γ-Aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the brain and plays a major role in determining the routing or sculpting of neural activity, as well as limiting excessive neuronal firing, via the fast-acting ionotropic GABA A receptors and the slow-acting metabotropic GABA B receptors. Functional GABA B receptors are heterodimers comprising the subunits GABA B1 and GABA B2 . Presynaptically, GABA B receptors inhibit voltage-gated Ca 2+ channels and suppress

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... release whereas postsynaptically they induce slow inhibitory postsynaptic potentials by activation of Kir3-type K + channels. Dysfunction of GABA B receptors in the CNS is believed to be involved in various nervous system disorders such as addiction, anxiety, epilepsy, chronic pain and depression. Elucidation of the regulation of GABA B receptor signalling is essential for the understanding of the contribution of GABA B receptors to pathophysiological conditions. In this respect, the dynamic control of cell surface expression of the receptors is a main factor regulating GABA B receptor signalling. The aim of this thesis was to analyze to which extent degradation mechanisms regulate cell surface expression of GABA B receptors. The research program was divided into three sub-projects. In project one we tested the hypothesis that the endoplasmic reticulum (ER)-associated protein degradation (ERAD) machinery may contribute to the regulation of GABA B receptor expression levels. We found that bidirectional modulation of proteasome activity increased and decreased, respectively, the expression level of GABA B receptors in cultured cortical neurons. Proteasomal degradation of GABA B receptors required K48-linked ubiquitination of two lysine residues in the C-terminal domain of GABA B2 . Blocking proteasome activity in neurons resulted in the accumulation of GABA B receptors in the endoplasmic reticulum, indicating that the degradation is mediated by the proteasome-dependent ERAD machinery. Pharmacological inhibition of ERAD or over-expression of a dominant-negative mutant form of VCP/p97, an essential protein of the ERAD complex, increased total as well as cell surface levels of GABA B receptors in neurons. In conclusion, our results indicate that the level of newly synthetized GABA B receptor in the ER available for cell surface trafficking is regulated by ERAD. In project two; we searched for GABA B receptor-interacting proteins involved in proteasomal degradation and tested the hypothesis that proteasomal degradation might be regulated by neuronal activity. We found that the C-terminus of GABA B2 interacts with Rpt6, one of the six AAA-ATPases of the 19S regulatory complex of the proteasome. Overexpression of Rpt6 in HEK293 cells strongly reduced the levels of co-expressed GABA B2 , whereas deletion of the C terminal domain of GABA B2 or a synthetic peptide comprising the GABA B2 interaction