NTRK2A203T , NTRK3E176D , and NTRK3L449F exhibit increased receptor dimerization in comparison with their respective WT receptor. (A) Schematic of NTRK dimerization studies. A total of 0.5 µg of FLAG (F)- and hemagglutinin (HA)-tagged NTRK WT or mutant constructs were cotransfected into HEK293T/17 cells and immunoprecipitated (IP) using anti-FLAG beads. FLAG immunoprecipitates were then probed with an anti-HA antibody to detect the coimmunoprecipitating receptor, suggestive of receptor dimerization. The level of HA and FLAG coimmunoprecipitation was quantified using ImageJ and normalized to input. A higher ratio of HA to FLAG immunoprecipitation was indicative of increased receptor dimerization. (B-C) Immunoblot analysis and quantification of WT and mutant NTRK2 constructs suggest that the NTRK2A203T mutation caused increased receptor dimerization. (D-E) Increased receptor dimerization was seen with both NTRK3 mutants relative to WT as evident by immunoblot and quantification analysis. For all experiments, statistical significance was assessed by a 1-way ANOVA followed by a Tukey multiple comparison test. All statistical comparisons shown are between WT and mutant receptors. Three biological replicates were performed for each condition. Each experiment was performed at least twice with consistent results. The average mean plus or minus SEM is shown. *P < .05; **P < .01; ***P < .001.