Bitter taste is one of the five basic taste modalities, and is mediated by 25 bitter taste receptors (T2Rs) in humans. How these few receptors recognize a wide range of structurally diverse bitter compounds is not known. To address this question, structural and functional studies on T2Rs are necessary. Quinine is a natural alkaloid and one of the most intense bitter tasting compounds. Previously it was shown that quinine activates T2R4, however, whether T2R4 has only one binding site for quinine, and the amino acids on the receptor involved in binding to quinine remain to be determined. In this study, the ligand binding pocket on T2R4 for quinine was characterized using a combination of approaches. These included molecular model guided site-directed mutagenesis, characterization of the expression of the mutants by flow cytometry, and functional characterization by cell based calcium imaging. Twelve mutations were made in T2R4 and their expression and function were characterized. Results show that the ligand binding pocket of T2R4 for quinine is situated on the extracellular side, and is formed by the residues present on the transmembrane regions TM3, TM4, and extracellular loop regions ECL2 and TM6-ECL3-TM7 interface. Further, this study identified the following amino acids : A90, F91, Y155 N173, T174, Y258 and K270 to play an important role in quinine binding to T2R4. The detailed study of residues interacting with ligand will help in understanding how various ligands interact with T2Rs, and facilitate the pharmacological characterization of potent antagonists or bitter taste blockers. The characterization of novel ligands, including bitter taste blockers will help in dissecting the signaling mechanism(s) of T2Rs, and help in the development of novel therapeutic tools for the food and drug industry.

La dégradation des ARN messagers (ARNm) est un processus universel extrêmement complexe. D’une manière semblable aux polymerases pour la transcription et ribosomes pour la traduction, les complexes de protéines effectuant la dégradation des ARNm sont précisément régulés. La dégradation des ARNm eucaryotes s’effectue selon un schéma conservé évolutivement qui est initié par la déadénylation résultant dans la formation de transcrits avec des queues polyA courtes. De tels intermédiaires sont alors dégradés par le clivage de leur coiffe suivi par une digestion exonucléolytique 5’-3’ effectuée par Xrn1, ou alternativement par une digestion 3 ’-5’ catalysée par l’exosome. Dans ma thèse je présente une dissection fonctionnelle du complexe de déadénylation CCR4-NOT basée sur son analyse structurale. Je me suis essentiellement intéressé à cinq questions fondamentales concernant ce complexe : La formation du complexe CCR4-NOT complexe est-elle requise pour la déadénylation ? Quel est le rôle moléculaire de sous-unités Not2/3/5 du complexe ? Pourquoi la protéine Not1 est-elle essentielle chez la levure ? Le complexe CCR4-NOT joue-t-il un rôle dans la répression de la traduction ? Comment le complexe CCR4-NOT est-il ciblé sur ses substrats ARNm ? / MRNA degradation is a highly complex and versatile process. In a manner similar to polymerase complexes in transcription and ribosomes in translation, protein complexes mediating mRNA decay are tightly regulated. Eukaryotic mRNA decay follows a conserved pathway initiated by deadenylation that generates transcripts with short polyA tails. The latter intermediates are degraded either by decapping followed with 5’-3’ trimming mediated by Xrn1, or by exosome-mediated digestion in the 3’-5’ direction. In my thesis I present a functional dissection of the Ccr4-Not deadenylase complex based on its structural analysis. Essentially, I addressed five fundamental questions related to this complex: Is CCR4-NOT complex formation required for deadenylation activity? What is the molecular role of associated Not2/3/5 subunits? Why is the Not1 protein essential in yeast? Does the CCR4-NOT complex play role in translation regulation? How is the CCR4-NOT complex targeted to its mRNA substrates?