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

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Abstract

UTP activates P2Y2 receptors in both 1321N1 cell transfectants expressing the P2Y2 receptor and human HT-29 epithelial cells expressing endogenous P2Y2 receptors with an EC50 of 0.2- 1.0 μM. 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 μM 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 P2Y,2 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 P2Y2 receptor agonists.

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Authors and Affiliations

  1. Department of Chemistry, University of Puerto Rico, Río Piedras Campus, San Juan, Puerto Rico, 00931, USA

    Miguel Otero, Betty Velázquez, Melvin G. Hernández-Pérez & Fernando A. González

  2. Department of Biochemistry, University of Missouri-Columbia, Columbia, MO, 65212, USA

    Richard C. Garrad, Laurie Erb & Gary A. Weisman

  3. Department of Pharmacology, University of Missouri-Columbia, Columbia, MO, 65212, USA

    Jean M. Camden & John T. Turner

  4. Veterinary Biomedical Sciences & Dalton Cardiovascular Research Center, University of Missouri-Columbia, Columbia, MO, 65212, USA

    Lane L. Clarke

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  1. Miguel Otero
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Otero, M., Garrad, R.C., Velázquez, B. et al. Mechanisms of agonist-dependent and -independent desensitization of a recombinant P2Y2 nucleotide receptor. Mol Cell Biochem 205, 115–123 (2000). https://doi.org/10.1023/A:1007018001735

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