Lys-63-linked Ubiquitination of γ-Aminobutyric Acid (GABA), Type B1, at Multiple Sites by the E3 Ligase Mind Bomb-2 Targets GABA(B) 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.