Effects of the A2AR C-terminus on receptor stability
July 16-20, 2017
G-protein coupled receptors (GPCRs) are seven-transmembrane domain membrane proteins which are targets for nearly half of all pharmaceuticals on the market. The adenosine A2A receptor (A2AR) is a class A GPCR that is often a drug target due to its involvement in neurodegenerative diseases, diabetes, inflammatory diseases, cancer, and heart disease. At present, the only crystal structures of A2AR are of a truncated variant of the receptor (A2AΔ316R). However, the full length C-terminus has been shown to be critical in downstream signaling. Here, we use ligand binding to investigate the effects of the C-terminus on A2AR stability by comparing full-length A2AR, A2AΔ316R, and Rag23, a thermostable A2AΔ316R variant with 5 point mutations favoring agonist binding (Magnani, Shibata, Serrano-Vega, & Tate, 2008). Receptors were overexpressed using a multicopy vector, pITy, purified in detergent micelles, and incubated with fluorescent agonist FITC-APEC. Fluorescence anisotropy was used to determine FITC-APEC binding after receptors were exposed to various conditions (Swonger & Robinson, 2017). We quantified equilibrium binding, temperature stability, kinetic rates, and competition with agonists and antagonists and find that beyond downstream signaling, the C-terminus contributes strongly to A2AR stability in micelles. Magnani, F., Shibata, Y., Serrano-Vega, M. J., & Tate, C. G. (2008). Co-evolving stability and conformational homogeneity of the human adenosine A2a receptor. Proceedings of the National Academy of Sciences, 105(31), 10744–10749. https://doi.org/10.1073/pnas.0804396105 Swonger, K. N., & Robinson, A. S. (2017). Using fluorescence anisotropy for ligand binding kinetics of membrane proteins. Current Protocols. Submitted
Kirsten N. Swonger and Annie Tir, "Effects of the A2AR C-terminus on receptor stability" in "Biochemical and Molecular Engineering XX", Wilfred Chen, University of Delaware, USA Nicole Borth, Universität für Bodenkultur, Vienna, Austria Stefanos Grammatikos, UCB Pharma, Belgium Eds, ECI Symposium Series, (2017). http://dc.engconfintl.org/biochem_xx/17
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