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. 2019 Aug 2;8:e44502. doi: 10.7554/eLife.44502

Figure 5. β-arrestins and Nedd4 regulate endocytosis of mGlu7 in neurons.

(A) The endocytosis of mGlu7 was analyzed by an antibody uptake internalization assay. myc-mGlu7 was co-transfected with Nedd4 WT, C867S mutant, or vector control (Vec) in cultured hippocampal neurons. Two days after transfection, neurons were labeled with anti-myc antibody for 10 min, and returned to conditioned media for 15 min at 37°C. Neurons were fixed and incubated with Alexa Fluor 568-conjugated secondary antibody (red) to label surface-expressed receptors before permeabilization. After permeabilization with 0.25% Trion X-100 for 5 min, neurons were then incubated with Alexa Fluor 488-conjugated secondary antibody (green) to label the internalized receptors. Merged images are presented in which the red signal represents the surface mGlu7 and the green signal represents the internalized mGlu7. Scale bar, 20 μm. (B) Summary histograms quantifying the internalized mGlu7 from panel A are present as the ratio of the internalized population compared with total (surface + internalized) population measured using Metamorph software. Scatter plots show mean ± SEM (Vec, 1.00 ± 0.04; Nedd4 WT, 1.23 ± 0.04; Nedd4 C867S, 1.02 ± 0.04; n > 35, **p<0.01, one-way ANOVA). (C) myc-mGlu7 was co-transfected with pSuper-Ctl shRNA (Ctl KD) or pSuper-Nedd4 shRNA (Nedd4 KD) in cultured hippocampal neurons. Internalization of mGlu7 was analyzed in the absence or presence of 400 μM L-AP4 for 15 min at 37°C. (D) Summary histograms quantifying the internalized mGlu7 from panel C. Scatter plots show mean ± SEM (Vec, 1.00 ± 0.06; Vec + L-AP4, 1.39 ± 0.14; Nedd4 KD, 0.86 ± 0.06; Nedd4 KD + L-AP4, 0.94 ± 0.09; n > 10, *p<0.05, n.s. indicates p>0.05, one-way ANOVA). (E) myc-mGlu7 and β-arrestin 1 or 2 were co-expressed and the internalized mGlu7 was analyzed as above. (F) Summary histograms quantifying the internalized mGlu7 from panel E. Scatter plots show mean ± SEM (Vec, 1.00 ± 0.05; β-arr1, 1.26 ± 0.07; β-arr2, 1.30 ± 0.07; n > 30, **p<0.01, one-way ANOVA).

Figure 5—source data 1. Endocytosis of mGlu7 by beta-arrestins and Nedd4.
elife-44502-fig5-data1.xlsx (12.7KB, xlsx)
DOI: 10.7554/eLife.44502.020

Figure 5.

Figure 5—figure supplement 1. Ubiquitination is required for endocytosis of endogenous mGlu7.

Figure 5—figure supplement 1.

Cortical neurons were treated with 0.1 μM MLN7243 for 18 hr to inhibit ubiquitination. After treatment with 800 μM L-AP4 for 15 min, surface biotinylation assay was performed. Treatment of MLN7243 in cortical neurons resulted in increased surface expression and decreased ubiquitination of endogenous mGlu7.
Figure 5—figure supplement 2. Separated images that supplement the merged images shown in Figure 5.

Figure 5—figure supplement 2.

Surface-expressed and internalized mGlu7 signals in Figures 5A, C and E were separated, and individual images were presented in A, B, and C, respectively.