Evidence that human P2Y1 and P2Y12 receptors form functional heterodimers

Roger Naughton, Kenneth Paton, Trevor Bushell, Charles Kennedy. University of Strathclyde, Glasgow, UK

P2Y receptors are a family of eight G protein-coupled receptors (GPCR) that are activated by endogenous nucleotides (Kennedy et al., 2013). Our previous data suggest that two ADP-sensitive P2Y receptors, the Gαq-coupled hP2Y1 receptor and the Gαi/o-coupled hP2Y12 receptor, may form heterodimers with novel pharmacological and signalling properties (Shakya-Shrestha et al., 2010). Formation of GPCR oligomers is now recognised as being common and perhaps even crucial for the trafficking, membrane expression and functional activity of GPCR (Milligan, 2013). The aim of this study was, therefore, to characterise the properties of this potentially novel P2Y1/P2Y12 receptor heterodimer.

Human tsA201 cells, a modified HEK293 cell line, were grown to confluence on 13 mm glass coverslips then transfected with hP2Y1-eYFP and/or hP2Y12-eCFP cDNA. To determine the role of pertussis toxin (PTX)-sensitive G proteins, cells were incubated with PTX (200 ng.ml-1) for at least 18 h. Following incubation with the Ca2+-sensitive dye, Fluo-4-AM (5 μM), the cells were continuously superfused with a HEPES-based buffer at 4 ml.min-1 at room temperature and fluorescence monitored in a Perkin Elmer LS50B spectrophotometer. Drugs were applied via the superfusate. Data were analysed using Student\'s paired t test or one way ANOVA with Tukey\'s comparison, as appropriate. Values of P<0.05 were considered to be statistically significant. P2Y receptor heterodimerisation was studued by Fluorescence Resonance Energy Transfer (FRET) imaging, using a Leica SP-5 laser scanning confocal microscope.

In untransfected cells ADP (10 nM-10 µM) evoked a concentration-dependent increase in fluorescence, (EC50=210 nM, 95% cl=155-285 nM, n=5), which was unaffected by pretreatment with PTX (EC50=208 nM, 95% cl=186-232 nM, n=4). The rise evoked by ADP (300 nM) was abolished by the selective P2Y1 antagonist, MRS2179 (10 µM) (n=3), but not inhibited by the selective P2Y12 antagonist, AR-C69913MX (1 µM) (n=5). These data are consistent with the presence of an endogenous P2Y1 receptor in tsA201 cells that mediates release of intracellular Ca2+ stores (Shakya-Shrestha et al., 2010). Incubating cells with hP2Y12-eCFP cDNA for 24 h led to the expression of fluorescent protein in approximately 60-70% of cells, but had no significant effect on the potency of ADP (EC50=264 nM, 95% cl=190-367 nM, n=4) or on the ability of MRS2179 (10 μM) to abolish its action (n=3). In contrast to the non-transfected cells, however, AR-C69913MX (1 µM) now significantly inhibited the ADP (300 nM)-evoked Ca2+ increase (47.5 ± 5.8% of control, n=4) (P<0.01). Finally, FRET analysis in cells co-expressing hP2Y1-eYFP and hP2Y12-eCFP indicated an interaction between the two proteins.

These results suggest that recombinant P2Y12 receptors heterodimerise with the native P2Y1 receptor in tsA201 cells. ADP appears to bind to both receptors to activate the complex and blockade of the ADP binding site on either prevents activation. Furthermore, the P2Y12 receptor acts through Gαi/o to elicit a rise in cytoplasmic Ca2+.

Kennedy C et al, Future Med Chem 5:431, 2013

Milligan G, Mol Pharmacol 84:158, 2013

Shakya-Shrestha S et al, Mol.Cell Neurosci 43:363, 2010