Fig. 5. Characterization of interactions of APP with contactins and NgCAM. (A) AP-fused APP domains were tested for binding to Fc-fused contactin 3 or 4 domains [for concentrations, see Materials and methods; for domain selection see Rader et al. (Rader et al., 1996)]. Binding activity localizes to the fibronectin (FN) domains of contactins, and to the N-terminal domain of APP. (B) APP association with NgCAM. Western blots with antibodies against NgCAM after immunoprecipitating for AP tag. Asterisks mark major bands specifically co-immunoprecipitated with AP-APPs, consistent with published molecular weights of NgCAM. (C) Amino acids 199-345 of APP are sufficient for association with NgCAM. Non-cleavable crosslinker BS3 used here to examine net molecular weight of immunoprecipitated complexes. Resulting spread of signal might indicate the presence of additional molecules in the crosslinked NgCAM-APP complexes. (D) Model for interactions among proteins. N-terminal domain of APP (amino acids 18-205, `E1') binds directly to fibronectin domains of contactin 4, whereas amino acids 199-345 of APP interact, directly or indirectly, with NgCAM. Amino acids 199-345 of APP encompass the acidic domain (oval, `A') and the N-terminal portion of the central APP domain, termed E2, which includes the RERMS peptide previously implicated in APPs function (Reinhard et al., 2005). (E) Co-transfection of APP-HA and indicated constructs, followed by anti-HA western blot. Contactin 4-Fc or NgCAM-Fc constructs increased the CTF level compared with Fc control. (F) Co-migration with an artificial CTF polypeptide confirms the identity of the APP cleavage fragment observed after co-expression with contactin 4-AP.