nvestigating functional and structural roles of G-protein coupled receptor oligomerization

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BackgroundGPCRs are the largest family of receptor proteins in eukaryotes and play essential roles in signal transduction in all physiological systems. To relay extracellular information to the cell, GPCRs undergo ligand-specific structural rearrangements that promote association with, and activation of intracellular transducers of different signalling pathways. Through differential post-translational modifications and allosteric interactions, these intracellular signalling proteins alter cell physiology; enzymatic activities, ion channel states, and gene expression levels. Thus, GPCRs act as sensors of the external environment, allowing extracellular ligands to modulate intracellular processes.Though originally thought to function exclusively as monomers, increasing evidence has shown that some GPCRs adopt homomeric and heteromeric assemblies with novel functional and pharmacological properties. Though structural information is available for GPCR homomers, our understanding of the mechanisms driving heteromer formation, the interfaces involved, their stability, allostery, and stoichiometry remain unclear. A high-resolution structure of a GPCR heteromer has never been reported and would facilitate the generation of heteromerisation-deficient receptors to unpick their specific contributions to in vivofunctions. More critically, structural data could be used for rational design of drugs biased towards or away from heteromer-specific signalling. Furthermore, computational approaches could be employed to enhance our understanding of the structure and function of other, related heteromers. AimsThis project aims to use molecular, computational, biophysical, cellular and super-resolution microscopy approaches to investigate the structure and function of the Adenosine A2A -Dopamine D2 heteromer complex. From a pharmacological perspective, understanding the differences between homomeric and heteromeric signalling modes of A2A and D2 receptors in may lead to novel strategies in the treatment of dopaminergic disorders such as Parkinson's Disease, addiction, and schizophrenia. MethodsIn particular, this project will aim to obtain sufficiently stable A2A-D2 oligomers for structural studies. To enhance the stability, mutational approaches will be used to generate cross-linked mutants, conformationally-stabilised mutants, and truncated forms. Mutagenesis approaches will be rationalised by computational modelling, and assessed by functional studies and super-resolution microscopy. The viability of additional tools such as ligands and stabilising proteins will also be assessed. Stable and functional forms of the A2A-D2 heteromer will be purified and biophysical/structural analyses will aim to elucidate the nature of protomer-protomer interactions and heteromer function. Outcomes The outcomes expected from this project are to stabilise the inter-protomer interactions in the A2A-D2 heteromer, in order to purify high quality protein, and in sufficient quantity, for high-resolution structure determination. This information would facilitate the rational design of pharmaceuticals that modify unique, oligomer-specific outcomes.