G protein-coupled receptors (GPCRs) are integral membrane proteins vital for cellular signaling and constitute one of the major drug targets. Despite their importance, relatively little information regarding their structure, folding, and stability is known. This work describes the impact that structural and environmental factors have on the expression, activity and structural stability of the human adenosine receptor, A2A (hA2AR).The four disulfide bonds present in the hA2AR were found to be critical for the high level expression of this receptor in yeast, but were found not to contribute significantly to the structural stability of the purified protein. In addition, we found that different ligand-binding profiles and oligomerization states were obtained when solubilizing hA2AR from yeast membrane preparations using different detergents and lipids. We have also utilized pressure denaturation to unfold the GPCR under various solubilization environments, and found that SDS stabilizes the protein fold at the concentrations tested.We intend to employ the results from these studies to develop improved methods to engineer GPCR expression in heterologous systems, as well as to improve the GPCR reconstitution environment for functional and structural characterization.

Exploring the stability and activity of a model GPCR in various biomimetic systems

245th National Meeting (2013)

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