Interrogating purinergic P2Y1 and P2Y12 heterodimerisation

ADP is an endogenous agonist for some G protein-coupled receptors (GPCRs) such as P2Y1 and P2Y12 purinoceptors. Previous studies demonstrated that AR-C69931MX, which is P2Y12 antagonist, inhibits ADP-induced intracellular Ca2+ in recombinant P2Y1 and P2Y12 receptors. In this study, we examined the effect of AR-C69931MX on native P2Y1 receptor signalling. Also, test the physical interaction between both receptors in the recombinant and native system. In the recombinant system (tSA201 cells), the physical interaction between the receptors was determined using co-immunoprecipitation (co-IP), proximity ligation assay (PLA) and the combination of lifetime and FRET (FLIM-FRET) technique that was developed and optimised with the cooperation with the physics department at the University of Strathclyde1. While in the native system (BV-2 microglial cells), PLA was used to determine the physical interaction. To investigate the influence of this dimer on receptors function, we measured the intracellular Ca2+ influx using Cal-520 dye in FlexStation Microplate Reader. We found that P2Y1 and P2Y12 can form a heterodimer that locates on the cell membrane in the recombinant system while it locates intracellularly in the native system. Native P2Y1 receptor signalling inhibited using P2Y12 antagonist, which similar finding from the Kennedy lab that used a recombinant system. Further work in underway to investigate the interaction between both receptors in disease models, and investigate the signalling relevance for the formed dimer.