The parasympathetic limb of the autonomic nervous system regulates the activity of multiple organ systems. Muscarinic receptors are G protein coupled receptors (GPCRs) that mediate the response to acetylcholine released from parasympathetic nerves.1–5 Their role in the unconscious regulation of organ and central nervous system function makes them potential therapeutic targets for a broad spectrum of diseases. The M2 muscarinic acetylcholine receptor (M2 receptor) is essential for the physiologic control of cardiovascular function through activation of G protein-coupled inwardly-rectifying potassium channels, and is of particular interest because of its extensive pharmacological characterization with both orthosteric and allosteric ligands. Here we report the structure of antagonist-bound M2 receptor, the first human acetylcholine receptor to be characterized structurally. The antagonist QNB binds in the middle of a long aqueous channel extending approximately two-thirds through the membrane. The orthosteric binding pocket is formed by amino acids that are identical in all 5 muscarinic receptor subtypes, and shares structural homology with other functionally unrelated acetylcholine binding proteins from different species. A layer of tyrosine residues forms an aromatic cap restricting dissociation of the bound ligand. A binding site for allosteric ligands has been mapped to residues at the entrance to the binding pocket near this aromatic cap. The M2 receptor structure provides insights into the challenges of developing subtype-selective ligands for muscarinic receptors and their propensity for allosteric regulation.

G protein-coupled receptors (GPCRs) are the largest class of cell-surface receptors, and these membrane proteins exist in equilibrium between inactive and active states.1-13 Conformational changes induced by extracellular ligands binding to GPCRs 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 brain.14 In this study, we have successfully raised a mouse monoclonal antibody against human A2AAR that prevents agonist but not antagonist binding to the extracellular ligand-binding pocket. The structure of the A2AAR-antibody Fab fragment (Fab2838) complex reveals that the fragment, unexpectedly, recognises 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 C-terminal fragment in the active opsin structure1 and to the CDR-3 of the nanobody in the active β2 adrenergic receptor structure2 but locks the A2AAR in an inactive conformation. These results shed light on a novel strategy to modulate GPCR activity.

Production of thromboxane (TX) A 2 and PG I 2 /prostacyclin (PGI 2 ) is increased in patients with atherosclerosis. However, their roles in atherogenesis have not been critically defined. To examine this issue, we cross-bred atherosclerosis-prone apoE-deficient mice with mice deficient in either the TXA receptor (TP) or the PGI receptor (IP). Although they showed levels of serum cholesterol and triglyceride similar to those of apoE-deficient mice, apoE -/-TP -/-mice exhibited a significant delay in atherogenesis, and apoE -/-IP -/-mice exhibited a significant acceleration in atherogenesis compared with mice deficient in apoE alone. The plaques in apoE -/-IP -/-mice showed partial endothelial disruption and exhibited enhanced expression of ICAM-1 and decreased expression of platelet endothelial cell adhesion molecule 1 (PECAM-1) in the overlying endothelial cells compared with those of apoE -/-TP -/-mice. Platelet activation with thrombin ex vivo revealed higher and lower sensitivity for surface P-selectin expression in platelets of apoE -/-IP -/-and apoE -/-TP -/-mice, respectively, than in those of apoE -/-mice. Intravital microscopy of the common carotid artery revealed a significantly greater number of leukocytes rolling on the vessel walls in apoE -/-IP -/-mice than in either apoE -/-TP -/-or apoE -/-mice. We conclude that TXA 2 promotes and PGI 2 prevents the initiation and progression of atherogenesis through control of platelet activation and leukocyte-endothelial cell interaction.

Production of thromboxane (TX) A 2 and PG I 2 /prostacyclin (PGI 2 ) is increased in patients with atherosclerosis. However, their roles in atherogenesis have not been critically defined. To examine this issue, we cross-bred atherosclerosis-prone apoE-deficient mice with mice deficient in either the TXA receptor (TP) or the PGI receptor (IP). Although they showed levels of serum cholesterol and triglyceride similar to those of apoE-deficient mice, apoE -/-TP -/-mice exhibited a significant delay in atherogenesis, and apoE -/-IP -/-mice exhibited a significant acceleration in atherogenesis compared with mice deficient in apoE alone. The plaques in apoE -/-IP -/-mice showed partial endothelial disruption and exhibited enhanced expression of ICAM-1 and decreased expression of platelet endothelial cell adhesion molecule 1 (PECAM-1) in the overlying endothelial cells compared with those of apoE -/-TP -/-mice. Platelet activation with thrombin ex vivo revealed higher and lower sensitivity for surface P-selectin expression in platelets of apoE -/-IP -/-and apoE -/-TP -/-mice, respectively, than in those of apoE -/-mice. Intravital microscopy of the common carotid artery revealed a significantly greater number of leukocytes rolling on the vessel walls in apoE -/-IP -/-mice than in either apoE -/-TP -/-or apoE -/-mice. We conclude that TXA 2 promotes and PGI 2 prevents the initiation and progression of atherogenesis through control of platelet activation and leukocyte-endothelial cell interaction.