Receptor G protein coupling and the cytoskeleton

The A_2A adenosine receptor is a prototypical G_s protein-coupled receptor; it has been proposed as a drug target in the treatment of Parkinson's disease, because there is a mutual antagonism between D₂ dopamine and A_2A receptors in striatal neurons. Because of their ability to stimulate endothelial cell proliferation, A_2A agonists are in clinical development for diabetic ulcers. Several modes of coupling have been proposed to account for the interaction of receptor and G proteins; these have been termed precoupling, restricted collision coupling etc. Here, we investigated the mode of coupling of the A_2A receptor by visualizing agonist-induced changes in mobility of the YFP-tagged receptor by FRAP (fluorescence recovery after photobleaching) microscopy. Agonist stimulation did not affect the mobility of the A_2A receptor; in contrast, agonist challenge induced a decrease in the mobility of the D₂ receptor. When coexpressed in the same cell, the A_2A receptor precluded the agonist-induced change in D₂ receptor mobility. Thus, the A_2A receptor does not only undergo restricted collision coupling but it also restricts the mobility of the D₂ receptor. Restricted mobility is not due to tethering to the actin cytoskeleton but is, in part, related to the cholesterol content of the membrane. Depletion of cholesterol increases receptor mobility, but blunts activation of adenylyl cyclase. We conclude that signalling of the A_2A receptor takes place in cholesterol-rich domains of the membrane.