Mechanisms of agonist-dependent and -independent desensitization of a recombinant P2Y2 nucleotide receptor


UTP activates P2Y receptors in both 1321N1 cell transfectants expressing the P2Y receptor and human HT-29 epithelial cells expressing endogenous P2Y receptors with an EC of 0.2-1.0 μM. Pretreatment of these cells with UTP diminished the effectiveness of a second dose of UTP (the IC for UTP-induced receptor desensitization was 0.3-1.0 μM for both systems). Desensitization and down-regulation of the P2Y nucleotide receptor may limit the effectiveness of UTP as a therapeutic agent. The present studies investigated the phenomenon of P2Y receptor desensitization in human 1321N1 astrocytoma cells expressing recombinant wild type and C- terminal truncation mutants of the P2Y receptor. In these cells, potent P2Y receptor desensitization was observed after a 5 min exposure to UTP. Full receptor responsiveness returned 5-10 min after removal of UTP. Thapsigargin, an inhibitor of Ca -ATPase in the endoplasmic reticulum, induced an increase in the intracellular free calcium concentration, [Ca ](i), after addition of desensitizing concentrations of UTP, indicating that P2Y receptor desensitization is not due to depletion of calcium from intracellular stores. Single cell measurements of increases in [Ca ](i) induced by UTP in 1321N1 cell transfectants expressing the P2Y receptor indicate that time- and UTP concentration-dependent desensitization occurred uniformly across a cell population. Other results suggest that P2Y receptor phosphorylation/dephosphorylation regulate receptor desensitization/resensitization. A 5 min preincubation of 1321N1 cell transfectants with the protein kinase C activator, phorbol 12-myristate 13- acetate (PMA), reduced the subsequent response to UTP by about 50%, whereas co-incubation of PMA with UTP caused a greater inhibition in the response. The protein phosphatases-1 and-2A inhibitor, okadaic acid, partially blocked resensitization of the receptor. Furthermore, C-terminal truncation mutants of the P2Y receptor that eliminated several potential phosphorylation sites including two for PKC were resistant to UTP-, but not phorbol ester-induced desensitization. Down regulation of protein kinase C isoforms prevented phorbol ester-induced desensitization but had no effect on agonist-induced desensitization of wild type or truncation mutant receptors. These results suggest that phosphorylation of the C-terminus of the P2Y receptor by protein kinases other than protein kinase C mediates agonist-induced receptor desensitization. A better understanding of the molecular mechanisms of P2Y nucleotide receptor desensitization may help optimize a promising cystic fibrosis pharmacotherapy based on the activation of anion secretion in airway epithelial cells by P2Y receptor agonists. 2 2 2 50 50 2 2 2 2 2 2 2 2 2 2 2 2+ 2+ 2+

Document Type





1321N1 astrocytoma, Cystic fibrosis, HT-29 epithelial cells, Protein kinase C isoforms, Uridine 5'-triphosphate

Publication Date


Journal Title

Molecular and Cellular Biochemistry