![]() D and E, in vitro immunoprecipitation assay for the binding of MAGUIN to s-afadin. C, the average ratio of MAGUIN-2 immunoprecipitated with each indicated Ab to MAGUIN-2 immunoprecipitated with the l/s-afadin pAb. B, the average ratio of MAGUIN-1 immunoprecipitated with each indicated Ab to MAGUIN-1 immunoprecipitated with the l/s-afadin pAb. B and C, quantification of band intensities in three independent experiments. Arrow, l-afadin arrowhead, s-afadin open arrow, MAGUIN-1 open arrowhead, MAGUIN-2. The lysate and the immunoprecipitated samples were subjected to SDS-PAGE, followed by Western blotting with the l/s-afadin pAb and the MAGUIN pAb recognizing both of MAGUIN-1 and MAGUIN-2. The lysate of the light membrane fraction of the mouse brains was immunoprecipitated by rabbit IgG, the l-afadin pAb, or the l/s-afadin pAb. A− C, in vivo immunoprecipitation assay for the binding of MAGUIN to s-afadin. A− E, preferential binding of MAGUIN to s-afadin over l-afadin. These results indicate that s-afadin binds to MAGUIN-1 more preferentially than l-afadin in vivo and in vitro.įigure 1 Binding of MAGUIN to s-afadin. V5-tagged MAGUIN-1 was immunoprecipitated with EGFP-tagged s-afadin, while V5-tagged MAGUIN-1 was hardly immunoprecipitated with EGFP-tagged l-afadin. This more preferential binding of MAGUIN to s-afadin than to l-afadin was confirmed by immunoprecipitation assays in which each of enhanced GFP (EGFP)-tagged afadin splice variants and V5-tagged MAGUIN-1 were co-expressed in human embryonic kidney (HEK) 293 cells ( Fig. The amounts of MAGUIN-1 and MAGUIN-2 immunoprecipitated by the l/s-afadin pAb were higher than those immunoprecipitated by the l-afadin pAb ( Fig. We found that MAGUIN-1 and MAGUIN-2 were co-immunoprecipitated with afadin. l-Afadin was immunoprecipitated with both the pAbs, whereas s-afadin was immunoprecipitated by the l/s-afadin pAb, but not the l-afadin pAb ( Fig. The immunoprecipitates were subjected to SDS-PAGE, followed by Western blotting. To elucidate the mechanism for the formation of the PSDs by l-afadin and s-afadin, we attempted to identify afadin-binding proteins using an immunoprecipitation assay with the l-afadin polyclonal antibody (pAb), and the l/s-afadin pAb recognizing both l-afadin and s-afadin in the crude membrane fraction prepared from the mouse brains. KeywordsĪbbreviations: aa ( amino acids), Ab ( antibody), AMPA ( α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic), AZs ( active zones), DDM ( dodecyl maltoside), EGFP ( enhanced GFP), ES ( embryonic stem), FAB ( F-actin-binding), GST ( glutathione S-transferase), HEK ( human embryonic kidney), mEPSC ( miniature excitatory postsynaptic current), NMDA ( N-methyl-D-aspartate), pAb ( polyclonal antibody), PAJ ( puncta adherentia junctions), PH ( pleckstrin homology), PSDs ( postsynaptic densities), RA ( Ras associating), TBS ( Tris buffered saline) These results indicate that s-afadin binds to MAGUIN and regulates the PSD-95-dependent cell surface localization of the AMPA receptor and glutamatergic synaptic responses in the hippocampal neurons and that MAGUIN is not involved in the induction of epileptic seizure by flurothyl in our mouse model. Furthermore, disruption of MAGUIN did not increase the seizure susceptibility to flurothyl, a GABA A receptor antagonist. Our electrophysiological analysis revealed that the postsynaptic response to glutamate, but not its release from the presynapse, was impaired in the MAGUIN-deficient cultured hippocampal neurons. Genetic ablation of MAGUIN impaired PSD-95 localization and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic (AMPA) receptor surface accumulation in cultured hippocampal neurons. ![]() MAGUIN/CNKSR2 is one of the causative genes for non-syndromic X-linked intellectual disability accompanied by epilepsy and aphasia. We found here that s-afadin more preferentially bound to MAGUIN (a product of the Cnksr2 gene) than l-afadin in vivo and in vitro. l-Afadin, but not s-afadin, regulates the formation of the PAJ, but the roles of s-afadin in synaptogenesis remain unknown. Afadin has two splice variants: l-afadin and s-afadin. We previously showed that the scaffolding protein afadin regulates the formation of the PAJs, PSDs, and active zones in the mossy fiber synapse. The postsynaptic densities (PSDs) are localized at the heads of each of these spines and faces to the presynaptic active zones. Glycobiology and Extracellular MatricesĪ hippocampal mossy fiber synapse implicated in learning and memory is a complex structure in which a presynaptic bouton attaches to the dendritic trunk by puncta adherentia junctions (PAJs) and wraps multiply branched spines.
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