Lys-63-linked Ubiquitination of γ-Aminobutyric Acid (GABA), Type B1, at Multiple Sites by the E3 Ligase Mind Bomb-2 Targets GABAB Receptors to Lysosomal Degradation

Abstract

GABA_B receptors are heterodimeric G protein-coupled receptors, which control neuronal excitability by mediating prolonged inhibition. The magnitude of GABA_B receptor-mediated inhibition essentially depends on the amount of receptors in the plasma membrane. However, the factors regulating cell surface expression of GABA_B receptors are poorly characterized. Cell surface GABA_B receptors are constitutively internalized and either recycled to the plasma membrane or degraded in lysosomes. The signal that sorts GABA_B receptors to lysosomes is currently unknown. Here we show that Mind bomb-2 (MIB2)-mediated Lys-63-linked ubiquitination of the GABA_B1 subunit at multiple sites is the lysosomal sorting signal for GABA_B receptors. We found that inhibition of lysosomal activity in cultured rat cortical neurons increased the fraction of Lys-63-linked ubiquitinated GABA_B receptors and enhanced the expression of total as well as cell surface GABA_B receptors. Mutational inactivation of four putative ubiquitination sites in the GABA_B1 subunit significantly diminished Lys-63-linked ubiquitination of GABA_B receptors and prevented their lysosomal degradation. We identified MIB2 as the E3 ligase triggering Lys-63-linked ubiquitination and lysosomal degradation of GABA_B receptors. Finally, we show that sustained activation of glutamate receptors, a condition occurring in brain ischemia that down-regulates GABA_B receptors, considerably increased the expression of MIB2 and Lys-63-linked ubiquitination of GABA_B receptors. Interfering with Lys-63-linked ubiquitination by overexpressing ubiquitin mutants or GABA_B1 mutants deficient in Lys-63-linked ubiquitination prevented glutamate-induced down-regulation of the receptors. These findings indicate that Lys-63-linked ubiquitination of GABA_B1 at multiple sites by MIB2 controls sorting of GABA_B receptors to lysosomes for degradation under physiological and pathological conditions.

Keywords: E3 ubiquitin ligase; GABA receptor; lysosome; protein degradation; ubiquitylation (ubiquitination).

FIGURE 1.

Expression level of GABA_B receptors is controlled by lysosomes. A, total expression level of GABA_B receptors is increased in neurons after blocking lysosomal activity. Cortical neurons were incubated for 12 h with 100 μm leupeptin (Leup) followed by immunostaining for total GABA_B1 and GABA_B2 as well as for actin using the in-cell Western technique. Neurons not treated with leupeptin served as controls (Ctrl). Right, representative images of an in-cell Western blot. Left, graph shows the quantification of fluorescence intensities normalized to the corresponding actin signals. Fluorescence intensities for GABA_B1 and GABA_B2 in control neurons were set to 100%. The data represent the mean ± S.E. of 30 cultures from three independent experiments. ***, p < 0.0001; two-tailed unpaired t test. B, expression of cell surface GABA_B receptors is increased in neurons after inhibiting lysosomal activity. Cortical neurons were treated as indicated in A and immunostained for cell surface GABA_B1 and GABA_B2. Left, representative images of the soma of stained neurons. Scale bar, 5 μm. Right, graphs show the quantification of fluorescence intensities. Fluorescence intensities for GABA_B1 and GABA_B2 in control neurons were set to 100%. The data represent the mean ± S.E. of 30–40 neurons from three independent experiments. ***, p < 0.0001; two-tailed unpaired t test.

FIGURE 2.

