Gamma-aminobutyric acid type B (GABAB) receptor is a G protein coupled receptor (GPCR) that mediates decreased neural activity. It has two subunits, GABAB1 and GABAB2. Previous complementary DNA (cDNA) microarray data showed strong GABAB1 signals from human prefrontal cortex using an intron 4 region probe, and these studies indicated that novel intron 4 containing GABAB1 splicing variants exist. We cloned GABAB1k, l, m, and n including mouse GABAB1j. Expression of these variants are much lower than other major known splicing variants, but GABAB1k, l, m, and n levels are similar across brain tissues. GABAB1l and GABAB1m impair GABAB receptor induced function. To better define GABAB1 splicing in alcoholic brains, whole transcriptome shortgun sequencing (RNA-seq) experiments were proposed. Due to the complexity of GABAB1 splicing, we used gene specific libraries as well as whole transcriptome libraries to maximize GABAB1 specific splicing junction search. The splicing junction search data found that GABAB1 gene is 2 to 3 times longer than the previous known gene length. Extremely low expression at 5’ end exons was found, and GABAB1 exons were grouped based on expression levels. Chronic alcohol altered exon/intron expression and splicing junctions more than overall gene expression. Decreased exon expression at a GABA binding site, a transmembrane domain (TM), and a microRNA (miRNA) binding site may diminish the normal GABAB1 transcript population and compromise signal transduction following chronic alcohol exposure. This may explain why GABAB receptor agonists have therapeutic benefit in treating alcoholism. During the sequence mapping, read pile-ups and gaps were found from whole transcriptome libraries in known exons. These may prevent single nucleotide polymorphism (SNP) and splicing junction identification and gene expression calculations. Sequence analysis found sequence biases from their mapped reads. The major sequence biases were from RNaseIII RNA fragmentation and T4 polynucleotide kinase (T4PNK) reaction. Heat fragmentation and OptiKinase treatment removed the read pile-ups and gaps including the sequence biases. The identification of RNaseIII target sequences can be incorporated into methods of miRNA gene prediction. These data showed the complexity of GABAB1 receptor splicing and the perturbation of splicing by chronic alcohol abuse demonstrate the power of RNA-seq to provide new insight into gene expression and the role of GABAB receptors in alcoholism. In addition, many other important brain genes may have unexplored splicing variants which will be important for alcoholism and other psychiatric diseases. Also, new RNA-seq library constructions improved the quality of gene expression studies. / text

Abstract The broad aim of this thesis is to structurally and functionally explore two α-conotoxins, from venomous sea snails, Vc1.1 and RgIA, in the hope of improving their journey to becoming analgesic therapeutics (introduction to conotoxins in Chapter 1). Vc1.1 is a two-disulfide peptide that is of interest as a potential therapeutic for the treatment of neuropathic pain. Despite investigations, limited structure-activity relationships have been conducted on this α-conotoxin. Consequently there is restricted insight into the interaction of this peptide with one of its analgesic targets, the α9α10 nicotinic acetylcholine receptor (nAChR). Late in this PhD project, the GABAB receptor was implicated as the possible target for conotoxins in neuropathic pain relief. However, there is still debate in the literature with regard to the true target of Vc1.1 and the α9α10 nAChR is still believed to be the target by some groups. This thesis predominantly focuses on the α9α10 nicotinic acetylcholine receptor. Chapter 4 of this thesis presents an extensive series of mutational studies in which all residues except the conserved cysteines were mutated separately to Ala, Asp or Lys (materials and methods described in Chapter 3) and examined using NMR spectroscopy (theory of NMR presented in Chapter 2), to determine the effects of the mutations on the structure of Vc1.1. The structural fold was found to be preserved in all peptides except where Pro was substituted. Chapter 5 explores the effect of these mutations on the blocking of acetylcholine (ACh)-evoked membrane currents at the α9α10 nAChR. Electrophysiological studies showed that the key residues for Vc1.1’s activity are Asp5-Arg7 and Asp11-Ile15, as changes at these positions resulted in the loss of activity at the α9α10 nAChR. Interestingly, the S4K and N9A analogs were more potent than Vc1.1 itself. Hence, Chapter 6 describes a second generation of mutants that was synthesized, namely N9G, N9I, N9L, S4R and S4K+N9A, all of which were more potent than Vc1.1 at both the rat α9α10 and the human α9/rat α10 hybrid receptor, providing a mechanistic insight into the key residues involved in eliciting the biological function of Vc1.1. The most potent analogs were also tested at the α3β2, α3β4 and α7 nAChR subtypes to determine their selectivity. All mutants tested were most selective for the α9α10 nAChR. These findings provide valuable insight into the interaction of Vc1.1 with the α9α10 nAChR subtype and will help in the further development of Vc1.1 or its analogs as drugs. However, despite peptides exhibiting high degrees of potency and selectivity, such as Vc1.1 and RgIA, they are potentially hindered in their development as drugs due to their stability and bioavailability limitations, leading to invasive delivery techniques. Chapter 7 presents a range of cyclic RgIA analogs, tested at their targets the α9α10 nAChR and the GABAB receptor, that retain their activity and increase their stability in human serum relative to non-cyclic RgIA. NMR spectroscopy was used to determine the structure of the non-cyclic peptide and the cyclic peptide to confirm similarities in the global fold of the peptide. Structural perturbations and reduced activities were observed for cyclic RgIA analogs cyclized via linkers composed of three and four residues. Analogs with five, six and seven residues showed no structural perturbations, but differences in their activities at the different receptors. Because cRgIA-6 showed high potency for the GABAB receptor and lower potency for the α9α10 nAChR, this study has identified a GABAB selective peptide. Additionally, because the cRgIA-7 showed high potency for the α9α10 nAChR and low potency for the GABAB receptor, a α9α10 nAChR selective analog has also been identified. With improvements in these peptides against enzymatic attack, they show great potential on their path to becoming orally available analgesics as they may be able to withstand enzymatic conditions in the stomach.

