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J. Biol. Chem., Vol. 283, Issue 14, 9276-9288, April 4, 2008

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Restricted Collision Coupling of the A_2A Receptor Revisited

EVIDENCE FOR PHYSICAL SEPARATION OF TWO SIGNALING CASCADES^*

From the Institute of Pharmacology, Center of Biomolecular Medicine and Pharmacology, Medical University of Vienna, Währinger Strasse 13a, A-1090 Vienna, Austria

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 G_s; 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 A_2A 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 A_2A receptor precluded the agonist-induced change in D₂ receptor mobility. Thus, the A_2A 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 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.

Received for publication, July 31, 2007 , and in revised form, December 27, 2007.

^* This work was supported in part by grants from Austrian Science Fund (to F. W. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Both authors contributed equally to this work.

² Supported by a DOC-fforte stipend from the Austrian Academy of Sciences.

³ To whom correspondence should be addressed. Tel.: 43-1-4277-64171; Fax: 43-1-4277-9641; E-mail: michael.freissmuth{at}meduniwien.ac.at.

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