Expression level of GABA_B receptors is regulated by Lys-63-linked ubiquitination. A, interference with Lys-63-linked ubiquitination increased the expression level of cell surface GABA_B receptors. Neurons were transfected with wild-type ubiquitin (Ub) or a ubiquitin mutant unable to form Lys-63-linked chains (Ub(K63R)) and analyzed for GABA_B receptor expression using GABA_B1 as well as GABA_B2 antibodies. Left, representative images of stained neuronal somata (scale bar, 5 μm). Right, quantification of fluorescence intensities. The fluorescence signal of neurons transfected with wild-type ubiquitin was set to 100%. The data represent the mean ± S.E. of 30–34 neurons from three (GABA_B1) and two (GABA_B2) independent experiments. **, p < 0.004; ***, p < 0.0001; two-tailed unpaired t test. B, inhibition of lysosomal activity enhanced Lys-63-linked ubiquitination of GABA_B receptors. Cortical neurons were incubated for 12 h with or without (control) 100 μm leupeptin (Leup) and analyzed for Lys-63-linked ubiquitination by in situ PLA using antibodies directed against GABA_B1 and Lys-63-linked ubiquitin (white dots in representative images, scale bar, 5 μm). Right, quantification of in situ PLA signals. The data represent the mean ± S.E. of 30–40 neurons from three independent experiments. ***, p < 0.00001; two-tailed unpaired t test. Ctrl, control. C, inhibition of lysosomal activity did not affect Lys-48-linked ubiquitination of GABA_B receptors. Cortical neurons were treated as in B and analyzed for Lys-48-linked ubiquitination by in situ PLA using antibodies directed against GABA_B1 and Lys-48-linked ubiquitin (white dots in representative images, scale bar, 5 μm). Right, quantification of in situ PLA signals. The data represent the mean ± S.E. of 27–37 neurons from three independent experiments; n.s., p > 0.05; two-tailed unpaired t test.

FIGURE 3.

Identification of Lys-63-linked ubiquitination sites in GABA_B1. A, GABA_B1 is the main target for Lys-63-linked ubiquitination. HEK293 cells were either transfected with a GABA_B1 mutant containing an inactivated ER retention signal (GABA_B1a(RSAR)), which permits ER exit and cell surface targeting of the subunit when expressed alone, or with GABA_B1 and GABA_B2 and tested for Lys-63-linked ubiquitination by in situ PLA using GABA_B1 antibodies in combination with an antibody detecting Lys-63-linked ubiquitin (white dots in representative images, scale bar, 7 μm). The data represent the mean ± S.E. of 47–49 neurons from three independent experiments. ns, p > 0.05; two-tailed unpaired t test. B, decreased Lys-63-linked ubiquitination of GABA_B1(Lys → Arg) mutants. Cortical neurons were transfected with HA-tagged wild-type GABA_B1a, HA-tagged GABA_B1a(K697R/K698R), HA-tagged GABA_B1a(K892R), or HA-tagged GABA_B1a(K960R) together with wild-type GABA_B2 and analyzed for Lys-63-linked ubiquitination by in situ PLA using antibodies directed against the HA tag and Lys-63-linked ubiquitin (white dots in representative images, scale bar, 7 μm). B′, quantification of in situ PLA signals. B″, schematic depicting the location of Lys → Arg mutations in GABA_B1. The data represent the mean ± S.E. of 26–35 neurons from three independent experiments. ns, p > 0.05; ***, p < 0.0001; one-way ANOVA, Bonferroni's Multiple Comparison test.

FIGURE 4.

GABA_B1(Lys → Arg) mutants exhibit increased total and cell surface expression. A, increased total expression levels of GABA_B1a(Lys → Arg) mutants. Neurons were transfected with HA-tagged wild-type GABA_B1a, HA-tagged GABA_B1a(K697R/K698R), HA-tagged GABA_B1a(K892R), or HA-tagged GABA_B1a(K960R) together with wild-type GABA_B2 and analyzed for the expression level of transfected GABA_B1 using antibodies directed against the HA tag. Left, representative images (scale bar, 7 μm). Right, quantification of fluorescence signals. The fluorescence signals of neurons transfected with wild-type GABA_B1 were set to 100%. The data represent the mean ± S.E. of 23–27 neurons per experimental condition derived from three independent experiments. ***, p < 0.0001; one-way ANOVA, Dunnett's Multiple Comparison test. B, increased cell surface expression levels of GABA_B1a(Lys → Arg) mutants. Neurons were transfected with HA-tagged wild-type GABA_B1a, HA-tagged GABA_B1a(K697R/K698R), HA-tagged GABA_B1a(K892R), or HA-tagged GABA_B1a(K960R) together with wild-type GABA_B2 and analyzed for cell surface expression levels of transfected GABA_B1 as well as transfected plus endogenous GABA_B2 using antibodies directed against the HA tag and GABA_B2, respectively. Left, representative images (scale bar, 7 μm). Right, quantification of fluorescence signals. The fluorescence signals of neurons transfected with wild-type GABA_B1a or wild-type GABA_B2, respectively, was set to 100%. The data represent the mean ± S.E. of 26–28 neurons per experimental condition derived from three independent experiments. **, p < 0.001; ***, p < 0.0001; one-way ANOVA, Dunnett's Multiple Comparison test.

