MDGA2 interacts with TrkB and suppresses TrkB activity.

(A) Primary hippocampal neurons derived from Mdga2^+/− (Het) mice and their wild-type (WT) littermates at DIV 7 were treated with different doses of BDNF for 30 min. Equal amounts of protein lysates were subjected to immunoprecipitation (IP) with an anti-TrkB antibody, followed by immunoblotting with an anti-phosphotyrosine (pTyr) antibody or the anti-TrkB antibody. The pTyr levels were quantified by densitometry, normalized to total TrkB levels, and then compared to those of WT controls. n = 3 per group. (B) WT mouse brain lysates were subjected to IP with an anti-MDGA2 antibody and a control IgG, and immunoblotted (IB) for the components indicated. mBDNF, mature BDNF; ImBDNF, immature BDNF. (C) Hippocampal neurons from WT mice at DIV 7 were immunostained with antibodies against MDGA2 (red) and TrkB (green), and then counter-stained with DAPI (blue). Images were acquired by a confocal microscope. Scale bar: 10 μm. (D) Surface plasmon resonance (SPR) analysis of the binding between recombinant MDGA2 protein and recombinant TrkB protein. TrkB was immobilized on a Biacore CM5 chip and tested for its binding with MDGA2 at the concentrations indicated. (E) Computer simulation of the binding mode of TrkB and MDGA2. The predicted binding domain of TrkB (the d5 domain) is highlighted by a red square and labeled in brown. The predicted binding domain of MDGA2 (the MAM domain) is highlighted by a red square and labeled in yellow. (F) HEK293T cells were co-transfected with TrkB-Flag and GFP-tagged full-length MDGA2 (MDGA2-GFP) or an MDGA2 deletion fragment lacking the entire MAM domain (ΔMAM-GFP). Cell lysates were subjected to IP with control IgG and antibodies against Flag or GFP, and immunoblotted for the components indicated. A scheme of full-length MDGA2 shows its six IgG domains, FNIII domain, MAM domain, and C-terminal GPI anchor. The scheme of MDGA2-ΔMAM is also shown. (G) HeLa cells were co-transfected with TrkB that is tagged with the N-terminal Venus fragment (TrkB-VN) and full-length MDGA2 or MDGA2-ΔMAM that are tagged with the C-terminal Venus fragment (MDGA2-VC or ΔMAM-VC) for 24 h. After immunostaining with antibodies against MDGA2/MDGA2-ΔMAM (pink) and TrkB (red), cells were observed by confocal microscopy. Venus signal (green) indicates the interaction between MDGA2 and TrkB. Schematic diagrams depicting the BiFC assay are also shown. Scale bars: 10 μm. (H, I) Equal amounts of protein lysates from primary neurons derived from Het, KO, and their WT littermate mice (H), and from WT primary neurons transfected with GFP control, MDGA2-GFP or ΔMAM-GFP (I) were subjected to IP with IgG and an anti-BDNF antibody, and then immunoblotted for the components indicated. Immunoprecipitated TrkB levels were quantified by densitometry, normalized to input levels, and compared to respective controls (set to one arbitrary units). n = 3 per group. (J) Neurons derived from KO mice were transfected with GFP control (Ctrl), MDGA2-GFP, or MDGA2-ΔMAM-GFP, and neurons derived from their WT littermates were transfected with GFP control (Ctrl). Equal amounts of protein lysates were subjected to immunoblotting for the proteins indicated. Levels of phosphorylated proteins were quantified by densitometry, normalized to respective total protein levels and compared to controls (set to one arbitrary units). n = 6 per group. Data represent mean ± SEM. P-values were determined by two-tailed unpaired Student t-test in (A) and one-way ANOVA with Tukey’s multiple comparisons test in (H, I, J). *p < 0.05, **p < 0.01, ***p < 0.001, ns: not significant. The data underlying this figure can be found in S1 Data, specifically in the sheet labeled “Figure 3”.