Date of Graduation

Spring 2011

Degree

Master of Science in Cell and Molecular Biology

Department

Biomedical Sciences

Committee Chair

Richard Garrad

Abstract

The P2Y2 receptor belongs to the P2 class of G protein-coupled receptor (GPCR) systems. P2 receptors are characterized by response to nucleotide ligands. Activation of the P2Y2 receptor in vivo results in fluid movement from the intracellular to extracellular compartment of respiratory epithelium, resulting in hydration of the mucous lining. Hydration of the respiratory epithelium is maintained by the Cystic Fibrosis Transmembrane Regulatory (CFTR) protein. Since CFTR is defective in Cystic Fibrosis, the P2Y2 receptor has emerged as a therapeutic target. However, the expected result of P2Y2 receptor activation in CF models lasts only seconds due to a desensitization mechanism mediated by cytoplasmic proteins. We proposed that the inhibition of these proteins including would prolong P2Y2 activation and that down regulation of these proteins can be achieved using anti-sense oligonucleotides. Experiments were performed using 1321N1 astrocytoma cells expressing the P2Y2 receptor system. Cells were transfected with antisense oligonucleotides and calcium flux was measured as a marker of activation. Transfection optimization was then performed by transfecting 1321N1 cells with oligonucletotides with a fluoroscein tag. Anti-sense oligonucleotides did not inhibit the P2Y2 desensitization pathway. However, epifluorescent microscopy demonstrated the presence of oligonucleotides within the cell. These results support the hypothesis that the transfection process is functional. However, lack of protein down-regulation is likely due to an inherent inability of the oligonucleotide to induce inhibition.

Keywords

P2Y[2] receptor, desensitization, G protein coupled receptor, cystic fibrosis, dynamin, β-arrestin

Subject Categories

Medical Molecular Biology

Copyright

© Lucas Harmon Bradley

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