Summary of binding competition of mutants versus biotinylated NT3.

Biotinylated NT3 ± competitorsBiotin-NT3 binding (% of untreated control)
No competitor100100100
NT3 wild-type60 ± 3.353 ± 7.456 ± 3.1
D52 ± 3.855 ± 3.157 ± 3.2
RK51 ± 5.883 ± 8.161 ± 5.3
DRK56 ± 6.685 ± 6.164 ± 2.5
BDNF wild-typeNot doneNot done58 ± 2.6
  • Cells transfected with the indicated receptor were preincubated for 15 minutes in binding buffer on ice without (untreated) or with competitors wild-type NF (control) or test mutants D, RK, or DRK (each at 380 nM). Then, saturating biotinylated NT3 was added for 15 minutes. The labeled secondary was fluorescein-avidin, and cells were analyzed by flowcytometry. The MCFs of bell-shaped histograms were standardized, with no competition = 100% binding. Values shown are the average ± S.D. (n = 3 biologic replicates; in each assay 5000 cells analyzed). Statistical analysis was done by one-way ANOVA with significance α < 0.05, followed by Bonferroni’s correction for multiple comparisons. In Trk-expressing cells, the three mutants, wild-type NT3, and wild-type BDNF significantly inhibited NT3-biotin binding (P < 0.001 in TrkC and TrkB). There are no significant differences in competition between NT3 wild-type versus the mutants or BDNF. In p75-expressing cells, wild-type NT3 and mutant D significantly inhibited NT3-biotin binding (P < 0.05). In p75-expressing cells, mutants RK and DRK did not significantly compete binding compared with untreated control, and binding remained significantly higher compared with control wild-type NT3 competition (P < 0.05).