ADP plays a central role in platelet activation by co-stimulation of two surface expressed G protein-coupled receptors P2Y₁ and P2Y₁₂. G protein-coupled receptor surface expression and coupling to intracellular signalling is tightly controlled. We hypothesised that the responsiveness of platelets to ADP is also regulated. In order to more fully elucidate the molecular mechanisms responsible for receptor desensitisation, in addition to studies using human platelets, we have studied the regulation of P2Y₁ and P2Y₁₂ receptors heterologously expressed in 1321N1 astrocytoma cells, a P2Y-null cell line.

In platelet studies, human platelets were prepared as previously described (Crosby & Poole 2003). In parallel experiments, 1321N1 cells stably transfected with either P2Y₁ or P2Y₁₂ receptor or vector alone were maintained in DMEM containing 10% fetal bovine serum and 400 µg/ml geneticin prior to use. In both experimental systems changes in the P2Y₁ Gq-coupled receptor activity were assessed by measurement of agonist-induced increases in cytosolic free calcium using the fluorescent Ca²⁺ indicator Fura-2. G_i-coupled P2Y₁₂ receptor responses were assessed in platelets and 1321N1 cells by measuring ADP-induced inhibition of forskolin-stimulated adenylyl cyclase. Further studies were also undertaken in receptor expressing 1321N1 cells transiently transfected with dominant negative mutant (DNM)-GRK2 and DNM-GRK6 using Lipofectamine 2000. DNM expression was assessed by western blotting.

Initial studies in human platelets showed that both P2Y₁- and P2Y₁₂-mediated responses desensitize rapidly and substantially in human platelets pre-treated with ADP. ADP (10 µM) stimulated G_q-coupled P2Y₁ receptor activity was reduced by 80 ± 5.3 % following agonist pre-treatment (ADP, 10 µM; 30 sec). Agonist (ADP; 10 µM) stimulated P2Y 12-dependent inhibition of adenylyl cyclase was also significantly reduced from 52.1 ± 7.1 % to 15.3 ± 4.2 % (p<0.05, Mann-Whitney U-test; n=4) following ADP (10 µM; 300 sec) pre-treatment. Responses to both P2Y₁ and P2Y₁₂ receptors also rapidly desensitized when each receptor was stably expressed in 1321N1 cells. Transient overexpression of DNM-GRK2 or DNM-GRK6 did not effect agonist-induced P2Y₁ receptor desensitisation whilst interestingly inhibition of protein kinase C activity with GF109203X (2 µM) did significantly attenuate the desensitisation of this receptor. Desensitisation of P2Y₁ receptor activity was significantly reduced from 38.4 ± 7.5% to 15.7 ± 6.5% following GF109203X treatment (p<0.05, Mann-Whitney U-test; n=5). In contrast, overexpression of DNM-GRK2 or DNM-GRK6 attenuated agonist-induced P2Y₁₂ receptor desensitization whilst pre-treatment with GF109203X had no effect. The percentage desensitisation of P2Y₁₂ activity was 74.3 ±12.4, 17.8 ±5.3*, 3.9 ±4.7* and 51.8 ±12.4 respectively in vector transfected control, DNM-GRK2, DNM-GRK6 and GF109203X treated cells (* indicates significant difference from vector transfected controls p<0.05, Mann-Whitney U-test; n=5).

In conclusion we have demonstrated for the first time that both P2Y₁ and P2Y₁₂ receptor responses undergo rapid homologous desensitization in human platelets. We also show that P2Y₁ and P2Y₁₂ receptors are differentially regulated: P2Y₁ is regulated in a PKC-dependent, GRK-independent manner whilst the reverse is true for P2Y₁₂ , which undergoes GRK-dependent, PKC-independent desensitization.

Crosby, D & Poole, AW (2003) J Biol Chem 278, 24533-24541.