Abstract
The G protein-coupled GABAB receptors, constituted from GABAB1 and GABAB2 subunits, are important regulators of neuronal excitability by mediating long-lasting inhibition. One factor that determines receptor availability and thereby the strength of inhibition is regulated protein degradation. GABAB receptors are constitutively internalized from the plasma membrane and are either recycled to the cell surface or degraded in lysosomes. Lys-63-linked ubiquitination mediated by the E3 ligase Mind bomb-2 (MIB2) is the signal that sorts GABAB receptors to lysosomes. However, it is unknown how Lys-63-linked ubiquitination and thereby lysosomal degradation of the receptors is regulated. Here, we show that Ca2+/calmodulin-dependent protein kinase II (CaMKII) promotes MIB2-mediated Lys-63-linked ubiquitination of GABAB receptors. We found that inhibition of CaMKII in cultured rat cortical neurons increased cell surface GABAB receptors, whereas overexpression of CaMKIIβ, but not CaMKIIα, decreased receptor levels. This effect was conveyed by Lys-63-linked ubiquitination of GABAB1 at multiple sites mediated by the E3 ligase MIB2. Inactivation of the CaMKII phosphorylation site on GABAB1(Ser-867) strongly reduced Lys-63-linked ubiquitination of GABAB receptors and increased their cell surface expression, whereas the phosphomimetic mutant GABAB1(S867D) exhibited strongly increased Lys-63-linked ubiquitination and reduced cell surface expression. Finally, triggering lysosomal degradation of GABAB receptors by sustained activation of glutamate receptors, a condition occurring in brain ischemia, was accompanied with a massive increase of GABAB1(Ser-867) phosphorylation-dependent Lys-63-linked ubiquitination of GABAB receptors. These findings indicate that CaMKIIβ-dependent Lys-63-linked ubiquitination of GABAB1 at multiple sites controls sorting of GABAB receptors to lysosomes for degradation under physiological and pathological condition.
Highlights
Introduction γAminobutyric acid (GABA), the main inhibitory neurotransmitter in the brain, activates the heterodimeric G protein-coupled GABAB receptors, which are assembled from GABAB1 and GABAB2 subunits
Phosphorylation of GABAB1 at Ser-867 by calmodulin-dependent protein kinase II (CaMKII) plays a key role in this mechanism [29]
Incubation of neurons with KN93 transiently increased the expression of GABAB1 and GABAB2, which peaked at about 7.5 min as tested by Western blotting (Fig. 1a)
Summary
Introduction γAminobutyric acid (GABA), the main inhibitory neurotransmitter in the brain, activates the heterodimeric G protein-coupled GABAB receptors, which are assembled from GABAB1 and GABAB2 subunits. A main factor regulating GABAB receptor signaling is the dynamic control of their cell surface expression. In this respect, protein degradation is one important mechanism that regulates receptor availability. The two main cellular protein degradation systems, which are proteasomes and lysosomes, contribute to the regulation of cell surface GABAB receptors in different cellular compartments. The activity state of the neuron controls the rate of Lys-48-linked ubiquitination of the GABAB2 subunit required for proteasomal receptor degradation [11]. Lysosomal degradation of GABAB receptors depends on Lys-63linked ubiquitination of the GABAB1 subunit at multiple sites mediated by the E3 ubiquitin ligase MIB2 [17]. The mechanism that regulates Lys-63-linked ubiquitination of GABAB1, and thereby lysosomal degradation of the receptors, was unknown. We show that phosphorylation of Ser-867 of GABAB1 by CaMKIIβ regulates the extent of MIB2-mediated K63-linked ubiquitination of GABAB1 and thereby the amount of lysosomal degradation
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