Restricted Collision Coupling of the A₂A Receptor Revisited: EVIDENCE FOR PHYSICAL SEPARATION OF TWO SIGNALING CASCADES

• Published in: The Journal of biological chemistry 2008-04, Vol.283 (14), p.9276-9288
• Main Authors: Charalambous, Christoforos, Gsandtner, Ingrid, Keuerleber, Simon, Milan-Lobo, Laura, Kudlacek, Oliver, Freissmuth, Michael, Zezula, Jürgen
• Format: Article
• Language: English
• Subjects: Adenosine A2 Receptor Agonists; Adenylyl Cyclases - metabolism; Animals; Cell Membrane - metabolism; Cholesterol - metabolism; GTP-Binding Protein alpha Subunits, Gs - metabolism; Humans; MAP Kinase Signaling System - physiology; Mitogen-Activated Protein Kinase Kinases - metabolism; PC12 Cells; Photobleaching; Protein Transport - physiology; Rats; Receptor, Adenosine A2A - metabolism; Receptors, Dopamine D2 - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M706275200

The A₂A-adenosine receptor is a prototypical Gs protein-coupled receptor but stimulates MAPK/ERK in a Gs-independent way. The A₂A 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 photobleaching microscopy. Stimulation with a specific A₂A receptor agonist did not affect receptor mobility. In contrast, stimulation with dopamine decreased the mobility of the D₂ receptor. When coexpressed in the same cell, the A₂A receptor precluded the agonist-induced change in D₂ receptor mobility. Thus, the A₂A receptor did not only undergo restricted collision coupling, but it also restricted the mobility of the D₂ 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 A₂A receptor engages Gs and thus signals to adenylyl cyclase in cholesterol-rich domains of the membrane. In contrast, stimulation of MAPK by the A₂A 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 A₂A 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.