Restricted collision coupling of the A2A receptor revisited -: Evidence for physical separation of two signaling cascades

The A(2A)-adenosine receptor is a prototypical G(s) protein-coupled receptor but stimulates MAPK/ERK in a G(s)-independent way. The A(2A) receptor has long been known to undergo restricted collision coupling with Gs; the mechanistic basis for this mode of coupling has remained elusive. Here we visualized agonist-induced changes in mobility of the yellow fluorescent protein-tagged receptor by fluorescence recovery after photo-bleaching microscopy. Stimulation with a specific A(2A) receptor agonist did not affect receptor mobility. In contrast, stimulation with dopamine decreased the mobility of the D-2 receptor. When coexpressed in the same cell, the A(2A) receptor precluded the agonist-induced change in D-2 receptor mobility. Thus, the A(2A) receptor did not only undergo restricted collision coupling, but it also restricted the mobility of the D-2 receptor. Restricted mobility was not due to tethering to the actin cytoskeleton but was, in part, related to the cholesterol content of the membrane. Depletion of cholesterol increased receptor mobility but blunted activation of adenylyl cyclase, which was accounted for by impaired formation of the ternary complex of agonist, receptor, and G protein. These observations support the conclusion that the A(2A) receptor engages G(s) and thus signals to adenylyl cyclase in cholesterol-rich domains of the membrane. In contrast, stimulation of MAPK by the A(2A) receptor was not impaired. These findings are consistent with a model where the recruitment of these two pathways occurs in physically segregated membrane microdomains. Thus, the A(2A) receptor is the first example of a G protein-coupled receptor documented to select signaling pathways in a manner dependent on the lipid microenvironment of the membrane.