Figure 1.
Identification of IL1RAPL1-ICD-binding proteins by affinity chromatography.
A, Negative staining of IL1RAPL1-binding proteins from the brain extracts, resolved by SDS-PAGE. Affinity chromatography of brain extracts was performed with maltose binding protein (MBP) conjugated with or without the cytoplasmic domain of IL1RAPL1. Protein bands specific to or thicker on the “MBP-IL1RAPL1 (+ brain extract)” lane (arrowheads 1–5) were excised and analyzed by LC-MS/MS. After subtraction of proteins detected in the control “MBP (+ brain extract)” lane, 9 candidate interactors were identified. B, List of identified proteins from each gel band. Numbers of identified peptides for each protein and scores of Mascot searches are shown.
Figure 2.
Analysis of IL1RAPL1-binding protein candidates (band #3).
A, Coimmunoprecipitation of YFP-IL1RAPL1 with FLAG-Bat3 in HEK 293T cells. Immunoprecipitation with anti-GFP antibody and total cell lysates, followed by western blotting with anti-FLAG antibody are shown. B, Colocalization of IL1RAPL1 (middle, green) and myc-Bat3 (left, red) in HEK 293T cells. Merged images are shown (right). Scale bar, 10 μm.
Figure 3.
Analysis of IL1RAPL1-binding protein candidates (band #4).
A, C, E, G, Coimmunoprecipitation of FLAG-IL1RAPL1 with myc-PLCβ1 (A) and myc-SNIP (E) and that of YFP-IL1RAPL1 with FLAG-Snap91 (C) and FLAG-Mcf2l (G) in HEK 293T cells. Immunoprecipitation with anti-FLAG antibody followed by western blotting with anti-Myc antibody (A, E) and that with anti-GFP antibody followed by western blotting with anti-FLAG antibody (C, G) are shown. B, D, F, H, Colocalization of IL1RAPL1 (middle, green) and myc-PLCβ1 (B), myc-Snap91 (D), myc-SNIP (F) and myc-Mcf2l (H) (left, red) in HEK 293T cells. Merged images are shown (right). Scale bar, 10 μm.
Figure 4.
Analysis of IL1RAPL1-binding protein candidates (band #5).
A, C, Coimmunoprecipitation of YFP-IL1RAPL1 with FLAG-Rasal1 (A) and FLAG-PKCε (C) in HEK 293T cells. Immunoprecipitation with anti-GFP antibody followed by western blotting with anti-FLAG antibody is shown. B, D, Colocalization of IL1RAPL1 (middle, green) and myc-Rasal1 (B) and myc-PKCε (D) (left, red) in HEK 293T cells. Merged images are shown (right). Scale bar, 10 μm.
Figure 5.
Pull down experiments of each IL1RAPL1 intracellular domain with interacting proteins.
A, Schematic structures (top) and SDS-PAGE followed by Coomassie Brilliant Blue staining (bottom) of purified MBP (1) and MBP-fusion proteins with the whole cytoplasmic region (2), the TIR domain (3) and the CT domain (4) of IL1RAPL1. B, Cell lysates from HEK 293T cells transfected with FLAG-Mcf2l, myc-PLCβ1, myc-SNIP, FLAG-Rasal1 or FLAG-PKCε were incubated with amylose resins coupled to MBP (1) or MBP-fusion protein with the whole cytoplasmic region (2), the TIR domain (3) or the CT domain (4) of IL1RAPL1. Precipitates were analyzed by SDS-PAGE followed by immunoblotting with anti-FLAG or anti-Myc antibody.
Figure 6.
Mcf2l and ROCK regulate IL1RAPL1-induced dendritic protrusion numbers in cortical neurons.
