Modulation of GABAB receptor signaling by associated proteins and phosphorylation

GABAB receptors are the G protein-coupled receptors (GPCRs) for y-aminobutyric acid (GABA), the main inhibitory neurotransmitter in the central nervous system. They are membrane receptors with a seven membrane-spanning domain and are composed of the principal subunits GABAB1 and GABAB2. Activated GABAB receptors regulate G protein-coupled inwardly rectifying K+ channels (GIRKs), voltage-gated Ca2+ channels (VGCCs) and adenylyl cyclases (ACs). The K+ channel tetramerization domain-containing (KCTD) proteins were shown to be auxiliary subunits of GABAB receptors that constitutively bind to the receptor via GABAB2 and regulate receptor signaling in a KCTD-subtype specific manner (Schwenk et al., 2010). The aim of this thesis was to better understand the KCTD12-mediated modulation of GABAB receptor signaling as well as to explore new mechanisms of receptor fine-tuning. The first part of this thesis uncovers the mechanism of GABAB receptor-activated K+ current desensitization induced by KCTD12. Besides the constitutive binding of all KCTDs to the G-protein, selectively KCTD12 has a second binding site on the activated Gß? subunit. A switch in binding uncouples Gß? from the K+ channels and induces K+ current desensitization. Native KCTD12 associates exclusively with GABAB receptors rendering this mechanism receptor specific. (Turecek et al., 2014) In the second part, we investigated the effects of GABAB receptor phosphorylation on KCTD12-induced K+ current desensitization. Phosphorylation of serine-892 (S892) in GABAB2 by protein kinase A (PKA) was previously described as a rather slow mechanism to regulate GABAB receptor desensitization (Couve et al., 2002). In contrast, KCTD12-induced desensitization of GABAB receptor-activated K+ currents represents a fast form of desensitization. We show that both mechanisms of desensitization influence each other. S892 phosphorylation slows KCTD12-induced K+ current desensitization by rearranging KCTD12 at the receptor. In turn, KCTD12 promotes tonic S892 phosphorylation by binding to GABAB receptors. This cross-regulation renders the signaling of GABAB receptors more precise and highly dependent on both the expression of KCTD12 and the activity of PKA. (Adelfinger et al., 2014) The third part reveals that all KCTDs exert marginal allosteric influence on the ligand binding affinity of GABAB receptors. However, we show that KCTD8, in contrast to KCTD12 and KCTD16, reduces the basal G-protein activation of GABAB receptors. (Rajalu et al., 2014) The fourth part summarizes an ongoing project in which we investigate the effects of 14-3-3 proteins and cullin3 (CUL3) on GABAB receptor signaling. 14-3-3 e and ? specifically interact with KCTD16 through its H2-domain. This binding regulates the expression of KCTD16 and GABAB2 resulting in altered Ga-signaling of GABAB receptors. Furthermore, we report a specific interaction between KCTD16 and CUL3, which is controlled by 14-3-3 proteins. In summary, we propose that 14-3-3 proteins determine the rate of lysosomal GABAB receptor degradation, which is induced upon binding of KCTD16 and CUL3. (Adelfinger et al., in preparation) Finally, in collaboration with the group of Jan Siemens from Heidelberg we studied how GABAB receptors reciprocally counteract the sensitization of the capsaicin receptor TRPV1. (Hanack et al., in preparation

Auxiliary GABAB Receptor Subunits Uncouple G Protein βγ Subunits from Effector Channels to Induce Desensitization

SummaryActivation of K+ channels by the G protein βγ subunits is an important signaling mechanism of G-protein-coupled receptors. Typically, receptor-activated K+ currents desensitize in the sustained presence of agonists to avoid excessive effects on cellular activity. The auxiliary GABAB receptor subunit KCTD12 induces fast and pronounced desensitization of the K+ current response. Using proteomic and electrophysiological approaches, we now show that KCTD12-induced desensitization results from a dual interaction with the G protein: constitutive binding stabilizes the heterotrimeric G protein at the receptor, whereas dynamic binding to the receptor-activated Gβγ subunits induces desensitization by uncoupling Gβγ from the effector K+ channel. While receptor-free KCTD12 desensitizes K+ currents activated by other GPCRs in vitro, native KCTD12 is exclusively associated with GABAB receptors. Accordingly, genetic ablation of KCTD12 specifically alters GABAB responses in the brain. Our results show that GABAB receptors are endowed with fast and reversible desensitization by harnessing KCTD12 that intercepts Gβγ signaling

GABA Blocks Pathological but Not Acute TRPV1 Pain Signals

SummarySensitization of the capsaicin receptor TRPV1 is central to the initiation of pathological forms of pain, and multiple signaling cascades are known to enhance TRPV1 activity under inflammatory conditions. How might detrimental escalation of TRPV1 activity be counteracted? Using a genetic-proteomic approach, we identify the GABAB1 receptor subunit as bona fide inhibitor of TRPV1 sensitization in the context of diverse inflammatory settings. We find that the endogenous GABAB agonist, GABA, is released from nociceptive nerve terminals, suggesting an autocrine feedback mechanism limiting TRPV1 sensitization. The effect of GABAB on TRPV1 is independent of canonical G protein signaling and rather relies on close juxtaposition of the GABAB1 receptor subunit and TRPV1. Activating the GABAB1 receptor subunit does not attenuate normal functioning of the capsaicin receptor but exclusively reverts its sensitized state. Thus, harnessing this mechanism for anti-pain therapy may prevent adverse effects associated with currently available TRPV1 blockers