Erbin is a recently described member of the LAP (leucine-rich repeat and PDZ domain) protein family. We used a C-terminally displayed phage peptide library to identify optimal ligands for the Erbin PDZ domain. Phage-selected peptides were type 1 PDZ ligands that bound with high affinity and specificity to the Erbin PDZ domain in vitro. These peptides most closely resembled the C-terminal PDZ domain-binding motifs of three p120-related catenins: ␦-catenin, ARVCF, and p0071 (DSWV-COOH). Analysis of the interactions of the Erbin PDZ domain with synthetic peptides matching the C termini of ARVCF or ␦-catenin also demonstrated specific high affinity binding. We characterized the interactions between the Erbin PDZ domain and both ARVCF and ␦-catenin in vitro and in vivo. The Erbin PDZ domain co-localized and coprecipitated with ARVCF or ␦-catenin complexed with -catenin and E/N-cadherin. Mutagenesis and peptide competition experiments showed that the association of Erbin with the cadherincatenin complex was mediated by the interaction of its PDZ domain with the C-terminal PDZ domain-binding motifs (DSWV-COOH) of ARVCF and ␦-catenin. Finally, we showed that endogenous ␦-catenin and Erbin co-localized in and co-immunoprecipitated from neurons. These results suggest that ␦-catenin and ARVCF may function to mediate the association of Erbin with the junctional cadherin-catenin complex. They also demonstrate that C-terminal phage-display technology can be used to predict physiologically relevant ligands for PDZ domains.PDZ 1 domains are 80 -100-amino acid compact globular motifs that are usually embedded in larger multidomain scaffolding proteins (1-3). PDZ domains predominantly mediate protein/protein interactions by recognizing the C termini of various intracellular and cell-surface proteins. Type 1 PDZ domains interact with the C-terminal consensus sequence X(S/ T)X(V/I/L)-COOH, whereas type 2 domains bind to the C-terminal consensus sequence X-hydrophobe-X-hydrophobe-COOH (3-5). Structural analyses of peptides bound to PDZ domains suggest necessary interactions at both positions 0 and Ϫ2 (6-8). However, among type 1 PDZ ligands, these residues are relatively invariant, indicating that other residues within the C terminus likely contribute to the specificity of PDZ domain/ ligand interactions (3). For example, two previous studies (9 -11), as well as those presented here, demonstrated the importance of residues Ϫ1 and Ϫ3 for binding specificity and affinity for some PDZ domain/ligand interactions.Genetic evidence supports a role for several families of PDZ domain-containing proteins as scaffolding molecules that target signaling complexes to various subcellular locations. For example, the multi-PDZ domain protein INAD was found to assemble components of the Drosophila visual transduction system to allow for efficient signaling in response to light (12, 13). Another study in the nematode Caenorhabditis elegans demonstrated that the basolateral localization of the LET-23 receptor tyrosine kinase is dependent upon direct b...
Although traditional roles ascribed to myelinating glial cells are structural and supportive, the importance of compact myelin for proper functioning of the nervous system can be inferred from mutations in myelin proteins and neuropathologies associated with loss of myelin. Myelinating Schwann cells are known to affect local properties of peripheral axons (de Waegh et al., 1992), but little is known about effects of oligodendrocytes on CNS axons. The shiverer mutant mouse has a deletion in the myelin basic protein gene that eliminates compact myelin in the CNS. In shiverer mice, both local axonal features like phosphorylation of cytoskeletal proteins and neuronal perikaryon functions like cytoskeletal gene expression are altered. This leads to changes in the organization and composition of the axonal cytoskeleton in shiverer unmyelinated axons relative to age-matched wild-type myelinated fibers, although connectivity and patterns of neuronal activity are comparable. Remarkably, transgenic shiverer mice with thin myelin sheaths display an intermediate phenotype indicating that CNS neurons are sensitive to myelin sheath thickness. These results indicate that formation of a normal compact myelin sheath is required for normal maturation of the neuronal cytoskeleton in large CNS neurons.
Altered axon-Schwann cell interactions in PNS myelin-deficient Trembler mice result in changed axonal transport rates, neurofilament and microtubule-associated protein phosphorylation, neurofilament density, and microtubule stability. To determine whether PNS and CNS myelination have equivalent effects on axons, neurofilaments, and microtubules in CNS, myelindeficient shiverer axons were examined. The genetic defect in shiverer is a deletion in the myelin basic protein (MBP) gene, an essential component of CNS myelin. As a result, shiverer mice have little or no compact CNS myelin. Slow axonal transport rates in shiverer CNS axons were significantly increased, in contrast to the slowing in demyelinated PNS nerves. Even more striking were substantial changes in the composition and properties of microtubules in shiverer CNS axons. The density of axonal microtubules is increased, reflecting increased expression of tubulin in shiverer, and the stability of microtubules is drastically reduced in shiverer axons. Shiverer transgenic mice with two copies of a wild-type myelin basic protein transgene have an intermediate level of compact myelin, making it possible to determine whether the actual level of compact myelin is an important regulator of axonal microtubules. Both increased microtubule density and reduced microtubule stability were still observed in transgenic mouse nerves, indicating that signals beyond synaptogenesis and the mere presence of compact myelin are required for normal regulation of the axonal microtubule cytoskeleton.Key words: axonal transport; glia; oligodendrocyte; myelin; shiverer; microtubule Vertebrates have two distinct paths for formation of compact myelin. CNS and PNS myelin serve comparable functions and are superficially similar, but they differ in embryonic origin, genetics, composition, and ultrastructure. In both CNS and PNS, myelination is a complex process in which glial cells extend processes that enwrap axons and generate compact myelin (Morell and Quarles, 1999). However, Schwann cells enwrap single axons in the PNS, whereas one oligodendrocyte myelinates 6 -10 different axons in the CNS. Myelin protein composition also differs. For example, myelin basic protein (MBP) comprises up to 40% of oligodendrocyte protein synthesis but is much lower in PNS. Although proteins responsible for compaction of CNS and PNS myelin are well characterized, less is known about proteins mediating axonglial cell interactions in either CNS or PNS. Equally important, neuronal responses to different CNS or PNS environments are virtually uncharacterized.The neuronal cytoskeleton is critical for neurite outgrowth during development and regeneration (Brady, 1993). This cytoskeleton comprises microtubules (MTs), neurofilaments (NFs), and microfilaments. Hallmark changes in isotype composition, posttranslational modification, and stability of these accompany neuronal development and regeneration Sahenk and Brady, 1987;Hoffman and Cleveland, 1988;Oblinger and Lasek, 1988;Tetzlaff et al., 1988;Oblinger et al.,...
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