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