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GABAB receptors are heterodimeric G protein-coupled receptors, which control neuronal excitability by mediating prolonged inhibition. The magnitude of GABAB receptor-mediated inhibition essentially depends on the amount of receptors in the plasma membrane. However, the factors regulating cell surface expression of GABAB receptors are poorly characterized. Cell surface GABAB receptors are constitutively internalized and either recycled to the plasma membrane or degraded in lysosomes. The signal that sorts GABAB receptors to lysosomes is currently unknown. Here we show that Mind bomb-2 (MIB2)-mediated Lys-63-linked ubiquitination of the GABAB1 subunit at multiple sites is the lysosomal sorting signal for GABAB receptors. We found that inhibition of lysosomal activity in cultured rat cortical neurons increased the fraction of Lys-63-linked ubiquitinated GABAB receptors and enhanced the expression of total as well as cell surface GABAB receptors. Mutational inactivation of four putative ubiquitination sites in the GABAB1 subunit significantly diminished Lys-63-linked ubiquitination of GABAB receptors and prevented their lysosomal degradation. We identified MIB2 as the E3 ligase triggering Lys-63-linked ubiquitination and lysosomal degradation of GABAB receptors. Finally, we show that sustained activation of glutamate receptors, a condition occurring in brain ischemia that down-regulates GABAB receptors, considerably increased the expression of MIB2 and Lys-63-linked ubiquitination of GABAB receptors. Interfering with Lys-63-linked ubiquitination by overexpressing ubiquitin mutants or GABAB1 mutants deficient in Lys-63-linked ubiquitination prevented glutamate-induced down-regulation of the receptors. These findings indicate that Lys-63-linked ubiquitination of GABAB1 at multiple sites by MIB2 controls sorting of GABAB receptors to lysosomes for degradation under physiological and pathological conditions.
Pubmed ID: 27573246 RIS Download
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View all literature mentionsA national mouse monoclonal antibody generating resource for biochemical and immunohistochemical applications in mammalian brain. NeuroMabs are generated from mice immunized with synthetic and recombinant immunogens corresponding to components of the neuronal proteome as predicted from genomic and other large-scale cloning efforts. Comprehensive biochemical and immunohistochemical analyses of human, primate and non-primate mammalian brain are incorporated into the initial NeuroMab screening procedure. This yields a subset of mouse mAbs that are optimized for use in brain (i.e. NeuroMabs): for immunocytochemical-based imaging studies of protein localization in adult, developing and pathological brain samples, for biochemical analyses of subunit composition and post-translational modifications of native brain proteins, and for proteomic analyses of native brain protein networks. The NeuroMab facility was initially funded with a five-year U24 cooperative grant from NINDS and NIMH. The initial goal of the facility for this funding period is to generate a library of novel NeuroMabs against neuronal proteins, initially focusing on membrane proteins (receptors/channels/transporters), synaptic proteins, other neuronal signaling molecules, and proteins with established links to disease states. The scope of the facility was expanded with supplements from the NIH Blueprint for Neuroscience Research to include neurodevelopmental targets, the NIH Roadmap for Medical Research to include epigenetics targets, and NIH Office of Rare Diseases Research to include rare disease targets. These NeuroMabs will then be produced on a large scale and made available to the neuroscience research community on an inexpensive basis as tissue culture supernatants or purified immunoglobulin by Antibodies Inc. The UC Davis/NIH NeuroMab Facility makes NeuroMabs available directly to end users and is unable to accommodate sales to distributors for third party distribution. Note, NeuroMab antibodies are now offered through antibodiesinc.
View all literature mentionsCommercial organization which provides life science services and products to researchers. They specialize in gene synthesis, peptide, protein, antibody and preclinical drug development service.
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