G-protein coupled receptors (GPCRs) represent the largest family of receptor proteins in the living world, having approximately 800 human genes predicted; however the high-resolution crystal structures of only 26 GPCRs have been reported (Ghosh et al., 2015). The first human GPCR to be crystallized was the β2-adrenergic receptor (β2AR) in 2007 (Cherezov et al., 2007; Rasmussen et al., 2007; Rosenbaum et al., 2007). Shortly thereafter an alternate crystal form of the β2AR revealed a specific cholesterol binding site between helices I, II, III and IV. From this work a cholesterol consensus motif (CCM) was established, which defined specific interactions between cholesterol and the receptor. Utilization of this CCM predicted that as many as 25% of all class A GPCRs could have a specific interaction with cholesterol (Hanson et al., 2008). Our focus is the effects of cholesterol on the activity of the Adenosine A2a receptor (A2aR), a class A GPCR. Membrane cholesterol concentrations were varied in Chinese hamster ovary (CHO) cells by two approaches. The first method utilized methyl- β-cyclodextrin (MβCD), which is capable of capturing cholesterol in its inner cavity (Pucadyil et al., 2006). The second method took advantage of a CHO cell variant, sterol regulatory deficient 12B (SRD-12B). This cell line contains a mutation in the sterol regulatory synthesis pathway- specifically site 1 protease (S1P), reducing the synthesis levels of cholesterol within the cell (Rawson et al., 1999). We tested the role that bulk cholesterol depletion played in expression, ligand binding and downstream synthesis of cyclic AMP (cAMP) in CHO cells, and found that decreased membrane cholesterol reduced A2aR’s activity. Continuing work from our lab will study the specific interaction between cholesterol and A2aR by making point mutations in the residues of the CCM. In A2aR 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.

Membrane cholesterol and the adenosine A2a receptor

251st National Meeting (2016)

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