FIGURE 5.

Expression levels of GABA_B1a(Lys → Arg) mutants are unaffected by inhibition of lysosomal degradation. A, total expression levels of GABA_B1a(Lys → Arg) mutants are unaffected by blocking lysosomal activity with leupeptin. Neurons were transfected with HA-tagged wild-type GABA_B1a or HA-tagged GABA_B1a(Lys → Arg) mutants together with GABA_B2, incubated with 100 μm leupeptin for 12 h, followed by immunostaining for transfected HA-tagged GABA_B1 using HA antibodies. Left, representative images of untreated neurons (control, left) and of neurons incubated with leupeptin (right, scale bar, 7 μm). Right, quantification of fluorescence intensities. The fluorescence intensity of GABA_B1a from untreated neurons (control) was set to 100%. The data represent the mean ± S.E. of 27–34 neurons per experimental condition derived from three independent experiments. ***, p < 0.0001, two-tailed unpaired t test. B, total expression levels of GABA_B1a(Lys → Arg) mutants are unaffected upon blocking lysosomal targeting by inactivation of Rab7. Neurons were transfected with HA-tagged wild-type GABA_B1a or GABA_B1a(Lys → Arg) mutants together with GABA_B2 and with either wild-type Rab7 or with a non-functional mutant of Rab7 (Rab7(DN)) and analyzed for total expression levels of transfected GABA_B1 using HA antibodies. Left, representative images depicting total expression of transfected GABA_B1a (scale bar, 7 μm). Right, quantification of fluorescence intensities. The fluorescence intensities of GABA_B1a coexpressed with wild-type Rab7 were set to 100%. The data represent the mean ± S.E. of 27–34 neurons derived from three independent experiments. ***, p < 0.0001, two-tailed unpaired t test.

FIGURE 6.

E3 ligase MIB2 colocalizes with GABA_B receptors and affects their expression level. A and B, overexpression of MIB2 in neurons reduced expression levels of GABA_B receptors. Neurons were either transfected with EGFP (controls) or MIB2 and analyzed for cell surface (A) or total (B) expression of GABA_B receptors. Left, representative images (scale bar, 5 μm). Right, quantification of fluorescence intensities. The data represent the mean ± S.E. of 30 (A) and 45 (B) neurons derived from two independent experiments. ***, p < 0.0001, two-tailed unpaired t test. C, MIB2 and GABA_B receptors extensively colocalize in cortical neurons. Neurons were simultaneously stained for GABA_B1 (red) and MIB2 (green). Scale bar, 5 μm. D, GABA_B receptors interact with MIB2. Neurons were stained for GABA_B1 and MIB2 and analyzed for interaction via in situ PLA. Scale bar, 5 μm.

FIGURE 7.

MIB2-induced down-regulation of GABA_B receptors is mediated by Lys-63-linked ubiquitination. A, MIB2 mediates Lys-63-linked ubiquitination of GABA_B receptors. Neurons were transfected with EGFP (control) or MIB2 and tested for Lys-63-linked ubiquitination of GABA_B receptors using in situ PLA using antibodies directed against GABA_B1 and Lys-63-linked ubiquitin (white dots in representative images, scale bar, 5 μm). The graph depicts quantification of the in situ PLA signals. The data represent the mean ± S.E. of 20 neurons per condition derived from two independent experiments. *, p < 0.05, two-tailed unpaired t test. B, MIB2-induced down-regulation of GABA_B receptors is mediated by Lys-63-linked ubiquitination. Neurons were transfected with either wild-type ubiquitin (WT Ub), mutant ubiquitin that cannot form Lys-63 linkages (Ub(K63R), mutant ubiquitin that can only form Lys-63 linkages (Ub(Lys-63), and mutant ubiquitin that is deficient in forming Lys-48 linkages (Ub(K48R)), and with or without (control) MIB2 followed by determination of cell surface GABA_B receptors using GABA_B1 antibodies. Left, representative images (scale bar, 5 μm). Right, quantification of fluorescence intensities. The data represent the mean ± S.E. of 20–22 neurons for each condition derived from two independent experiments. ns, p > 0.05; **, p < 0.005; ***, p < 0.0005, two-tailed unpaired t test; ns, p > 0.05.

