PMC

(A) Interaction of APPcyt with X11L and X11L PI/PTB domain mutants derived from sorbitol treated cells. HEK293 cells (~2 × 10⁵) were transiently transfected with 0.5 μg of pcDNA3.1-FLAG-X11L (WT), pcDNA3.1-FLAG-X11L F520V (F520V), or pcDNA3 alone (−), and treated with (+) or without (−) 0.5 M sorbitol for 45 min. The cell lysates were incubated with GST-APPcyt, and FLAG-X11L bound to GST-APPcyt (bound) and the lysates (input) were analyzed by immunoblotting with anti-FLAG M2 antibody.
(B) Interaction of APPcyt carrying a mutation in the NPTY element with X11L derived from sorbitol treated cells. HEK293 cells (~2 × 10⁵) were transiently transfected with 0.5 μg of pcDNA3-HA-X11L (+), and treated with (+) or without (−) 0.5 M sorbitol for 45 min. The cell lysates were incubated with GST alone, GST-APPcyt, or GST-APPcytAAAA in which the NPTY sequence was altered to AAAA. HA-X11L bound to APPcyt (bound) and the lysates (input) were analyzed by immunoblotting with anti-HA 12CA5 antibody.

(A) Sequences of amino acid substitution mutants at Ser236 and/or Ser238. Alanyl residues substituted for Ser236 (S236A) and Ser238 (S238A) are underlined. WT indicates the wild-type X11L. PI indicates the PI/PTB domain.
(B) Association of APPcyt with X11L mutants derived from sorbitol treated cells. HEK293 cells (~2 × 10⁵) were transiently transfected with 0.5 μg of pcDNA3-HA-X11L (WT), pcDNA3-HA-X11L S236A (S236A), pcDNA3-HA-X11L S238A (S238A), or pcDNA3-HA-X11L S236A/S238A (S236A/S238A), and treated with (+) or without (−) 0.5 M sorbitol for 45 min. The cell lysates were incubated with GST-APPcyt, and HA-X11L proteins bound to APPcyt (bound) and the lysates (input) were analyzed by immunoblotting with anti-HA 12CA5 antibody.
(C) Quantification of X11L proteins bound to APPcyt. X11L proteins bound to APPcyt were quantified with the VersaDoc imaging system (Bio-Rad). The data are presented as the ratio (sorbitol (+)/sorbitol (−)) relative to the level of wild type X11L-binding (WT, 1.0). Results represent means ± s.d. for n=4 experiments. *, p<0.05, **, p<0.01 by Student’s t-test.

(A) Schematic structures of X11L, X11 and X11L2. Human X11 family proteins are shown. PI indicates the PI/PTB domain, which associates with APP. Numbers indicate amino acid positions.
(B) Amino acid sequence in the highly conserved regulatory region of X11L^221-250 and X11^265-294. Positions of Ser236 and Ser238 in X11L^221-250 and Ser280 and Ser282 in X11^265-294 are shown. The protein sequences are presented using the single-letter amino acid code. Amino acid residues of X11 identical (*) or similar (:) to X11L are indicated.
(C) Association of APPcyt with X11, X11L and X11L2 derived from sorbitol treated cells. HEK293 cells (~2 × 10⁵) were transiently transfected with 0.5 μg of pcDNA3-HA-X11L (X11L), pcDNA3.1-HA-X11 (X11), or pcDNA3.1-HA-X11L2 (X11L2), and treated with (+) or without (−) 0.5 M sorbitol for 45 min. The cell lysates were incubated with GST-APPcyt, and HA-X11 family proteins bound to APPcyt (bound) and the lysates (input) were analyzed by immunoblotting with anti-HA 12CA5 antibody. Numbers indicate molecular weight standards (kDa).

(A) Genomic map of Drosophila X11L (CG5675, cytological location 16B7). The P element insertion (triangle), X11L coding and the untranslated regions (dark and shaded rectangles, respectively) are illustrated. X11L encodes a protein with a PTB and two PDZ domains that are conserved in human X11 proteins. (B–M) Scanning electron micrographs (SEM) of adult fly eyes of various genotypes. GAMAREP flies exhibit small, rough eyes (C) as compared to the wildtype (B). Overexpression of X11L (GMR-X11L) shows no external eye phenotypes (E), but strongly suppresses GAMAREP eye phenotypes (F). Expression of human X11α (GMR-X11α), which by itself does not produce any visible eye phenotypes (H), suppresses GAMAREP phenotypes as effectively as fly X11L overexpression (I). X11L's ability to suppress GAMAREP is dependent on its PTB domain, since overexpression of X11LΔPTB (GMR-X11LΔPTB) (K) fails to suppress GAMAREP eye phenotype (L). The ability of X11 to suppress GAMAREP eye phenotype is specific, since overexpression of X11L, X11ΔPTB or human X11 fails to suppress the eye phenotype of the false-positive reporter (GMR-Gal4, UAS-grim/reaper) (D, G, J and M). The X11LΔPTB transgene expresses a truncated protein as detected by anti-X11L antibodies . Of note, strong overexpression of X11L (multiple copies of GMR-X11L or GMR-Gal4, UAS-X11L) can result in a rough eye phenotype (data not shown), consistent with previous reports .