Date of Graduation

Summer 2014

Degree

Master of Science in Cell and Molecular Biology

Department

Biomedical Sciences

Committee Chair

Richard Garrad

Keywords

β-arrestin 1, P2Y[2] receptor, desensitization, internalization, signal transduction

Subject Categories

Medical Molecular Biology

Abstract

The scaffold protein β-arrestin is involved in many essential cell-signaling pathways. Arrestin was first discovered for its regulatory effect on rhodopsin, a well-studied G protein-coupled receptor (GPCR), and specifically for its involvement in receptor desensitization. Upon activation of the receptor, β-arrestin is recruited to the phosphorylated receptor, uncoupling its interaction with the G protein and resulting in desensitization. Further investigation of the protein has revealed its involvement in GPCR internalization, through interaction with AP2 and clathrin. The internalized receptor-β- arrestin complex functions as a signaling molecule, that can facilitate the activation of a number of cell signaling pathways, such as its canonical activation of MAPK through Src. The P2Y2 nucleotide receptor, Gq/i-coupled receptor, has been shown to be desensitized via β-arrestin involvement. Inhibition of P2Y2 receptor desensitization is pivotal as a potential therapeutic treatment for patients with Cystic Fibrosis in airway epithelia. Desensitization was assessed using Western blot, and calcium assay in 1321N1 astrocytoma cells, which were stably transfected with P2Y2 receptor. To study the role of β-arrestin 1 in P2Y2 receptor desensitization, the cells were transfected with 100nM of β- arrestin 1 siRNA to knock down its expression level. The transcript of β-arrestin 1, ARRB1, was successfully knocked down, as revealed by a Real-time RT-PCR assay. However, there were no differences in the calcium responses to UTP between wild type and β-arrestin 1 knocked down cells. Future directions are to assess the effects of P2Y2 receptor desensitization in response to concomitant knockdown of β-arrestin 1 and β- arrestin 2.

Copyright

© Katie Deann McLemore

Campus Only

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