Distinct requirements of the ICD ^N and TM ^N domains for NRG3 association with Rab5+ early endosomal and Rab4+ transport vesicles. (A) Schematic representation of NRG3 variants and mutants used for deletion experiments. All constructs harbor a V5 epitope tag upstream of the EGF-L domain used for detection. Note that hFB-NRG3 is based on a naturally occurring variant encoding a single-pass TM protein that results from the use of an alternative 5′ exon lacking ICD^N and TM^N sequences (Carteron et al., 2006). The truncated NRG3^Q360* mutant essentially encompasses the NTF following BACE1 cleavage. N and C termini are indicated. (B) Representative images (above) and corresponding line scan densitometry (below) of MAP2+ dendrites showing Rab5 co-localization with NRG3 mutants harboring the TM^N domain (NRG3^Q360*, ICD^N-TM^N, TM^N-ICD^C) but not with variants/mutants lacking the TM^N (hFB-NRG3, ICD^N-TM^C, TM^C-ICD^C). (C) Summary analysis of experiments shown in B. Data are plotted as Mander’s overlap coefficients and represent the mean ± SEM of three independent experiments (n = 7 ROIs). (D and E) mCherry was C-terminally fused to either the NRG3 TM^N (D) or TM^C (E). Representative images and line scan densitometry show co-localization of TM^N-mCherry (mCh) but not TM^C-mCherry in MAP2+ dendrites (blue in merged images). (F) Summary analysis of data shown in D and E; data plotted as described in C, representing the mean ± SEM of three independent experiments (n = 16 ROIs). (G) Representative images and line scan densitometry illustrating co-localization of NRG3 mutants harboring the TM^N and ICD^N, but not of NRG3 mutants lacking the ICD^N, with axonal Rab4+ vesicles. (H) Summary analysis of data shown in G. Data are plotted as integrated density values and represent as mean ± SEM of three independent experiments. ****, P < 0.001 (C and H: one-way ANOVA with Tukey’s post-hoc test; F: unpaired t test). Scale bars: 5 μm.