Probing Cholesterol-Dependence of Integrin-Urokinase Receptor Complex Formation using Confocal Dual-Color Fluorescence Intensity Analysis

Abstract

Our focus is the effects of cholesterol on the activity of the Adenosine A 2a receptor (A 2a R), a class A GPCR. Membrane cholesterol concentrations were varied in Human embryonic kidney (HEK-293) cells by the use of methylb-cyclodextrin (MbCD), which is capable of capturing cholesterol in its inner cavity (Pucadyil et al., 2006). We tested the role that bulk cholesterol depletion played in expression, ligand binding and downstream synthesis of cyclic AMP (cAMP) in HEK-293 cells. Continuing work from our lab will study the specific interaction between cholesterol and A 2a R by making point mutations in the residues of the CCM. In A 2a R those residues include Tyr43(2.41), Ser47(2.45), Lys122(4.43), Ile125(4.46) and Trp129(4.50) (Lee et al., 2013). Our lab will investigate whether these point mutations effect the interaction between cholesterol and purified receptor, as well as the receptors activity in both yeast and mammalian cell model systems.