A, Test of siRNA knock-down efficiency for Mcf2l in cultured cortical neurons. Mcf2l-EGFP and mCherry were cotransfected with siRNAs. Representative patterns of Mcf2l-EGFP and mCherry expression are shown (left). The ratios of fluorescence intensities of Mcf2l-EGFP (green) to mCherry (red) were calculated (right, n = 5, n = 3, n = 5, n = 5, n = 7 [left to right bars]). B, Effects of endogenous Mcf2l knock-down on IL1RAPL1-mediated increase of protrusion numbers along the dendrites. Typical images are shown as representative (top). Six bars [EGFP]: n = 16, n = 10, n = 13, [EGFP+IL1RAPL1]: n = 10, n = 4, n = 11, F = 4.92, p<0.001, ANOVA (bottom). C, Three independent siRNA knock-down experiments for endogenous Mcf2l on IL1RAPL1-mediated increase of protrusion numbers along the dendrites. Ten bars [EGFP]: n = 16, n = 10, n = 5, n = 5, n = 5, [EGFP+IL1RAPL1]: n = 10, n = 4, n = 5, n = 5, n = 5, p<0.001, ANOVA (bottom). D, Effects of Y27632 on IL1RAPL1-mediated increase of protrusion numbers along the dendrites. Typical images are shown as representative (top). Four bars [EGFP]: n = 27, n = 32, [EGFP+IL1RAPL1]: n = 32, n = 25, F = 12.5, p<0.0001, ANOVA (bottom). **, p<0.01; ***, p<0.001. Error bars represent s.e.m.
Figure 7.
IL1RAPL1 regulates AMPA receptor newly insertion to surface in cortical neurons.
A, pHluorin fluorescence of pH-GluA in neurons. pHluorin signals are invisible in Golgi and endosome (in low pH) and weakly visible in the endoplasmic reticulum (ER, pH ∼7.0). Bright punctate signals of fluorescence increase when pH-GluA is inserted to surface and the pHluorin tag is exposed to the extracellular space (pH 7.4). B, Representative real time visualization of typical pH-GluA1 insertion events. Signal position around a neuron (y-axis, 83 μm) and time (x-axis, 5 min). Each ‘comet-like’ event is indicated by a white arrowhead. The sudden rising and disappear in fluorescence represents individual surface expression of pH-GluA1. C, E–G, Effects of IL1RAPL1 overexpression on the insertion frequency of pH-GluA1 (n = 9, n = 7) (C), pH-GluA2/GluA3 (n = 10, respectively) (E), pH-GluA2 (n = 10, respectively) (F) and pH-GluA3 (n = 10, respectively) (G). D, Longer observation of IL1RAPL1 effects on the pH-GluA1 insertion frequency (n = 4, respectively). Signals existing on surface over 1 min were calculated. Student t-test. *, p<0.05; **, p<0.01. Error bars represent s.e.m.
Figure 8.
Effects of IL-1R1 and IL-1R1/IL1RAPL1 chimeras overexpression on pH-GluA1 insertion.
Schematic structures of FLAG-IL1RAPL1, FLAG-IL-1R1 and their swap mutants are shown. The frequency of pH-GluA1 insertion was measured in cortical neurons transfected with IL1RAPL1, IL-1R1 or their swap mutants, n = 8, respectively, F = 7.98, p<0.0001, ANOVA). ***, p<0.001. Error bars represent s.e.m.
Figure 9.
RhoA signaling pathway regulates IL1RAPL1-mediated AMPA receptor newly insertion to surface in cultured cortical neurons.
A, Effects of knock-down of endogenous Mcf2l on IL1RAPL1-mediated suppression of pH-GluA1 insertion (n = 9, n = 9, n = 10, n = 10, F = 3.54, p<0.05, ANOVA). B, Effects of Y27632 on IL1RAPL1-mediated suppression of pH-GluA1 insertion (n = 10, respectively, F = 10.4, p<0.0001, ANOVA). C, Effects of Y27632 on IL1RAPL1-induced increase of pH-GluA2 insertion (n = 10, respectively, F = 5.21, p<0.005, ANOVA). D, Influences of NLGN1 overexpression on pH-GluA1 insertion with or without Y27632 (n = 8, respectively, F = 9.36, p<0.0002, ANOVA). E, Influences of NLGN1 overexpression on pH-GluA2 insertion with or without Y27632 (n = 8, respectively, F = 13.1, p<0.0001, ANOVA). Cultured cortical neurons were treated with 10 μM Y27632 for 3 days. *, p<0.05; **, p<0.01; ***, p<0.001. Error bars represent s.e.m.