Endocrine Abstracts

The D2 dopamine receptor (D2R) is a G protein-coupled receptor (GPCR) known to have key roles in neurotransmission, hypothalamic-pituitary hormone release and glucose homeostasis. Thus, this receptor is a therapeutic target in schizophrenia, neurodegenerative disorders, endocrine tumours and even diabetes. As for many GPCRs, D2R can associate with itself as homodimers or heterodimers with distinct receptors. The balance between monomers, homodimers and heterodimers of D2R has also been implicated in several diseases. However, the lack of high-resolution dimer structures hinders our understanding of these complexes and ability to exploit these interactions therapeutically. Therefore, our aim was to stabilise D2R homomers, assess the impact on functional activity and develop pathways for high resolution structures. Molecular modelling identified residues that had the potential to stabilise and/or form disulphide bridges at the predicted homodimer interface. The impact of these mutations on homodimer stability was determined using bioluminescence resonance energy transfer, western blot analysis and super-resolution single molecule microscopy. While their impact on functional activity was assessed via agonist-mediated Gai-signalling, recruitment of a key GPCR adaptor protein, β-arrestin-2, MAPK signalling and receptor internalisation. D2R (Val96Cys) and D2R (Val96Ser/Val97Cys) mutations increased the amount and proximity of protomers within a dimer. Interestingly, these mutations reduced the efficacy of the D2R agonist quinpirole to activate Gai signalling, while promoting faster recruitment (Val96Cys) or higher basal associations (Val96Ser/Val97Cys) of β-arrestin-2. This potential bias towards β-arrestin-2 was supported by enhanced basal and agonist-induced internalization, and a ‘switch’ to β-arrestin-mediated MAPK signalling. Overall, these findings that modulating D2R homodimer associations and/or stability highlights a selective role in modulating interactions with β-arrestin-2, which in turn promote β-arrestin functions in directing signal activity. These modified receptors could not only form the basis of future structural studies on these homomers, but also identify new potential therapeutic targeting strategies.