Several antidepressant drugs activate tropomyosin-related kinase B (TRKB) receptor, but it remains unclear whether these compounds employ a common mechanism forTRKBactivation. Here, using MS, we found that a single intraperitoneal injection of fluoxetine disrupts the interaction of several proteins with TRKB in the hippocampus of mice. These proteins included members of adaptor protein complex-2 (AP-2) involved in vesicular endocytosis. The interaction of TRKB with the cargodocking μ subunit of the AP-2 complex (AP2M) was confirmed to be disrupted by both acute and repeated fluoxetine treatments. Of note, fluoxetine disrupted the coupling between fulllength TRKB and AP2M, but not the interaction betweenAP2M and the TRKB C-terminal region, indicating that the fluoxetinebinding site in TRKB lies outside the TRKB:AP2M interface. ELISA experiments revealed that in addition to fluoxetine, other chemically diverse antidepressants, such as imipramine, rolipram, phenelzine, ketamine, and its metabolite 2R, 6R-hydroxynorketamine, also decreased the interaction between TRKB andAP2Min vitro. Silencing the expression ofAP2Min a TRKB-expressing mouse fibroblast cell line (MG87.TRKB) increased cell-surface expression of TRKB and facilitated its activation by brain-derived neurotrophic factor (BDNF), observed as levels of phosphorylated TRKB. Moreover, animals haploinsufficient for the Ap2m1 gene displayed increased levels of active TRKB, along with enhanced cell-surface expression of the receptor in cultured hippocampal neurons. Taken together, our results suggest that disruption of the TRKB:AP2M interaction is a common mechanism underlying TRKB activation by several chemically diverse antidepressants. [ABSTRACT FROM AUTHOR]