G-protein coupled receptors (GPCRs) are the largest family of membrane proteins and remain popular drug targets; yet structure-based drug design has been hindered by a lack of high-resolution structural data. Problems with the biophysical characterization of these proteins stem from a general inability to express at high levels and maintain the proper conformation post-purification. Through heterologous expression in Saccharomyces cerevisiae, the human adenosine A2a receptor has been expressed functionally at high levels with the aid of a multi-integrating vector and a pre-pro leader sequence to introduce the receptor into the ER membrane. Overall, we have been able to purify approximately 6 milligrams per liter of culture A2aR. After the addition of a xanthine ligand affinity chromatography purification step, we found that all full-length purified protein was in an active conformation. Currently, this represents the highest total and functional yields reported for this receptor from any expression system.

High-level expression and purification of the human adenosine A2a receptor has enabled the characterization of this receptor using biophysical techniques. GPCRs, as with all membrane proteins, require surfactants to stabilize their conformation outside the plasma membrane. Isolation of this receptor and reconstitution in surfactant micelles requires the addition of mammalian cholesterol to retain ligand binding activity in vitro. This interaction may hold the 7-alpha helical domains of the GPCR more rigid, in an active conformation within the micelle. Functionality of A2aR is conserved in dodecyl-beta-D-maltoside under this condition, yet surfactants which share common structure and differ by only one carbon length show marked differences in their ability to stabilize A2aR, as assessed through radioligand binding. Thermal denaturation studies have shown that the secondary structure of A2aR is quite thermally stable, and that reduction of the putative disulfide bond conserved in all GPCRs causes aggregation upon temperature incubation. Current efforts are underway to further monitor changes in A2aR secondary and tertiary structure through chemical denaturation as measured by circular dichroism and intrinsic fluorescence. The effect of different ligand analogs on the receptor stability will also be addressed. These studies will enable us to better understand the factors that influence receptor stability, as well as develop approaches to identify improved experimental conditions for high-resolution structural efforts.