Development of a Microplate-Based Calcium Assay for Studying Desensitization of the P2Y2 Nucleotide Receptor

Extracellular nucleotides mediate cellular responses in a variety of tissues by binding to either the ionotropic P2X family or metabotropic P2Y family of nucleotide receptors. The P2Y2 receptor (P2Y2R) regulates several important processes, such as inflammation, neuroprotection, and NaCl balance on the airway surface and in the kidneys. Prolonged stimulation of the P2Y2R leads to desensitization of the intracellular signal. This regulatory mechanism is critically mediated by members of the G protein-coupled receptor kinase (GRK) family and the β-arrestins. As GRK2 and β-arrestin-1 have been demonstrated to mediate P2Y2R desensitization in rat arterial smooth muscle cells, it was hypothesized that the human homologs would mediate P2Y2R desensitization in human 1321N1 cells. To test this hypothesis, a microplate-based calcium assay was developed and employed to functionally assess P2Y2R desensitization in a high-throughput manner. The mRNA expression of GRK2 and β-arrestin-1 was knocked down using siRNA, and knockdown was verified via qRT-PCR. However, knockdown of GRK2 or β-arrestin-1 mRNA was not sufficient to functionally inhibit P2Y2R desensitization vs. vehicle-only control (P = 0.4853 for GRK2, P = 0.0502 for β-arrestin-1). Interestingly, the dose-response activation curve for β-arrestin-1 knockdown exhibited a significant rightward shift vs. control (P = 0.0004). While these data do not support the initial hypothesis, they may illuminate a potentiating role for β-arrestin-1 in P2Y2R activation.

Name thesis:degree_name

Master of Science in Biology

Level thesis:degree_level

Masters

Discipline thesis:degree_discipline

Biology

Year

2014

Author dc:creator

• Louiselle, Daniel Allen

Contributors dc:contributor

• Richard Garrad

• P2Y[2] receptor
• G protein-coupled receptor
• calcium signaling
• nucleotide signaling
• desensitization
• Biology

Statement dc:rights

• © Daniel Allen Louiselle

Repository record dc:identifier

https://bearworks.missouristate.edu/theses/1337

OAI identifier oai:identifier

oai:bearworks.missouristate.edu:theses-2338