Mechanisms of agonist-dependent and -independent desensitization of a recombinant P2Y2 nucleotide receptor.
UTP activates P2Y, receptors in both 1321N1 cell transfectants expressing the P2Y2 receptor and human HT-29 epithelial cells expressing endogenous P2Y, receptors with an EC50 of 0.2-1.0 microM. Pretreatment of these cells with UTP diminished the effectiveness of a second dose of UTP (the IC50 for UTP-induced receptor desensitization was 0.3-1.0 microM for both systems). Desensitization and down-regulation of the P2Y2 nucleotide receptor may limit the effectiveness of UTP as a therapeutic agent. The present studies investigated the phenomenon of P2Y2 receptor desensitization in human 1321N1 astrocytoma cells expressing recombinant wild type and C-terminal truncation mutants of the P2Y2 receptor. In these cells, potent P2Y2 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 Ca2+-ATPase in the endoplasmic reticulum, induced an increase in the intracellular free calcium concentration, [Ca2+]i, after addition of desensitizing concentrations of UTP, indicating that P2Y2 receptor desensitization is not due to depletion of calcium from intracellular stores. Single cell measurements of increases in [Ca2+]i induced by UTP in 1321N1 cell transfectants expressing the P2Y2 receptor indicate that time- and UTP concentration-dependent desensitization occurred uniformly across a cell population. Other results suggest that P2Y2 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 P2Y2 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 P2Y2 receptor by protein kinases other than protein kinase C mediates agonist-induced receptor desensitization. A better understanding of the molecular mechanisms of P2Y2 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.
Otero
M
M
Department of Chemistry, University of Puerto Rico, San Juan 00931-3346, USA.
Garrad
R C
RC
Velázquez
B
B
Hernández-Pérez
M G
MG
Camden
J M
JM
Erb
L
L
Clarke
L L
LL
Turner
J T
JT
Weisman
G A
GA
González
F A
FA
D001254:Astrocytoma / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002448:Cell Adhesion; D002460:Cell Line; D003106:Colon / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004848:Epithelium / Q000378:metabolism; D006801:Humans; D007295:Inositol Phosphates / Q000378:metabolism; D007476:Ionophores / Q000494:pharmacology; D016296:Mutagenesis; D019319:Okadaic Acid / Q000494:pharmacology; D010703:Phorbol Esters / Q000494:pharmacology; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism; D058910:Purinergic P2 Receptor Agonists; D018048:Receptors, Purinergic P2 / Q000737:chemistry / Q000378:metabolism*; D058473:Receptors, Purinergic P2Y2; D011994:Recombinant Proteins / Q000378:metabolism; D013997:Time Factors; D014407:Tumor Cells, Cultured; D014544:Uridine Triphosphate / Q000494:pharmacology
D007295:Inositol Phosphates; D007476:Ionophores; C546385:P2RY2 protein, human; D010703:Phorbol Esters; D058910:Purinergic P2 Receptor Agonists; D018048:Receptors, Purinergic P2; D058473:Receptors, Purinergic P2Y2; D011994:Recombinant Proteins; D019319:Okadaic Acid; D011493:Protein Kinase C; D010744:Phosphoric Monoester Hydrolases; D002118:Calcium; D014544:Uridine Triphosphate