Summary of binding competition of mutants versus biotinylated NT3.
| Biotinylated NT3 ± competitors | Biotin-NT3 binding (% of untreated control) | ||
|---|---|---|---|
| TrkC | p75 | TrkB | |
| No competitor | 100 | 100 | 100 |
| NT3 wild-type | 60 ± 3.3 | 53 ± 7.4 | 56 ± 3.1 |
| D | 52 ± 3.8 | 55 ± 3.1 | 57 ± 3.2 |
| RK | 51 ± 5.8 | 83 ± 8.1 | 61 ± 5.3 |
| DRK | 56 ± 6.6 | 85 ± 6.1 | 64 ± 2.5 |
| BDNF wild-type | Not done | Not done | 58 ± 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).