Functional characterization of GABAB receptor-associated proteins

GABAB receptors (GBRs) are the G-protein coupled receptors (GPCRs) for γ‐aminobutyric acid (GABA), the main inhibitory neurotransmitter in the central nervous system (Roberts & Frankel, 1950). GBRs are membrane receptors with a seven membrane‐spanning domain composed of the principal subunits GABAB1 and GABAB2. Activated GBRs regulate G protein‐coupled inwardly rectifying K+ channels (GIRKs), voltage‐gated Ca2+ channels (VGCCs), and adenylate cyclases (ACs) (Kaupmann et al., 1998; Schwenk et al., 2010). Native GBRs form macromolecular complexes with auxiliary subunits and various constituents, which impart distinct functional properties to GBRs (Schwenk et al., 2016). One of the aims of my Ph.D. was to understand better the physiological role of previously identified proteins associated with the GBR core. In the first part of this thesis, we show that Synaptotagmin-11 (Syt11), a poorly characterized Synaptotagmin isoform with inactive calcium-binding sites (Von Poser et al., 1997), coordinates the trafficking of GBRs and Cav2.2 in hippocampal neurons. Syt11 can interact with GBRs and Cav2.2 through KCTD16 and APP (Dinamarca et al., 2019; Norstrom et al., 2010; Schwenk et al., 2016) and facilitates the co-transport of GBRs and Cav2.2 into post- Golgi vesicles. Furthermore, we found that Syt11 remains associated with GBR-complexes at the plasma membrane and reduces constitutive internalization of GBRs and Cav2.2. Finally, Syt11-deficient neurons exhibit impaired GBR-mediated presynaptic inhibition demonstrating an essential role for Syt11 in GBR physiology (Trovò et al., in preparation). In the second part, we investigated the controversial role of sAPP. Indeed, a recent publication demonstrates that the soluble fraction of APP (sAPPα) and a 17 amino acids long peptide (APP17) containing the GBR binding site, activates presynaptic GBR, reducing synaptic vesicle release (Rice et al., 2019). However, another manuscript indicated that sAPPα does not influence GBR-mediated G-protein activation (Dinamarca et al., 2019). Thus, after confirming that APP17 binds with nanomolar affinity to GBRs, we performed an in-depth investigation on the function of sAPP and APP17 in GBR physiology. Interestingly, biochemical and electrophysiological assays indicate that sAPP and APP17 do not influence the activity of recombinant GBRs. Furthermore, we found no evidence for APP17 regulating Kir3 channel activity in cultured neurons, neurotransmitter release in brain slices, or neuronal activity in vivo. Altogether, our results indicate that sAPP and APP17 do not regulate neuronal activity through GBRs (Rem et al., submitted). The third part of this thesis moves the focus to the metabotropic Glutamate receptors (mGlur). Although historically, mGluRs were known to form functional homodimers, several proofs of heterodimerization are emerging (in vitro) (Doumazane et al., 2011). Here, we demonstrated the existence of mGlu1/5 heterodimers in neurons in vivo (Werthmann et al., 2021)