Figure 1.

Cholesterol sensing by TRKB

(A) Identification of CARC motif (red) in the TM domain of TRKB, but not TRKA or TRKC.

(B) Cholesterol promotes the effects of BDNF on TRKB autophosphorylation (TRKB:pY) at moderate, but inhibits BDNF at low or high concentrations (interaction: F[5,84] = 5.654, p = 0.0002; n = 6/group). Cultured cortical cells received cholesterol (15 min) followed by BDNF or cholesterol (15 min) and were submitted to ELISA for TRKB:pY.

(C) β-cyclodextrin (bCDX, 2 mM, 30 min) prevents BDNF-induced increase in TRKB-PLC-γ1 interaction (TRK:PLCg1) (interaction: F[1,20] = 9.608, p = 0.0056, n = 6/group).

(D) Pravastatin (1 μM, 3 days) also blocks the BDNF-induced increase in TRKB:PLC-γ1 interaction (interaction: F[1,19] = 11.23, p = 0.003; n = 5–6). ^∗p < 0.05 from the ctrl/ctrl group, #p < 0.05 from ctrl/chol0 group, data expressed as mean ± SEM of percentage from control group.

(E) Microscale thermophoresis demonstrated direct interaction between GFP-tagged TRKB and cholesterol (15 min) in lysates from GFP-TRKB expressing HEK293T cells; mutation of Y433F blocks this interaction in MST (interaction: F[11,72] = 15.25, p < 0.0001, n = 4).

(F) Fluoxetine-induced increase in TRKB surface exposure is blocked by bCDX (interaction: F[1,73] = 7.022, p = 0.0099, n = 19–20).

(G–J) Structure of wild-type TRKB (G) in the absence of cholesterol and (H) at cholesterol concentrations of 20 mol% and (I) 40 mol%, and (J) for the heterodimer of TRKB.wt and TRKB.Y433F at 20 mol %. Related to systems 5–8 in Table S1 and Figure S2 for distance and α values between C termini.

See also Figures S1, S2, and S3.