Abstract
Delta-catenin is a synaptic adherens junction protein pivotally positioned to serve as a signaling sensor and integrator. Expression of delta-catenin induces filopodia-like protrusions in neurons. Here we show that the small GTPases of the Rho family act coordinately as downstream effectors of delta-catenin. A dominant negative Rac prevented delta-catenin-induced protrusions, and Cdc42 activity was dramatically increased by delta-catenin expression. A kinase dead LIMK (LIM kinase) and a mutant Cofilin also prevented delta-catenin-induced protrusions. To link the effects of delta-catenin to a physiological pathway, we noted that (S)-3,5-dihydroxyphenylglycine (DHPG) activation of metabotropic glutamate receptors induced dendritic protrusions that are very similar to those induced by delta-catenin. Furthermore, delta-catenin RNA-mediated interference can block the induction of dendritic protrusions by DHPG. Interestingly, DHPG dissociated PSD-95 and N-cadherin from the delta-catenin complex, increased the association of delta-catenin with Cortactin, and induced the phosphorylation of delta-catenin within the sites that bind to these protein partners.
Highlights
␦-Catenin is a component of the synaptic adherens junction that is necessary for normal learning and memory [1]
The core functions of this protein family are stabilization of cadherins by binding to a highly conserved sequence in the juxtamembrane region and regulatory coordination over Rho GTPases [7]. ␦-Catenin is localized to the post-synaptic adherens junction, collaborates with Rho GTPases to set a balance between neurite elongation and branching, and robustly induces dendritic protrusions [8]
Through the versatility of this domain, the multiple complex interactions of ␦-catenin with the synapse arise. ␦-Catenin binds to the synaptic scaffolding molecule (S-SCAM) [10] and to PSD-95 [11, 12], both of which contain multiple PDZ domains. ␦-Catenin binds via a PDZ interaction to ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor-binding protein and the related glutamate receptor (GluR)-interacting protein [12]
Summary
Cell Culture and DHPG Treatment—Primary cultures were prepared from hippocampi of embryonic day 18 Sprague-Dawley rats as described previously [19]. They were maintained in Neurobasal medium (Invitrogen) containing B27 supplement (Invitrogen) and 0.5 mM L-glutamine. Cortical neurons were treated with DHPG for 30 min. Antibodies—The affinity-purified antibody raised against a ␦-catenin peptide corresponding to amino acids 434 –530 in rabbit was described previously [20]. The cells were incubated with primary antibody for 90 min at room temperature or overnight at 4͉°C. A nano- DHPG/RNAi Quantification—After DHPG treatment the scale reverse-phase HPLC capillary column was created by neurons were stained with ␦-catenin and PSD-95 antibodies. The Rac-GTP quantification was developed using an enzyme-linked immunosorbent assay according to the manufacturer’s instructions (G-LISATM Rac activation assay biochem kit-BK125, Cytoskeleton, Denver, CO)
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