Desensitization and phosphorylation of the human P2Y(2) nucleotide receptor

Understanding the molecular basis of the P2Y₂ nucleotide receptor desensitization process may facilitate on-going studies aimed at elucidating the role of nucleotides in the inflammatory response of immune cells such as monocytes and macrophages. In this investigation, we studied activation of the P2Y₂ receptor by measuring changes in intracellular free Ca2+ levels of fura-2 labeled U-937 cells stimulated with agonist, and found that both UTP and ATP mediated rapid and reversible homologous desensitization of the P2Y₂ receptor. Activation of PKC with phorbol 12,13-dibutyrate caused heterologous desensitization of the P2Y₂ receptor in U-937 cells which was only partially inhibited by the PKC inhibitor GF 109203X, suggesting a role for both PKC-dependent and PKC-independent pathways in the desensitization. Prolonged treatment (≥60min) of U-937 cells with 100 μM UTP caused persistent desensitization which correlated with a significant decrease in levels of P2Y₂ receptor mRNA. Okadaic acid pretreatment prevented recovery of P2Y₂ receptor function in UTP-desensitized U-937 cells, implicating receptor protein phosphorylation during agonist-induced desensitization. Further studies were performed using the astrocytoma 1321N1 cells transfected with the HA-tagged human P2Y ₂ receptor, since no antibodies against the P2Y₂ receptor are currently available. Immunoprecipitation of the HA-P2Y₂ receptor revealed a heterogeneous complex glycosylation of the receptor. UTP mediated rapid homologous desensitization of the HA-P2Y₂ receptors. Okadaic acid inhibited the recovery of receptor activity from UTP-induced desensitization, and phosphorylation studies of intact cells indicated a 3.8 ± 0.2-fold increase in [32P]-radioactivity in P2Y₂-transfected 1321N1 cells treated for 15 minutes with 100 μM UTP prior to immunoprecipitation, as compared to untreated cells. Sequestration studies by flow cytometry revealed that 40% of the surface HA-P2Y₂ receptors were internalized after a 15-minute stimulation with 100 μM UTP, which reached to 60% when UTP-exposure was increased to 60 minutes. Taken together, our results indicate that P2Y ₂ receptor desensitization involves an increase in receptor phosphorylation of the sequestered receptors. GRK-mediated phosphorylation of the agonist-activated P2Y₂ nucleotide receptors is presumed, however PKC involvement in the desensitization could not be ruled out completely. Additional studies are currently underway to determine the receptor phosphorylation sites to confirm the involvement of kinases, such as GRKs, in the UTP-mediated P2Y ₂ receptor desensitization.