title: Agonist-induced phosphorylation and desensitization of the P2Y2 nucleotide receptor.
abstract: Purification of HA-tagged P2Y2 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 P2Y2 receptor phosphorylation in desensitization. Isolation of HA-tagged P2Y2 nucleotide receptors from metabolically [32P]-labelled cells indicated a (3.8 +/- 0.2)-fold increase in the [32P]-content of the receptor after 15 min of treatment with 100 microM UTP, as compared to immunoprecipitated receptors from untreated control cells. Receptor sequestration studies indicated that approximately 40% of the surface receptors were internalized after a 15-min stimulation with 100 microM UTP. Point mutation of three potential GRK and PKC phosphorylation sites in the third intracellular loop and C-terminal tail of the P2Y2 receptor (namely, S243A, T344A, and S356A) extinguished agonist-induced receptor phosphorylation, caused a marked reduction in the efficacy of UTP to desensitize P2Y2 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 P2Y2 receptor phosphorylation. Our results indicate a role for receptor phosphorylation by phorbol-insensitive protein kinases in agonist-induced desensitization of the P2Y2 nucleotide receptor.
authors.affiliation: Department of Chemistry, University of Puerto Rico, Río Piedras Campus, San Juan, PR, USA.
authors.forename: Rosa V
authors.initials: RV
authors.lastname: Flores
authors.forename: Melvin G
authors.initials: MG
authors.lastname: Hernández-Pérez
authors.forename: Edna
authors.initials: E
authors.lastname: Aquino
authors.forename: Richard C
authors.initials: RC
authors.lastname: Garrad
authors.forename: Gary A
authors.initials: GA
authors.lastname: Weisman
authors.forename: Fernando A
authors.initials: FA
authors.lastname: Gonzalez
mesh_terms: D006801:Humans; D047468:Immunoprecipitation; D019319:Okadaic Acid / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000378:metabolism; D021381:Protein Transport; D058910:Purinergic P2 Receptor Agonists; D018048:Receptors, Purinergic P2 / Q000378:metabolism*; D058473:Receptors, Purinergic P2Y2; D011994:Recombinant Proteins; D013755:Tetradecanoylphorbol Acetate / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured; D014544:Uridine Triphosphate / Q000494:pharmacology
chemical_list: C546385:P2RY2 protein, human; D058910:Purinergic P2 Receptor Agonists; D018048:Receptors, Purinergic P2; D058473:Receptors, Purinergic P2Y2; D011994:Recombinant Proteins; D019319:Okadaic Acid; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D014544:Uridine Triphosphate