(588d) Investigating Changes in Structural Conformation for Human Adenosine A2a Receptor Complexes upon Ligand Binding

G-protein coupled receptors (GPCRs) and their oligomers are viable drug targets for the treatment of disease, including those involving the central nervous system. However, design of structurally-inspired pharmaceuticals to target specific oligomers has been limited by the low abundance of GPCRs in native tissues, as well as a poor understanding of functional consequences of homo- and hetero-oligmerization. As such, many therapeutics engage in nonspecific interactions with off-target oligomers, giving rise to unwanted side effects. Toward our goal to elucidate structure-function relationships of oligomers, we have overexpressed the human dopamine receptors D1, D2, and the adenosine A2a receptor in yeast for biophysical study with spin label electron paramagnetic resonance (EPR). The D2 receptor is known to create hetero-dimers with both D1 and A2a with different functional implications for potential treatment of schizophrenia and Parkinson’s disease. 

To investigate the mechanism by which different drugs elicit different cellular responses, we employ site-directed spin labeling (SDSL) and EPR to explore the relationship between ligand binding and changes in structural conformation of cytoplasmic loop sites in these GPCRs and their oligomers. Typical practices to spin label a unique cysteine involve creating a cysteine-free mutant protein via site-directed mutagenesis, and reintroducing a single cysteine at a site of interest. However, for many GPCRs, several native cysteine residues are essential for receptor stability and function, and their mutation can result in receptor misfolding. To circumvent this limitation, we have examined the effects of binding different types of A2a ligands (agonist and antagonist) on structural dynamics of spin labeled C394S-A2a mutant relative to a wild-type control, and found that both agonist (NECA) and antagonist (XAC) alter which cysteine residues are accessible to spin labeling. This differential labeling indicates changes in structural conformation due to ligand binding, and further suggests that the C-terminal C394 residue is a buried site that is not accessible to spin label. By extending this approach to A2a, D1, D2 and the D2-A2a heterodimer, we will gain a better understanding of ligand mediated changes in receptor and oligomer conformations, and how this may be altered when a receptor is a part of a dimer or higher order oligomer.