Mécanismes de régulation du trafic et de l’activité du récepteur GABAB

γ-aminobutyric acid (GABA) is the principal inhibitory neurotransmitter of the central nervous system and is involved in diverse pathologies such as epilepsy, anxiety, depression and drug addiction.  GABAergic modulation of neuronal activity involves two different subsets of receptors: the GABAA receptor chlorine channel and the heterodimer of G protein coupled receptors (GPCR) GABAB.  Each of these receptors is responsible for mediating distinct parts of the GABA-induced signaling.  Upon stimulation, it is vital for the cell to control the signaling input and prevent overstimulation, using mechanisms such as functional desensitization and down-regulation to achieve this.  The processes controlling GABAB receptor activity are atypical for a GPCR and have yet to be fully characterized.  The aim of this thesis is to elucidate the mechanisms controlling GABAB activity by discovering novel proteins interactions mediating receptor desensitization, internalization and ubiquitination.  In the first study, we identified the N-ethylmaleimide sensitive factor (NSF) as a GABAB interacting protein and characterized its interaction site as the coiled-coil structure on both GABAB sub-units. We also showed that this interaction is sensitive to the ATPase state of NSF and that agonist treatment of GABAB led to dissociation of NSF from the receptor in a protein kinase C (PKC) dependent manner.  Interestingly, GABA-induced PKC activation was dependent on the NSF-GABAB interaction, suggesting a feedback mechanism for PKC.  Both PKC and NSF were involved in mediating receptor desensitization, suggesting a novel role of NSF in receptor signaling regulation.  In the proposed model, NSF interacts with GABAB at the basal state, and upon agonist stimulation, PKC is activated and can phosphorylate the receptor, promoting NSF dissociation and GABAB desensitization.  We then studied constitutive GABAB ubiquitination and degradation and its regulation by PKC and the deubiquitinating enzyme USP14 (Ubiquitin-specific protease 14).  GABAB shows a high constitutive ubiquitination and internalization level.  Activation of PKC promotes both phenomena and accelerates the rate of lysosomal receptor degradation.  In contrast, USP14 promotes post-endocytic deubiquitination of the receptor, but also accelerates receptor degradation in a catalytically-independent manner.  Our results suggest a mechanism where PKC-induced cell surface ubiquitination promotes GABAB endocytosis and USP14 interaction promotes endosomal sorting toward lysosomal degradation.  In the third study, we identified the growth factor receptor-bound protein 2 (Grb2) as a protein interacting with the PEST (proline, glutamate, serine, threonine rich) sequence of GABAB1 through a SH3-domain interaction and forming a ternary complex with the functional GABAB heterodimer.  We showed that Grb2 can regulate cell surface density of GABAB by decreasing constitutive endocytosis, suggesting that this interaction can compete for binding of the PEST sequence with proteins such as the GABAA γ2S sub-unit or the COPI complex (1, 2), promoting higher cell surface stability.    In proposing novel molecular mechanisms controlling GABAB signaling and cell surface expression through NSF, PKC, USP14 and Grb2, this thesis highlights the complex regulation of GABAB activity by its functional desensitization, ubiquitination, endocytosis and degradation.

Keyphrases: G protein coupled receptors (GPCR), desensitization, deubiquitination, Gamma-aminobutyric acid (GABA), Growth receptor bound protein 2 (Grb2), Ubiquitin-specific protease 14 (USP14), N-ethylmaleimide sensitive factor (NSF), Metabotropic GABA receptor (GABA(B)), ubiquitination, Protein kinase C (PKC)