In this work, the detailed analysis of GABAB-R/G protein coupling in the course of pre- and postnatal development of rat brain cortex indicated the significant intrinsic efficacy of GABAB-receptors already shortly after the birth: at postnatal day 1 and 2. Subsequently, both baclofen and SKF97541-stimulated G protein activity, measured as the high-affinity [35 S]GTPγS binding, was increased. The highest level of agonist-stimulated [35 S]GTPγS binding was detected at postnatal days 14 and 15. In older rats, the efficacy, i.e. the maximum response of baclofen- and SKF97541-stimulated [35 S]GTPγS binding was continuously decreased so, that the level in adult, 90-days old rats was not different from that in newborn animals. The potency of G protein response to baclofen stimulation, characterized by EC50 values, was also high at birth but unchanged by further development. The individual variance among the agonists was observed in this respect, as the potency of SKF97541 response was decreased when compared in 2-days old and adult rats. The highest plasma membrane density of GABAB-R, determined by saturation binding assay with specific antagonist [3 H]CGP54626AA, was observed in 1-day old animals. The further development was reflected in decrease of receptor number. The adult level was ≈3- fold lower than...

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Les récepteurs couplés aux protéines G (RCPG) constituent la plus grande famille de récepteurs transmembranaires. Ils sont impliqués dans une large variété de processus physiologiques et par conséquent ils représentent une cible thérapeutique d'intérêt pour le développement de médicaments. Plusieurs études ont démontré que les RCPGs sont capables d'interagir entre eux pour former des complexes oligomériques. Cependant, leur existence in vivo et leur rôle fonctionnel reste sujet à débats. Afin de mieux appréhender ce phénomène, nous avons utilisé un RCPG de classe C comme modèle d'étude, le récepteur de l'acide γ-aminobutyrique (GABAB), qui est impliqué dans une grande variété de désordres neurologiques et psychiatriques. Son originalité réside dans le fait qu'il est un hétérodimère obligatoire composé de deux sous-unités : GABAB1 et GABAB2 (GB1 et GB2). La liaison de l'agoniste sur GB1 conduit à l'activation de GB2. Au cours de ma thèse, nous avons montré en utilisant une nouvelle approche biophysique basée sur un marquage fluorescent enzymatique appelé Snap-tag que, contrairement aux récepteurs métabotropiques du glutamate, le récepteur GABAB forme des dimères de dimères (tétramères). Cette organisation hétéro-oligomérique est assurée par des contacts stables entre les domaines extracellulaires des sous-unités GB1. De plus, nous avons apporté des données en faveur de l'existence physiologique de cet assemblage en utilisant des membranes de cerveau de rat et de souris. Dans une seconde partie, nous avons souhaité déterminer les conséquences fonctionnelles de cette organisation. Nos résultats suggèrent une efficacité de couplage à la protéine G réduite du récepteur GABAB lorsqu'il est associé en dimères de dimères. Collectivement, nos données rapportent pour la première fois, l'existence de larges complexes allostériques de RCPGs dans le cerveau. / The G-protein coupled receptors (GPCR) constitute the main family of transmembrane receptors. They are involved in many physiological processes and, as a consequence, they represent a therapeutic target of interest for the development of new drugs. Few studies have demonstrated that GPCRs are able to interact with each other to form oligomeric complexes. However, the existence in vivo and the functional interest of these oligomers remain a subject of intense debates. To address this issue, we have used a class C GPCR as a model, the γ-aminobutyrate B receptor (GABAB), which is involved in a wide variety of neurological and psychiatric disorders. This receptor has the particularity to be an obligatory heterodimer composed of two subunits GABAB1 and GABAB2 (GB1 and GB2). Agonist binding on GB1 leads to G-protein activation by GB2. During my thesis, we developed a new biophysical approach based on an enzyme-mediated fluorescent labeling calle d Snap-Tag and showed that, unlike metabotropic glutamate receptors, GABAB forms dimers of dimers (tetramers). This oligo-heterodimers organization is mediated via stable contacts between extracellular domains of GB1 subunits. Furthermore, we brought evidence of the physiological reality of this assembly using rat and mouse brain membranes. Then, we aimed at assessing what would be the functional rational of the GABAB dimer of heterodimers. Our results suggest that the GABAB receptor has a lower G protein-coupling efficacy when associated into dimers of dimers. Altogether, our data report for the first time, the existence of large allosteric GPCR complexes in the brain.

