Fig. 2: GPR50 binds to LC3 via the LIR motif, which is attenuated by ASD mutations.
From: GPR50 regulates neuronal development as a mitophagy receptor
A BiCAP interactome analysis of GPR50-V1 and GPR50-V2 homodimers after GFP-Trap for LC-MS. B The heat map shows the interaction affinity of proteins enriched in “mitophagy” from the Go analysis. C Analysis of cellular components and biological processes of GPR50 interaction partners by Gene ontology (Go). D The conserved sequence of peptides containing the LIR motif (Y305WTI308) and its mutation sites. E Schematic description of the location of LIR Y305WTI308 in GPR50. F ELISA analysis of the binding of WT or mutant LIR motif containing-peptides to recombinant LC3. G Proximity Ligation Assay for HeLa cells transfected with either GPR50-FLAG WT, mLIR, Δ502-505, or T532A using antibodies against LC3 and FLAG. Scale bars: 10 μm. H Numbers of DUOLink+ puncta per cell were quantified. One-way ANOVA (F4, 980 = 922.910, p = 0.000) followed by Dunnett T3 post hoc tests for GPR50 WT versus mLIR (p = 0.000), GPR50 WT versus GPR50 Δ502-505 (p = 0.000), GPR50 WT versus GPR50 T532A (p = 0.000), mLIR versus GPR50 Δ502-505 (p = 0.000), mLIR versus GPR50 T532A (p = 0.000), GPR50 Δ502-505 versus GPR50 T532A (p = 0.395). n = 178–208 cells per group from 3 independent experiments (G). I Co-immunoprecipitation in HEK293T cells co-transfected with GFP-LC3 and GPR50-FLAG WT, mLIR, Δ502-505 or T532A. Cell lysates were immunoprecipitated with anti-FLAG and detected with antibodies against GFP and FLAG. J The relative levels of GFP-LC3 pulled down by GPR50-FLAG were quantified. One-way ANOVA (F3, 12 = 7.817, p = 0.004) followed by LSD post hoc tests for GPR50 WT versus mLIR (p = 0.001), GPR50 WT versus GPR50 Δ502-505 (p = 0.003), GPR50 WT versus GPR50 T532A (p = 0.005). n = 4 independent experiments (I). K Lysates of adult mouse brains were immunoprecipitated with antibodies against endo GPR50 and LC3 and detected with antibodies against GPR50 and LC3, respectively. Data are presented as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001. n.s, non-significance.
