Agonist-induced phosphorylation and desensitization of the P2Y2 nucleotide receptor

Purification of HA-tagged P2Y(2) receptors from transfected human 1321N1 astrocytoma cells yielded a protein with a molecular size determined by SDS-PAGE to be in the range of 57-76 kDa, which is typical of membrane glycoproteins with heterogeneous complex glycosylation. The protein phosphatase inhibitor, okadaic acid, attenuated the recovery of receptor activity from the agonist-induced desensitized state, suggesting a role for P2Y(2) receptor phosphorylation in desensitization. Isolation of HA-tagged P2Y(2) nucleotide receptors from metabolically [P-32]-labelled cells indicated a (3.8 +/- 0.2)-fold increase in the [P-32]-content of the receptor after 15 min of treatment with 100 mu M UTP, as compared to immunoprecipitated receptors from untreated control cells. Receptor sequestration studies indicated that similar to 40% of the surface receptors were internalized after a 15-min stimulation with 100 mu M UTP. Point mutation of three potential GRK and PKC phosphorylation sites in the third intracellular loop and C-terminal tail of the P2Y(2) receptor (namely, S243A, T344A, and S356A) extinguished agonist-induced receptor phosphorylation, caused a marked reduction in the efficacy of UTP to desensitize P2Y(2) receptor signalling to intracellular calcium mobilization, and impaired agonist-induced receptor internalization. Activation of PKC isoforms with phorbol 12-myristate 13-acetate that caused heterologous receptor desensitization did not increase the level of P2Y(2) receptor phosphorylation. Our results indicate a role for receptor phosphorylation by phorbol-insensitive protein kinases in agonist-induced desensitization of the P2Y(2) nucleotide receptor.