FIGURE 8.

MIB2 mediates Lys-63-linked ubiquitination of GABA_B receptors. GABA_B1a(Lys → Arg) mutants, which are partially resistant to Lys-63-linked ubiquitination, are not affected by overexpression of MIB2. Neurons were transfected with HA-tagged wild-type GABA_B1a (WT GABA_B1), GABA_B1a(K697/698R), GABA_B1a(K892R) or GABA_B1a(K960R) with (+MIB2) or without (control) MIB2 and analyzed for cell surface expression of wild-type and mutant GABA_B1a using HA antibodies. Left, representative images, scale bar, 5 μm. Right, quantification of fluorescence intensities. The data represent the mean ± S.E. of 19–24 neurons per experimental condition derived from two independent experiments. ns, p > 0.05; **, p < 0.005, two-tailed unpaired t test.

FIGURE 9.

GABA_B1a(Lys → Arg) mutants are resistant to glutamate-induced down-regulation. Neurons transfected with HA-tagged wild-type GABA_B1a or HA-tagged GABA_B1a(Lys → Arg) mutants along with GABA_B2 were incubated in the presence (glutamate) or absence (control) of 50 μm glutamate for 90 min followed by cell surface staining for transfected GABA_B1a using HA antibodies. Left, representative images, scale bar, 5 μm. Right, quantification of fluorescence intensities. The fluorescence intensity of neurons not treated with glutamate was set to 100%. The data represent the mean ± S.E. of 20–25 neurons per experimental condition derived from two independent experiments. ***, p < 0.0005, two-tailed unpaired t test.

FIGURE 10.

Glutamate-induced down-regulation of GABA_B receptors is mediated by Lys-63-linked ubiquitination. A, sustained activation of glutamate receptors enhanced Lys-63-linked ubiquitination of GABA_B receptors. Neurons were incubated for 60 min in the absence (control) or presence of 50 μm glutamate and analyzed for Lys-63-linked ubiquitination by in situ PLA using antibodies directed against GABA_B1 and Lys-63-linked ubiquitin (white dots in representative images, scale bar, 5 μm). The graph depicts quantification of the in situ PLA signals. The data represent the mean ± S.E. of 20 neurons derived from two independent experiments. **, p < 0.01; one-way ANOVA, Dunnett's Multiple Comparison test. B, preventing Lys-63-linked ubiquitination rendered GABA_B receptors resistant to glutamate-induced down-regulation. Neurons were transfected with wild-type ubiquitin (Ub(WT)), and mutants of ubiquitin that either permits only Lys-63-linked ubiquitination (Ub(Lys-63)) or prevents any kind of ubiquitin chain generation (Ub(KO)). Neurons were incubated for 90 min in the absence (control) or presence of 50 μm glutamate followed by determination of cell surface GABA_B receptors using GABA_B1 antibodies. Left, representative images, scale bar, 5 μm. Right, quantification of fluorescence intensities. The data represent the mean ± S.E. of 30–36 neurons from three independent experiments. ***, p < 0.0001; two-tailed unpaired t test; ns, p > 0.05.

FIGURE 11.

Glutamate exposure increases the expression level of MIB2 and the MIB2-GABA_B receptor interaction. A, increased MIB2 expression after glutamate exposure. Neurons were treated either for 15 or 30 min with glutamate and analyzed for MIB2 expression. Left, representative images, scale bar, 10 μm. Right, quantification of fluorescence intensities. The data represent the mean ± S.E. of 30 neurons from two independent experiments. ***, p < 0.0001; one-way ANOVA, Dunnett's Multiple Comparison test. B, increased interaction of GABA_B receptors with MIB2 after glutamate exposure. Neurons were treated either for 15 or 30 min with glutamate and analyzed for the interaction of MIB2 with GABA_B receptors using in situ PLA. Left, representative images, scale bar, 5 μm. Right, quantification of the in situ PLA signals. The data represent the mean ± S.E. of 14 neurons from two independent experiments. ***, p < 0.0003; one-way ANOVA, Dunnett's Multiple Comparison test.