Abstract

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 has been published.This work describes the impact of disulfide bonds on the expression and structural stability of the human adenosine receptor, A2A (hA2AR). The crystal structure of this receptor revealed four disulfide bonds present in extracellular loops that could contribute to expression, stability, or ligand binding or to a combination of these.To test the role of these residues, cysteine to alanine mutants of hA2AR were created; expression and ligand-binding activity of the constructs were tested in mammalian (HEK293) and yeast (Saccharomyces cerevisiae) cells. Once purified from yeast, unfolding of the hA2AR through thermal and chemical means was monitored via intrinsic tryptophan fluorescence and circular dichroism. The effect of ligand addition and reduction of disulfide bonds was also investigated.Thermal and chemical denaturation were not reversible, yet clear differences in the unfolding behavior were observed upon ligand binding via circular dichroism and fluorescence spectrometry. We found that the stability of hA2AR was increased upon incubation with the agonist N6-cyclohexyladenosine or the antagonist theophylline. When extracellular disulfide bonds were reduced with a chemical reducing agent, the ligand binding activity decreased by ∼40%, but reduction of these bonds did not compromise the unfolding transition observed via urea denaturation. Overall, these approaches offer a general strategy for characterizing the effect of disulfide bonds and ligand effects on the stability of GPCRs.

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