hybrid system to identify a novel ␣-dystrobrevin-binding partner called syncoilin. Syncoilin is a new member of the intermediate filament superfamily and is highly expressed in skeletal and cardiac muscle. In normal skeletal muscle, syncoilin is concentrated at the neuromuscular junction, where it colocalizes and coimmunoprecipitates with ␣-dystrobrevin-1. Expression studies in mammalian cells demonstrate that, while ␣-dystrobrevin and syncoilin associate directly, overexpression of syncoilin does not result in the self-assembly of intermediate filaments.Finally, unlike many components of the dystrophin protein complex, we show that syncoilin expression is upregulated in dystrophin-deficient muscle. These data suggest that ␣-dystrobrevin provides a link between the dystrophin protein complex and the intermediate filament network at the neuromuscular junction, which may be important for the maintenance and maturation of the synapse.

Gephyrin is an essential component of the postsynaptic cortical protein network of inhibitory synapses. Gephyrin-based scaffolds participate in the assembly as well as the dynamics of receptor clusters by connecting the cytoplasmic domains of glycine and GABA(A) receptor polypeptides to two cytoskeletal systems, microtubules and microfilaments. Although there is evidence for a physical linkage between gephyrin and microtubules, the interaction between gephyrin and microfilaments is not well understood so far. Here, we show that neuronal gephyrin interacts directly with key regulators of microfilament dynamics, profilin I and neuronal profilin IIa, and with microfilament adaptors of the mammalian enabled (Mena)/vasodilator stimulated phosphoprotein (VASP) family, including neuronal Mena. Profilin and Mena/VASP coprecipitate with gephyrin from tissue and cells, and complex formation requires the E-domain of gephyrin, not the proline-rich central domain. Consequently, gephyrin is not a ligand for the proline-binding motif of profilins, as suspected previously. Instead, it competes with G-actin and phospholipids for the same binding site on profilin. Gephyrin, profilin, and Mena/VASP colocalize at synapses of rat spinal cord and cultivated neurons and in gephyrin clusters expressed in transfected cells. Thus, Mena/VASP and profilin can contribute to the postulated linkage between receptors, gephyrin scaffolds, and the microfilament system and may regulate the microfilament-dependent receptor packing density and dynamics at inhibitory synapses.