Structural characterisation of A2A adenosine- D2 dopamine receptor heteromer

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G-protein coupled receptors (GPCRs) are the largest family of protein receptors and it is now widelyaccepted that many can not only function as monomers but also as oligomers with distinct biologicalfunctions (Maurice et al., 2011). However, there is a lack of structural information about these oligomers,particularly at the interface between protomers. A2A adenosine receptor (A2AR) and D2 dopamine receptor(D2R) are Class A GPCRs which are co- expressed in striatopallidal GABA neurons in the central nervoussystem (Fuxe et al., 2010; Schiffmann et al., 1991). A2AR allosterically inhibits D2R signalling in the brain,thus A2AR-D2R heteromers have been implicated in disordered dopamine signalling such as in Parkinson's,schizophrenia and cocaine addiction (Ferre et al., 1991; Fuxe et al., 2010). Evidence to suggest theexistence of an A2AR-D2R heteromers was found through coimmunoprecipitation, BRET (Bioluminescenceresonance energy transfer) and FRET (florescence resonance energy transfer) analyses (Canals et al.,2003; Hillion et al., 2002). Since then, studies to investigate the interface and overall structure of theheteromer have been carried out (Borroto-Escuela et al., 2018). in However, the precise molecular basisof the interactions between protomers remain unclear. The characterisation of receptor interface along witha high-resolution structure of the A2AR-D2R heteromer would allow the development of novel drugs to targetthe receptors in a heteromer- specific manner. Furthermore, the structure of this heteromer may allow agreater understanding of the nature of oligomer formation and regulation and other similar Class A GPCRoligomers.Since GPCRs are membrane proteins, they are notoriously difficult to isolate for structural studies due totheir dynamic flexibility (Zhao and Wu, 2012). Therefore, methods to engineer the GPCR to increase itsstability are common and widely used. This is even more relevant in the case of the receptor heterodimers,however methods to stabilise these complexes are less well developed.Therefore, the overall aim of the project is to stabilise the A2AR and D2R heteromer in mammalian systemsfor high resolution structural analysis. This optimally stable heteromer will be obtained by:1. Applying mutagenesis to introduce cross-linkages2. Screening of conformationally restricted mutants3. Use of nanobodiesWhile these strategies aim to obtain optimal constructs for structural studies, these experiments alone willprovide vital information on the precise molecular interactions between protomers.Site directed mutagenesis of A2AR and D2R will be carried out to try to establish cysteine cross-linkingbetween the two receptors. Our collaborator used molecular modelling to predict the heteromer interfaceand identify the optimal residues to create cross-linkages (Francesca Fanelli, unpublished). This hasidentified 4 candidate residue pairs for mutagenesis to cysteine. These cysteine mutant pairings will beassessed individually using BRET to determine their effect on interprotomer affinity and proximity. Mutantpairs which have successfully cross-linked and result in an increased affinity at the interface between A2AR and D2R will be combined to create a more stable heteromer. This will not only increase heteromer stability for structural analysis but will give further evidence for the predicted receptor interface, which is currently poorly understood. Previous work has also identified nanobodies which are effective in stabilising the A2A homomer (Thomas Diaz, unpublished). This will be applied to the cysteine mutated A2A-D2 heteromer to investigate if these further alter associations by BRET analysis. These strategies for stabilisation along with the use of thermostabilised A2A and D2 mutants will be used for isolation, purification and structural studies with the heteromer.