Journal: The Journal of Biological Chemistry
Article Title: ?-Arrestin Promotes Wnt-induced Low Density Lipoprotein Receptor-related Protein 6 (Lrp6) Phosphorylation via Increased Membrane Recruitment of Amer1 Protein *
Figure Lengend Snippet: Amer1 interacts with β-arrestin2. A , HEK293T cells were transfected with FLAG-Amer1 and HA-β-arrestin2 individually or together as indicated. Cell lysates were immunoprecipitated ( IP ) with anti-FLAG or anti-HA antibody. Immunoblotting ( WB ) was done with mouse M2 anti-FLAG antibody and with mouse anti-HA antibody. TCL was used as a loading control. B , NB4 cells were stimulated for 2 h with Wnt3a (or control) CM, and cytoplasmic and membrane fractions were harvested. Left panel , NB4 cells are Wnt3a-responsive as shown by the enhanced Lrp6 phosphorylation at Ser-1490. Both Amer1 and β-arrestin2 are isolated preferentially in the cytoplasmic fraction. However, upon Wnt3a stimulation, Amer1 is recruited to the membrane and/or associates more tightly with the membrane and is therefore isolated also in the membrane fraction of NB4 cells. Right panel , Amer1 co-immunoprecipitated with A1CT and A2CT anti β-arrestin antibodies, but not with anti-IgG control antibody and anti-β-arrestin2 cs-3857 antibody, which poorly precipitates endogenous β-arrestin2. C , domain mapping of the Amer1/β-arrestin2 interaction using full-length β-arrestin2 and deletion mutants of Amer1 shows schematic view of Amer1 mutants. The interaction with β-arrestin2 is indicated with +; M1/M2 indicate regions interacting with membrane via PtdIns(4,5)P 2 ; A1/A2/A3 are regions required for the interaction with Apc. Full blots are shown in supplemental Fig. 1, A and B . D , domain mapping of the Amer1/β-arrestin2 interaction uses full-length Amer1 and deletion mutants of β-arrestin2. The mutual interaction is indicated with +. Full blots are shown in supplemental Fig. 1 C . E , the interaction between the C-terminal region of Amer1 (amino acids 942–1135) and the central part of β-arrestin2 is demonstrated by co-immunoprecipitation.
Article Snippet: The following antibodies were used: mouse anti-FLAG antibody (F1804; Sigma-Aldrich), rat anti-GFP antibody (3H9; Chromotek), mouse HA.11 (MMS-101R; Covance), rabbit anti-HA (ab9110; Abcam), anti-Dvl3 (sc-8027; Santa Cruz Biotechnology), anti-p(S1490)-Lrp6 (2568; Cell Signaling), anti-β-catenin (610153; BD Biosciences), anti-β-actin (sc-1615; Santa Cruz Biotechnology), anti-Myc antibody (sc-40; Santa Cruz Biotechnology), anti-α-catenin (sc-7894; Santa Cruz Biotechnology) mouse anti-Amer1 , rabbit anti-Amer1 (AP17838PU-N; Tocris), rabbit anti-β-arrestin (3857; Cell Signaling), anti-β-arrestin (A1CT and A2CT), a kind gift from R. J. Lefkowitz), rabbit anti-PI4KII (a kind gift from P. De Camilli), and rabbit anti-IgG control antibody (3900; Cell Signaling). siRNA sequences targeting β-arrestin1/2 ( ) and PI4KII ( ) were described earlier.
Techniques: Transfection, Immunoprecipitation, Western Blot, Isolation