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. 2021 Mar 17;41(11):2344–2359. doi: 10.1523/JNEUROSCI.2108-20.2021

Figure 2.

Figure 2.

Degradation pathways and surface expression of mGlu7 pathologic variants. A, Primary cortical neurons were infected with rescue lentivirus expressing human myc-mGlu7 WT, I154T, or R658W/T675K. At DIV17, cortical neurons were incubated with 5 μm MG132, 100 μm chloroquine (CQ), or 100 nm bafilomycin-A1 (Baf-A1) for 15 h. Expression of exogenous mGlu7 was analyzed by Western blotting. LC3B and ubiquitin (Ub) blotting was performed to confirm the integrity of the assay. Bar graph with scatter plot represents the mean ± SEM normalized to the vehicle band intensity. *p = 0.0012, **p < 0.0001, ***p = 0.0010. B, Neuronal surface expression of mGlu7 variants was examined by a cell surface biotinylation assay. Cortical neurons were infected with rescue lentiviruses expressing myc-mGlu7 WT, R622Q, I154T, or R658W/T675K. At DIV17, the neurons were biotinylated using membrane-impermeable sulfo-NHS-SS-biotin and pulled down using streptavidin-agarose resin. Western blotting was conducted using the indicated antibodies. The short-exposed blot to x-ray film is presented for the myc-mGlu7 WT and R622Q. Na+/K+ ATPase α1 blot indicates a loading control for total and surface-expressed receptors, and α-tubulin blot shows the integrity of the assay. Bar graph with scatter plot represents a ratio of surface-to-total expression levels and is displayed as the mean ± SEM normalized to the WT level; n.s., not significant, *p = 0.0005, **p < 0.0001. C, EGFP-DNM1-K44A was cotransfected with myc-mGlu7 WT, I154T, or R658W/T675K in HEK 293T cells. Thirty-six hours after transfection, the surface-expressed receptors were analyzed by a cell surface biotinylation assay. TfR, transferrin receptor. D, Gel migration properties of the Endo Hf-treated or PNGase F-treated mGlu7 variants. Cortical neurons were infected by rescue lentivirus expressing myc-mGlu7 WT, R622Q, I154T, or R658W/T675K. At DIV17, neuronal lysates were immunoprecipitated with anti-myc antibody. The immunoprecipitates were incubated overnight with Endo Hf or PNGase F at 37°C and subjected to Western blotting using anti-myc antibody. Arrow, Endo Hf-resistant mature mGlu7; arrowhead, Endo Hf-sensitive immature mGlu7.