ED Sciences Chimiques
Dopamine D₂ receptor (D₂R) isolation, characterization and heteromerization
by Nejma RABHI (Institut de Chimie & de Biologie des Membranes & des Nano-objets)
The defense will take place at 13h30 - Amphithéâtre IECB, 2 Rue Robert Escarpit, 33600, Pessac
in front of the jury composed of
- Sylvie CLAEYSEN - Chargée de recherche - Université de Montpellier - Rapporteur
- Astrid WALRANT - Maîtresse de conférences - Sorbonne Université - Rapporteur
- Olivier LAMBERT - Directeur de recherche - Université de Bordeaux - Examinateur
- Birgit HABENSTEIN - Directrice de recherche - Université de Bordeaux - Examinateur
This PhD thesis investigates the role of membrane lipid composition in the heteromerization, dynamics, and signaling of the dopamine D₂ receptor (D₂R), a key GPCR implicated in schizophrenia and other neurological disorders. The work is structured around two main axes: (i) the expression, isolation, and characterization of functional D₂R in various membrane systems (in cellulo, ex cellulo, in vitro), and (ii) the in cellulo study of D₂R heteromers, particularly D₂-CB₁, focusing on their localization, dynamics, and intracellular signaling. To address the challenges of GPCR instability outside their native lipid environment, multiple strategies were employed for D₂R extraction and purification. Detergent-based methods (e.g., LMNG/CHS, DDM/CHAPS/CHS) were optimized to balance purity and functionality, though they often disrupted the receptor's native lipid surroundings. As an alternative, copolymer-based nanodiscs (DIBMA 12) were explored, preserving the receptor's lipid environment and yielding higher functional retention, as confirmed by GTPase Glo Assay and fluorescence polarization (FP). These nanodiscs allowed for controlled lipid composition and enabled structural and functional studies in a near-native context. Functional characterization of D₂R was achieved through FP and the innovative GTPase Glo Assay, which measures G protein activation upon ligand stimulation, overcoming limitations of traditional FP methods. Additionally, TIRF microscopy and BRET assays were used to study D₂-CB₁ heteromer dynamics and signaling in live cells, providing insights into their localization, interactions, and downstream effects. This work highlights the critical role of lipids in modulating D₂R structure, stability, and signaling, offering new perspectives for understanding GPCR function in physiological and pathological contexts, such as schizophrenia. The optimized methods for isolating functional D₂R in controlled lipid environments pave the way for advanced structural and pharmacological studies, ultimately aiming to improve therapeutic strategies for neurological disorders.