G-protein-coupled receptor inactivation by an allosteric inverse-agonist antibody

Abstract

Determination of the crystal structure of the human A2A adenosine receptor in complex with an inverse-agonist antibody shows that the allosteric site of the receptor inhibits agonist binding and that the antibody locks the receptor in an inactive conformation. G-protein-coupled receptors are the largest class of cell-surface receptors, and many of them are considered drug targets. The crystal structure of the human A2A adenosine receptor in complex with a mouse antibody fragment has now been determined. This structure includes the intracellular loop 3, critical for G-protein binding. In addition, the use of an antibody fragment to stabilize the receptor in an inactive conformation suggests a new strategy to modulate the activity of G-protein-coupled receptors. G-protein-coupled receptors are the largest class of cell-surface receptors, and these membrane proteins exist in equilibrium between inactive and active states1,2,3,4,5,6,7,8,9,10,11,12,13. Conformational changes induced by extracellular ligands binding to G-protein-coupled receptors result in a cellular response through the activation of G proteins. The A2A adenosine receptor (A2AAR) is responsible for regulating blood flow to the cardiac muscle and is important in the regulation of glutamate and dopamine release in the brain14. Here we report the raising of a mouse monoclonal antibody against human A2AAR that prevents agonist but not antagonist binding to the extracellular ligand-binding pocket, and describe the structure of A2AAR in complex with the antibody Fab fragment (Fab2838). This structure reveals that Fab2838 recognizes the intracellular surface of A2AAR and that its complementarity-determining region, CDR-H3, penetrates into the receptor. CDR-H3 is located in a similar position to the G-protein carboxy-terminal fragment in the active opsin structure1 and to CDR-3 of the nanobody in the active β2-adrenergic receptor structure2, but locks A2AAR in an inactive conformation. These results suggest a new strategy to modulate the activity of G-protein-coupled receptors.