To confirm the postsynaptic localization of ARMS, we denervated rat gastrocnemius muscle and examined the distribution of ARMS on muscle fibers as much as 20 d after dener vation, once the presynaptic axon terminals must have degen erated and retracted. Major ARMS staining could nevertheless be observed at kinase inhibitor AZD2171 junctional web-sites twenty d right after denervation, indicating that ARMS was localized postsynaptically. Additionally, we identified that ARMS was colocalized with EphA4 and TrkB recep tors, that are two RTKs that were previously reported to clus ter postsynaptically at the NMJ at all around P8. These findings recommend that TrkB, EphA4, and ARMS are all clustered at postsynaptic areas and are similarly regulated during improvement. Library screening identifies syntrophin as an ARMS interacting protein To identify which PDZ proteins could possibly interact with ARMS in the NMJ, we performed a yeast two hybrid screening implementing the COOH terminus of ARMS that con tained the prospective PDZ binding motif as bait to display a P12 mouse muscle cDNA library.
31 clones have been recognized PTC124 as pu tative candidates, and 20 of them contained the total coding sequence in the PDZ protein syntrophin. We confirmed the authenticity from the clones by transforming them back into yeast with all the ARMS COOH terminal fragment. In addition to syntrophin, two other isoforms, 1 and two syntrophin, are also expressed in muscle. These syntrophins share large homology in their protein domains with syntro phin. We identified that all 3 syntrophin isoforms bound for the ARMS COOH terminal fragment in yeast, despite the fact that to diverse extents. The PDZ binding motif RESIL with the ARMS cytoplasmic tail con tains the consensus sequence S/T X V/L that preferentially in teracts together with the class I PDZ domain present in syntrophin.
The fundamental amino acid at the four place and acidic residue at the 3 place might even further enhance the binding affinity amongst the two mo tifs. We found that ARMS lacking the

final three amino acids within the RESIL motif failed to interact with syntrophin in yeast, demonstrating that the PDZ binding motif is required for ARMS syntrophin interaction. Conversely, whenever we mutated the syntrophin PDZ domain by substituting two tremendously conserved residues during the PDZ GLGI loop with alanines, the ARMS syntrophin binding was eradicated. So, the syntro phin PDZ domain was also necessary for ARMS syntrophin interaction. We confirmed the wild form and mutated proteins were expressed at equivalent ranges in these experiments. In quantitative assays, galac tosidase exercise was detected only during the presence of the two intact ARMS PDZ binding motifs and wild form syntrophin PDZ do mains. Together with all the observation of cell growth on His /Trp /Leu selective plates, these results show that ARMS and syntrophin bind to one another via PDZ domain mediated interactions in yeast and that syntrophin binds to ARMS extra strongly than syntrophins.