FIGURE 3.

Lysine 28 is a critical determinate of the final size of secreted Aβ. Point mutations (shown in red) were made in the GSNK (25 to 28) region of the juxtamembrane region of C99-GVP by swapping the corresponding amino acid from the APLP2 region (SLSS, Fig. 2A). All mutants produced similar levels of total Aβ (1-X) compared with the wild-type (GSNK) control. Two point mutations showed decreased Aβ 40 (X-40), despite making comparable total amounts of Aβ. GSNS had the greatest Aβ 40 decrease and was similar to SLSS. Both total Aβ and Aβ 40 were completely inhibited by treatment with the γ-secretase inhibitor N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester DAPT (20 μm, not shown). As illustrated in Fig. 2, a decrease in Aβ 40 in this assay is a surrogate marker for a shift in Aβ toward smaller Aβ species. This result suggests that Lys-28 is the major residue regulating Aβ length in this region. The same transfected cells were used for the collection of conditioned media for the detection of 1-X and X-40 within each N (3), providing an internal control. All values are percentages (mean ± S.D.) of signals from the wild-type GSNK construct (set to 100%). One-way analysis of variance with post hoc Tukey's t test; ***, p < 0.001; *, p < 0.05. n.s., not significant.