Journal: bioRxiv

Article Title: Mutation in the TRKB cholesterol recognition site that blocks antidepressant binding does not influence the basal or BDNF-stimulated activation of TRKB

doi: 10.1101/2022.08.26.505413

Figure Lengend Snippet: (A) The sequence of TRKB transmembrane (TM) domain from rat (UniProt: Q63604, residues 430-453) was mutated at Y433 residue (Y433F) or the entire motif was substituted by the rat sequence of TRKA TM (P35739, residues 419-442). (B) The interaction between biotinylated BDNF (bBDNF) and TRKB is not affected by the TRKB.Y433F mutation or in TRKB/TRKA.TM constructs. (C) TRKB.Y433F does not prevent the BDNF-induced dimerization of TRKB. The constructs used allow the formation of TRKB.wt homodimer or TRKB.wt/Y433F heterodimer. (D , E) Analysis of TRKB phosphorylation shows that, cortical cultures from TRKB.Y433F heterozygous mouse embryos respond to BDNF similarly to the the TRKB.wt littermates, regardless of the tyrosine residue tested ( D : Y515, E : Y816).

Article Snippet: The plates were then washed 3x with PBS buffer, and various concentrations of biotinylated BDNF (Alomone Labs, cat#B-250, 0.1 – 100 pM) was added for 1h at RT.

Techniques: Sequencing, Mutagenesis, Construct

Journal: Cell

Article Title: Antidepressant drugs act by directly binding to TRKB neurotrophin receptors

doi: 10.1016/j.cell.2021.01.034

Figure Lengend Snippet: Antidepressants bind to TRKB transmembrane domain (A) Fluoxetine (10 μM/15 min) and ketamine (10 μM/15 min) increased pTRKB.Y816 in cortical neurons immunoprecipitated with anti-PLC-γ1 (F[2,45] = 11.03, p = 0.0001, n = 16/group). (B) Fluoxetine facilitates BDNF-induced activation of TRKB under high cholesterol concentrations (interaction: F[2,132] = 5.15, p = 0.0070, n = 12/group) in cultured cortical cells. (C and D) Biotinylated fluoxetine binds to TRKB in lysates of TRKB expressing HEK cells (interaction: F[7,153] = 16.18, p < 0.0001; n = 6–14), but not (C) to TRKB.Y433F mutant or (D) to TRKB carrying the TMD of TRKA (TRKB/TRKA.TM) (interaction: F[7,80] = 43.75, p < 0.0001, n = 6/group). (E and F) Binding of biotinylated R,R-HNK (interaction: F[7,160] = 14.91, p < 0.0001; n = 6–14) (E) and tritiated imipramine (interaction: F[7,16] = 106.1, p < 0.0001; n = 2) (F) to TRKB, but not to TRKB.Y433F. Data expressed mean ± SEM of percentage of binding at 100 μM for fluoxetine and R,R-HNK or at 30 μM for imipramine. (G) Esketamine displaces the interaction of biotinylated fluoxetine (1 μM) with TRKB (n = 8/group). (H and I) Cholesterol facilitates the interaction of (H) biotinylated fluoxetine (F[5,30] = 7.198, p = 0.0002, n = 6/group)and (I) R,R-HNK (F[5,30] = 4.592, p = 0.0031, n = 6/group) with TRKB. (J) In situ PLA demonstrates close proximity between biotinylated fluoxetine and TRKB on TRKB-expressing N2A cells (red dots). (K) No PLA signal is seen in cells not expressing TRKB. Blue, DAPI; scale bar, 10 μm. (L) MST demonstrated direct interaction between fluoxetine and GFP-tagged TRKB (15 min) in lysates from GFP-TRKB expressing HEK293T cells (n = 4/group). Experimental traces depicted in the inset, vertical bars: blue, fluorescence cold; red, fluorescence hot. See also , , and .

Article Snippet: biotinylated rh-BDNF , Alomone Labs , Cat#B-250.

Techniques: Immunoprecipitation, Activation Assay, Cell Culture, Expressing, Mutagenesis, Binding Assay, In Situ, Fluorescence

Journal: Cell

Article Title: Antidepressant drugs act by directly binding to TRKB neurotrophin receptors

doi: 10.1016/j.cell.2021.01.034

Figure Lengend Snippet: Antidepressants bind to TRKB transmembrane domain, related to Figure 2 Lysate from HEK293T cells expressing TRKB were submitted to ligand binding assays. (A) Schematic representation of the biotinylated fluoxetine interaction with immobilized TRKB. (B-J) Biotinylated fluoxetine (1 μM) interaction with TRKB is reduced by non-biotinylated (B) fluoxetine (n = 6/group), (C) imipramine (n = 8/group), (D) moclobemide (n = 10/group), (E) venlafaxine (n = 6/group), (F) ketamine (n = 8/group), (G) R,R-HNK (n = 8/group), but not reduced by (H) S,S-HNK (n = 8/group), (I) chlorpromazine (n = 8/group), isoproterenol (n = 8/group) or diphenhydramine (n = 8/group), or (J) BDNF (n = 6/group). (K) Biotinylated R,R-HNK (1 μM) interaction with TRKB is not reduced by S,S-HNK (n = 12/group). (L,M) Biotinylated fluoxetine interaction with (L) TRKA (n = 7/group) from MG87 cells, or (M) lysates from non-transfected HEK cells (n = 10/group) are negligible compared to TRKB. The interaction of biotinylated fluoxetine is not altered in (N) TRKB lacking most of the intra and extracellular domains (TRKB.T1ΔEC, n = 12/group), but it is reduced by (O) V437A and Y433F mutations, and partially attenuated by S440A (n = 6/group).

Article Snippet: biotinylated rh-BDNF , Alomone Labs , Cat#B-250.

Techniques: Expressing, Ligand Binding Assay, Transfection

Journal: Cell

Article Title: Antidepressant drugs act by directly binding to TRKB neurotrophin receptors

doi: 10.1016/j.cell.2021.01.034

Figure Lengend Snippet: Antidepressants bind to TRKB in the intact cells, related to Figure 2 (A) Schematic representation of the in situ proximity ligation assay (PLA) between TRKB and biotinylated fluoxetine. (B-G) HEK cells were transfected to express TRKB and farnesylated GFP and were exposed to biotinylated fluoxetine (10uM/15min). The cells were fixed in PFA and the PLA reaction was conducted in permeabilized cells. (B-D) No signal from TRKB-FLX interaction was observed when cells were not transfected to express TRKB. (E-G) positive signal of TRKB-FLX (PLA). Scale bar: 20 μm. Zoom in square: 2.5x.

Article Snippet: biotinylated rh-BDNF , Alomone Labs , Cat#B-250.

Techniques: In Situ, Proximity Ligation Assay, Transfection

Journal: Cell

Article Title: Antidepressant drugs act by directly binding to TRKB neurotrophin receptors

doi: 10.1016/j.cell.2021.01.034

Figure Lengend Snippet:

Article Snippet: biotinylated rh-BDNF , Alomone Labs , Cat#B-250.

Techniques: Recombinant, In Situ, Stable Transfection, Expressing, Plasmid Preparation, Software