GABAB receptor-associated KCTD proteins as molecular linkers to downstream signaling complexes

Berner, David. GABAB receptor-associated KCTD proteins as molecular linkers to downstream signaling complexes. 2016, Doctoral Thesis, University of Basel, Faculty of Science.

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Official URL: http://edoc.unibas.ch/diss/DissB_12630

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GABAB receptors are metabotropic receptors of the prevalent inhibitory neurotransmitter gammaaminobutyric acid (GABA), playing important roles in modulating overall neurotransmission and synaptic plasticity processes. The two principal subunits GABAB1 and GABAB2 form an obligate heterodimeric receptor, which is coupled to Gi/o (reviewed in Gassmann and Bettler, 2012). Additionally, GABAB receptors are found in complexes with members of a subfamily of potassium channel tetramerization domain proteins (KCTDs). Precisely, KCTD8, KCTD12, KCTD12b and KCTD16 function as auxiliary subunits, further increasing molecular diversity of GABAB receptors and specifically modulating receptor responses, e.g. KCTD12‐mediated desensitization of GABAB responses (Schwenk et al., 2010, Turecek et al., 2014). It has been described that GABAB receptors together with their KCTD subunits are present in large signaling complexes, e.g. in conjunction with the presynaptic N‐type voltage‐gated calcium channels and elements of the synaptic release machinery (Müller et al., 2010). However, the precise function of KCTDs in these complexes is not fully understood at the moment. This thesis aims to study some of the roles that the KCTD subunits play in GABAB signaling, especially with regard to the interconnection of GABAB receptors to downstream signaling complexes. The main hypothesis was that KCTDs are molecular scaffolds for these protein complexes, thereby enlarging the functional repertoire of GABAB receptors. The thesis is divided in two independent chapters. In the first chapter, I found that KCTD8 and KCTD16 are novel interactors of Cullin 3‐RING E3 ubiquitin ligases (CRL3). The hypothesis that the GABAB-associated KCTDs may bind to Cullin 3 was based on the fact that other KCTD family members were shown to be CRL3 substrate adaptors (Skoblov et al., 2013). Surprisingly, these newly‐discovered interactions with Cullin 3 depended on the homology 2 (H2) domains of KCTD16 and KCTD8, even though Cullin 3‐interactions of other KCTD family members are mediated by so‐called Bric‐a‐brac, Tramtrack, Broad‐complex (BTB) motifs found in their tetramerization (T1) domains (Furukawa et al., 2003, Skoblov et al., 2013). In the case of KCTD8 and KCTD16, their T1 domains were shown to lack Cullin 3‐binding. Similar to other BTB substrate adaptors, KCTD16 was found to bind to the N‐terminus of Cullin 3. The unusual Cullin 3‐binding domains of KCTD16 and KCTD8 were confirmed by BRET measurements. Finally, I found that KCTD16 provides a linker between the GABAB receptor and the Cullin 3 complex. Coexpression of KCTD16 with GABAB receptors did not down‐regulate GABAB receptors. Thus, the functional consequences of these novel CRL3 complexes are still unknown. In the second chapter, I studied the function of the KCTDs in the complex of GABAB receptors and N‐type voltage‐gated calcium channels (VGCCs). I discovered that the CaV2.2 α1 subunit specifically interacts with KCTD16 but not with KCTD12 and KCTD8. Interaction domain‐mapping showed that this CaV2.2-KCTD16 interaction relied on the H2 domain of KCTD16. Strikingly, the CaV2.2‐binding property could be transferred to KCTD12 by fusing the H2 domain of KCTD16 to KCTD12. Interestingly, the Gβγ‐binding intracellular loop I‐II of CaV2.2 was sufficient for the association with KCTD16. Finally, I confirmed the protein‐protein interactions of GABAB receptors with both CaV2.2 and the synaptic protein syntaxin‐1 in mouse brain tissue in co‐IP experiments. To understand the physiological relevance of this direct CaV2.2‐KCTD16 interaction, its electrophysiological effects were characterized. It was found that KCTD16 changes the biophysical properties of N‐type VGCCs in several ways. First, KCTD16 shifts the voltage‐dependence of the channel to more hyperpolarized potentials. Second, KCTD16 increases the permeability of N‐type VGCCs for divalent cations. Third, KCTD16 accelerates the kinetics of the channel activation. Perhaps most important for in vivo function, KCTD16 decreases the sensitivity and speed of response of N‐type VGCCs to GABAB‐mediated inhibition. In conclusion, the results of this thesis corroborate the concept that GABAB receptor‐associated KCTDs act as molecular linkers of GABAB receptors to downstream signaling complexes, as shown here for CRL3 and N‐type VGCCs. Furthermore, the results presented here also have functional implications for GABAB-modulation of presynaptic neurotransmitter release.
Advisors:Bettler, Bernhard and Rüegg, Markus A.
Faculties and Departments:03 Faculty of Medicine > Departement Biomedizin > Division of Physiology > Molecular Neurobiology Synaptic Plasticity (Bettler)
05 Faculty of Science > Departement Biozentrum
UniBasel Contributors:Bettler, Bernhard and Rüegg, Markus A.
Item Type:Thesis
Thesis Subtype:Doctoral Thesis
Thesis no:12630
Thesis status:Complete
Number of Pages:1 Online-Ressource
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edoc DOI:
Last Modified:01 Jul 2020 12:49
Deposited On:06 Jun 2018 12:44

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