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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.
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