GABAB receptors are G-protein coupled receptors for the major inhibitory neurotransmitter in the mammalian central nervous system, γ-aminobutyric acid (GABA). The receptor is linked to a variety of disorders including epilepsy, pain, spasticity, drug addiction and cognitive impairment and is, therefore of major importance for drug discovery. The most abundant receptor isoforms GABABR1a and R1b differ by the presence in R1a of a pair of Nterminal extracellular complement control protein modules (CCP1 and CCP2) which - in other proteins - are generally involved in mediating specific protein-protein recognition. The CCP1 module contains disulphides but is natively disordered. In the current work, the yeast two-hybrid system was used to confirm an interaction of CCP1 of GABABR1a with the extracellular protein fibulin-2. Further work with the yeast twohybrid system extablished the novel interaction of the abundant extracellular matrix protein laminin, with GABABR1a CCP1, via its laminin globular (LG) domains. The laminin interaction was further characterised by surface plasmon resonance, demonstrating that several different domains are involved in the binding to the GABAB receptor CCPs. The primary binding site is located on laminin α5 LG4-5, but the E10 domains of the β1 chain and LG1-3 on α1 may also be involved. The pharmacological properties of the GABABR1a and R1b isoforms were studied by transient expression in Xenopus laevis oocytes. It was demonstrated that the agonist baclofen, as well as the antagonist CGP55845, appear to be more potent at GABABR1b compared to GABABR1a. Intriguingly, when recorded in the precence of laminin, GABABR1b/R2 expressing oocytes exhibited an increased baclofen-evoked response while the response in GABABR1a/R2 was completely abolished. In conclusion, the work demonstrates that laminin is a binding partner for GABABR1a CCPs. Such an interaction between the metabotropic GABA receptor and the extracellular matrix may lie behind the recently reported roles of GABA in neuronal migration and the laying down of neuronal circuitry during the development of parts of the central nervous system.

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Les récepteurs couplés aux protéines G (RCPG) sont les cibles de 50% des médicaments actuellement sur le marché pharmaceutique. La compréhension de leur mode de fonctionnement est donc essentielle au développement de nouvelles molécules capables de cibler spécifiquement ces récepteurs. Ces dix dernières années, les technologies de transfert d'énergie ont permis de révéler la capacité des RCPG, longtemps considérés comme monomériques, à s'organiser en dimères voire en structures oligomériques plus grandes. Toutefois, la caractérisation précise de cette stœchiométrie d'assemblage implique la mise au point de méthodes adaptées. <br />Au cours de ce travail de thèse nous avons développé une approche de FRET en temps résolu permettant de mettre en évidence, à l'aide d'anticorps marqués, des interactions de sous-unités de RCPG à la surface de cellules vivantes. En choisissant le récepteur GABAB comme modèle d'étude, cette approche a permis de révéler l'homo- et l'hétérodimérisation de ce récepteur à la surface cellulaire. De plus, en condition de perméabilisation des cellules, l'oligomérisation de la sous-unité GABAB1 retenue dans les compartiments intracellulaires a pu être caractérisée par cette même approche.<br />Afin d'analyser plus précisément l'organisation du récepteur GABAB, nous avons mis au point une deuxième méthode permettant de marquer irréversiblement à l'aide de fluorophores les sous-unités GABAB1 et GABAB2 présentes à la surface cellulaire. La combinaison de cette méthode de marquage (SNAP-tag) avec une analyse de FRET en temps résolu a permis de caractériser l'organisation oligomérique de ce récepteur. Ainsi, le récepteur GABAB, connu pour être un hétérodimère obligatoire, semble capable de former des oligomères via la sous-unité GABAB1 qui représente un point de contact entre deux hétérodimères. Le rôle d'une telle organisation sur la fonction de ce récepteur reste toutefois indéterminé.

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