The Effect Of C-Terminal Tail Mutations On Desensitization Of The Murine P2Y2‚ Nucleotide Receptor

Date of Graduation

Summer 2004

Degree

Master of Science in Cell and Molecular Biology

Department

Biomedical Sciences

Committee Chair

Richard Garrad

Abstract

Cystic fibrosis (CF) is one of the most prevalent genetic disorders in Caucasians. CF is caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator gene encoding the protein that regulates the secretion of chloride ions across the pulmonary epithelium. Present in pulmonary epithelium are P2Y₂ nucleotide receptors of the G protein-coupled receptor (GPCR) family. Activation of the receptor by the agonist (ATP or UTP) leads to an increase in the concentration of intracellular calcium, thereby mediating the opening of calcium-dependent chloride channels and serving as an alternate pathway for chloride secretion. On repeated exposure to the P2Y₂ agonist, the receptor becomes desensitized and is unable to continue to elicit its original response within the cell. The focus of this study is to investigate the effects of mutations of potential phosphorylation sites in the C-terminal tail of the P2Y₂ receptor on desensitization. A better understanding of the process of desensitization could contribute to improved treatments for CF patients. Mutant P2Y₂ receptors have been constructed and expressed in a cell line normally devoid of the receptor, 1321N1 astrocytoma cells. Effects of these mutations on P2Y₂ receptor signaling and desensitization were studied utilizing spectrofluorometric analysis, SDS-PAGE, and western blotting techniques. Results showed that the S352A mutation may play a role in receptor endocytosis and the T359A mutation may play a role in receptor desensitization.

Keywords

P2Y₂ receptor, desensitization, protein kinase C, G protein-coupled receptors, cystic fibrosis

Subject Categories

Medical Molecular Biology

Copyright

© Hillary M. Glauser

Citation-only

Dissertation/Thesis

Share

COinS