The functional relevance of P2Y1 and P2Y12 receptor heterodimerization

Introduction: 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 signaling. Also, test the physical interaction between both receptors in the recombinant and native system.

Method: tSA201 cells, which are a transformed human kidney cell line, were transfected or co-transfected with hP2Y1 and hP2Y12 receptors tagged with HA (N-terminal) or a fluorescent protein (C-terminal). Other experiments used BV-2 cells, which are mouse microglial cells. The combination of lifetime and FRET (FLIM-FRET) technique using (CFP/YFP tagged receptors) was used to detect the physical interaction for P2Y1-P2Y12 heterodimerization in the recombinant system, while proximity ligation assay (PLA) used to determine the heterodimer in the recombinant and native system. Intracellular Ca2+ influx was determined using Cal-520 dye in FlexStation Microplate Reader.

Result: In tSA201 cells, PLA indicated that the physical interaction for both receptors locate mainly on the cell membrane (n=3). The measured fluorescence lifetime in cells transfected only with the donor (P2Y12-eCFP) (1.55 ns) was significantly higher compared to cells co-transfected with the acceptor (P2Y1-eYFP) (2.21 ns), providing a strong evidence for P2Y1-P2Y12 dimerization (n=3). In BV-2 cells, PLA detected the interaction between both receptors natively, which was located intracellularly (n=3). Interestingly, AR-C69931MX (1 μM) significantly reduced the ADP (300nM)-induced Ca2+ influx in the BV-2 cells (n=4).

Conclusion: P2Y1 and P2Y12 heterodimer locates on the cell membrane in the recombinant system while it locates intracellularly in the native system. Native P2Y1 receptor signaling inhibited using P2Y12 antagonist, which similar finding from the Kennedy lab that used recombinant system. Further work in under way to investigate the interaction between both receptors in disease models, and investigate the signaling relevance for the formed dimer.