LRP-ST61-97 sequence does not self-activate unlike LRP-ST in the yeast two-hybrid system. (A) Yeast transformed with pGB-LRP-ST1-97 and pGAD vector and plated onto synthetic dropout medium lacking leucine and tryptophan but containing kanamycin and X-gal (SD-LT + Kan + X-gal) turned blue indicating that the ST1-97 self-activates in this assay. (B) But yeast transformed with pGB-LRP-ST61-97 (LRP-C37) and pGAD vector and plated onto the same synthetic dropout medium does not turn blue indicating that LRP-C37 does not self-activate and therefore can be used in the yeast two-hybrid screens.

The dileucine motif mediates LRP-ST61-97-induced amyloidogenic processing of APP. (A) Conditioned media from cells in Figure 3B were immunoprecipitated and immunoblotted for Aβ (6E10). Note that ST61-97ΔELL mutant nearly abrogates the increase in the secretion of Aβ by ST61-97, indicating the dileucine motif within ST61-97 is crucial for the generation of Aβ. (B) HEK293FT cells were transiently cotransfected with APP751 and myc-vector, myc-ST-61-97 or myc-ST61-97ΔELL, and lysates were subjected to immunoblotting to verify the expression levels of APP-FL, APP-CTFs and LRP-ST variants.

FE65/APP binding is not required for LRP-ST-mediated amyloidogenic processing of APP (A) HEK293FT cells were transfected with combinations of FE65, APP751-wt or APP751-G737A mutant. Equal protein amounts were immunoprecipitated for APP (IG7). In the lower two panels, equal amounts of lysates were immunoblotted for FE65 (anti-FE65) APP-FL (CT15) and APP-CTFs (CT15). Note that FE65 does not bind to APP751-G737A mutant and APP-CTF levels were also unchanged. (B) HEK293FT cells were transiently transfected with either APP751-wt or APP751-G737A mutant and Aβ (6E10) detected in the conditioned media was not altered. Lysates were immunoblotted for APP-FL and APP-CTFs (CT15). (C) HEK293FT cells were cotransfected with APP-751-GA mutant along with empty myc-vector, myc-LRP-ST1-97 or LRP-ST61-97. Both LRP-ST1-97 and LRP-ST61-97 increased Aβ in the conditioned medium similar to that seen in APP-wt. Upper panel shows equal amounts of APP-FL while the levels of APP-CTFs were reduced by LRP-ST61-97.

LRP-ST61-97 sequence (C37 region) represents a novel protein interaction region with multiple binding partners. (A) pGB-ST61-97 or pGB-empty vector were cotransformed with pGAD-Filamin A or pGAD-SH3 protein into AH109 yeast strain and the resulting colonies were re-streaked onto high-stringency plates lacking histidine, adenine, leucine and tryptophan, but containing kanamycin and X-gal as a substrate for the target gene β-galactosidase. The robust growth of the colony and the change in colour to blue indicated the physical interaction between LRP-ST61-97 and Filamin A or SH3 protein (Accession: AK030242). However, the colonies failed to grow on the quadruple drop out plates when ST61-97 was replaced with either empty vector or vector expressing lamin as control. (B) Same as panel (A) but pGB-ST61-97 or pGB-empty vector were cotransformed with pGAD-Snapin or pGAD-RanBPM, demonstrating specific interactions of LRP-ST61-97 also with Snapin and RanBPM. (C) HEK293FT cells were cotransfected with combinations of LRP-CT, myc-Filamin A or myc-vector and lysates were subjected to immunoprecipitation for myc-Filamin A (9E10). Both the endogenous LRP (upper band) and the exogenously expressed LRP-CT (anti-LRP, 1704) were specifically pulled-down only when myc-Filamin was cotransfected but not with the myc vector. (D) Similar experiment as in panel (C), but cells were transfected with SH3 protein (AK030242).

LRP-ST61-97 lacking NPxY motifs is sufficient to robustly enhance amyloidogenic processing of APP. (A) Schematic illustration of amino acid sequence of LRP-ST1-97 to highlight the C37 region, NPxY-based motifs and dileucine motif. All LRP-ST variants were C-terminally fused with 6x myc tag. (B) HEK293FT cells were transiently cotransfected with APP751 and myc-tagged LRP-ST variants. Aβ (6E10), sAPPα (6E10) and sAPP total (63d) from the conditioned media were detected with their respective antibodies by straight Western blotting (for sAPPα & sAPP total) or by immunoprecipitation followed by Western blotting (for Aβ). Note the large increase in Aβ, reduction in sAPPα and no change in sAPP total by ST61-97. (C) Graph shows the mean Aβ levels (n = 6 each group) normalized to vector control from densitometric quantitations of blots using the Image J software. Error bars represent SEM. (D) HEK293FT cells were transiently cotransfected with APP751 and myc-tagged LRP-ST variants, and equal amount of lysates were immunoblotted for full-length APP (CT15), APP CTFs (CT15) and LRP-ST variants (9E10). Note the marked reduction in APP CTFs by ST61-97 (lane 2) and ST45-97 (lane 3) compared to vector control (lane 1) without altering APP-FL. The lower panel shows expression of various deletion mutants of LRP-ST.