DESENSITIZATION OF P2Y2 NUCLEOTIDE RECEPTOR & CF THERAPY

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Description (Adapted from Application): Extracellular nucleotides activate P2Y2 receptors that modulate important biological processes, such as ion secretion, cellular growth, and vasodilatation, among others. UTP (a P2Y2 receptor agonist) has been shown to induce calcium-dependent anion secretion in airway epithelial cells expressing a defective cystic fibrosis transmembrane conductance regulator, a cAMP-dependent chloride channel. This observation suggests that nucleotides may be efficacious in the treatment of cystic fibrosis. One potential limitation of this therapy is that prolonged exposure to nucleotides causes desensitization of the P2Y2 receptor leading to receptor sequestration and downregulation. Considering the therapeutic potential for P2Y2 receptor activation in vivo, the investigators will attempt to understand the molecular mechanisms underlying receptor desensitization. Preliminary results suggest that desensitization is caused by phosphorylation of amino acid residues in the intracellular C-terminal domain of the receptor. In the present application, the PI proposes to apply molecular, biochemical and pharmacological approaches to study the mechanisms of desensitization of a recombinant P2Y2 receptor expressed in human 1321N1J astrocytoma cells that lack endogenous nucleotide receptors. This transfection system will be used to express a variety of P2Y2 nucleotide receptor constructs (e.g., deletion and point mutants) to identify the specific phosphorylation sites and protein kinases that regulate agonist-induced desensitization and sequestration (i.e., uncoupling and internalization). To accomplish this goal they will express a series of epitope-tagged P2Y2 receptor mutants in 1321N1J cells followed by metabolic labeling of the cells and purification of receptor protein to directly determine the sites of phosphorylation. After identifying the P2Y2 receptor phosphorylation sites and kinases that regulate agonist-induced and heterologous desensitization, they will use the accumulated information to address the hypothesis that P2Y2 receptor uncoupling and internalization are distinct biochemical events. Therefore, they will determine how specific P2Y2 receptor domains and phosphorylation sites relate to receptor activation, signaling, desensitization (uncoupling), and sequestration (internalization). The information generated by the proposed experiments will provide an understanding of the molecular mechanisms for desensitization of P2Y2 receptors after acute and chronic exposure to nucleotides These results will be important for the future development of nucleotide therapies for cystic fibrosis and other diseases by elucidating means to optimize the beneficial effects of drug administration (i.e., signaling and anion secretion) while minimizing or eliminating the deleterious uncoupling and internalization of the receptor.