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. 2019 Feb 21;11:3. doi: 10.3389/fnsyn.2019.00003

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Copyright © 2019 Kang, Wang, Sanders, Zuo, Hayden and Raymond.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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HIP14 and HIP14L differentially modulate trafficking of GluN2B in COS-7 cells. Flag-tagged HIP14 or HIP14L construct, together with GFP-tagged GluN2B WT, GluN2B 5CS or GluN2B 3CS, and HA-tagged GluN1-1A, were transfected into COS-7 cells. 2B-NMDAR and/or HIP14/HIP14L subcellular distribution was assessed by immunostaining with anti-GFP (green channel, GluN2B), anti-Flag (red channel, HIP14 or HIP14L) and cis- and medial-Golgi marker, GOLPH4 (blue channel) 36 h after transfection. (A) GFP-tagged GluN2B alone localizes to the cytoplasm in a diffuse pattern. Exogenous HIP14 promotes the accumulation of GluN2B WT and GluN2B 5CS into the perinuclear region (indicated by GOLPH4). (B) Exogenous HIP14L preferentially promotes the accumulation of GluN2B 3CS into the perinuclear region. Scale bars, 20 μm. (C) Examples of line scan profiles in perinuclear region for GluN2B WT-GFP cotransfected with HIP14-Flag. (D) Examples of line scan profiles in perinuclear region for GluN2B WT-GFP co-transfected with HIP14L-Flag. (E) Line scan analysis for perinuclear region distribution was obtained from 30 cells/condition with or without HIP14 co-transfection. Consistent with images, HIP14 overexpression increases the percentage of cells showing perinuclear colocalization of GluN2B WT and 5CS with GOLPH4. Data was obtained from 3 independent experiments and bars represent means ± SD. Two-way ANOVA, p = 0.013 for interaction, p = 0.016 for mutant, ^∗∗∗p < 0.0001 by Bonferroni post hoc tests. (F) Line scan analysis for perinuclear region distribution was obtained from 30 cells/condition with or without HIP14L co-transfection. HIP14L overexpression enhances the percentage of cells showing perinuclear colocalization of GluN2B 3CS with GOLPH4 in perinuclear regions. Data was obtained from 3 independent experiments and bars represent means ± SD. Two-way ANOVA, p = 0.001 for interaction, p = 0.0728 for mutant, ^∗∗∗p < 0.0001 by Bonferroni post hoc tests.