(61g) G-Protein Coupled Receptors: Protein Engineering Approaches to Improve Expression and Determine Protein-Protein Interactions

The adenosine receptor subfamily of G-protein coupled receptors is an important family of membrane receptors that modulate blood pressure, and more recently have been implicated in cancer, neurodegenerative diseases, and diabetes, making them a significant fraction of drug discovery efforts. As multipass membrane proteins, G protein coupled receptors remain challenging but important targets of drug discovery, and our efforts have enabled the high-level expression of the adenosine family of receptors and in-depth biophysical characterization of their binding to G-proteins and lipids, which I will highlight in this talk. Adenosine A_2A receptor (A_2AR), one of four subfamily members, has a longer C-terminus than the other adenosine receptor subtypes, which may contribute to its exceptional trafficking to the plasma membrane, but its flexibility has been attributed to protein-protein interactions. Chimeric A₁ and A₃ receptors with A_2AR C-termini showed improved localization to the plasma membrane and are functionally active, with greater than three-fold higher yields than previously reported from other heterologous expression systems. Interestingly, these chimeras are able to bind to their native G proteins, suggesting the specificity of interaction does not rely on the C-terminus. To investigate this further using purified A_2AR and Gαs, we determined the affinity and binding rates via surface plasmon resonance (SPR). Together with in vivo signaling data, our results suggest that loss of downstream signaling for the truncation may result from a reduced GPCR-G protein complex interaction, which may be important for interpretation of structure-based drug discovery screening. We have also investigated the interactions with lipids and cholesterol, in light of the cholesterol binding motif that has been identified in Class A GPCRs, and which has been implicated in a role in allosteric binding. Using site-directed mutagenesis and both in vivo and in vitro binding studies, we determined that cholesterol has a specific effect on signaling, as well as a bulk effect on A_2AR function.