Engineering Stabilized GPCRs for Biochemical and Structural Studies

G-protein-coupled receptors (GPCRs) mediate most of our physiological responses to different stimulants, and have great potential as therapeutic targets for a broad spectrum of diseases. The rate of GPCR-targeting drugs entering the market has reduced and pharmaceutical companies are seeking structural and biochemical insights into GPCR function. Unfortunately, their structural characterization has been hampered by the low expression and their inherent instability in detergents which made protein engineering indispensable for structural and biophysical characterization.

Here we develop an innovative directed-evolution approach utilizing the unique biological properties of yeast expression system to improve GPCR stability in the presence of short-chain detergents. Human adenosine A2a-receptor (A2aR) is the model protein for these studies. To obtain detergent stabilized-GPCRs we use directed-evolution method in yeast, which involves creating a DNA library encoding millions of mutants of the protein of interest with the use of error-prone PCR.

At first we showed by using A2AR-GFP that our protein is localized in the Yeast membrane. Then we showed what happens to the membrane and the WT-protein once we use detergent. We performed detergent screening assay with octylglucoside- a short-chain-detergent. The treatment disrupted the membrane. We then performed Ligand binding assay to show our ligand binds the A2AR-WT as a baseline to compere to the mutant libraries. Next we showed disappearance of this binding once we use detergents on the expressing cells. Therefore we could conclude that treatment with octylglucoside disrupt the membrane, A2AR become instable and there is no ligand binding.

We are now in the process of creating a DNA library of mutant A2AR, in the aim of finding detergent-stable-A2AR for biochemical and structural studies and for future drug development.