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.

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.

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