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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 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.In the fluid mosaic model, the lipid bilayer is an isotropic milieu, in which membrane-embedded proteins diffuse in two dimensions and thus collide at random with each other (1). When applied to G protein-coupled receptors, the model predicts that G protein-coupled receptors move in a random walk, and, upon activation, this allows them to engage their cognate G proteins. This "collision coupling" mode of activation was validated in studies using the ␤-adrenergic receptor and its coupling to the effector enzyme adenylyl cyclase in turkey erythrocytes (2). However, experiments with the A 2 -adenosine receptor in turkey erythrocytes revealed kinetic properties of adenylyl cyclase activation that were incompatible with the collision coupling model. The results indicated a tight coupling of the adenosine receptor to G␣ s (3, 4); the term "restricted collision coupling" was coined to account for this altered mode of coupling. Restricted collision is not a feature unique to the avian A 2 -adenosine receptor, because it was also documented for the human A 2A receptor in platelet membranes (5). In addition, the A 2A -adenosine receptor has the unusual feature of forming a tight complex with G s , which persists in detergent solution, which is resistant to guanine nucleotides and which re...

G protein-coupled receptors have been proposed to exist in signalosomes subject to agonist-driven shifts in the assembly disassembly equilibrium, affected by stabilizing membrane lipids and/or cortical actin restricting mobility. We investigated the highly homologous corticotropin-releasing factor receptors (CRFRs), CRFR1 and -2, which are different within their hydrophobic core. Agonist stimulation of CRFR1 and CRFR2 gave rise to similar concentration-response curves for cAMP accumulation, but CRFR2 underwent restricted collision coupling. Both CRFR1 and CRFR2 formed constitutive oligomers at the cell surface and recruited ␤-arrestin upon agonist activation (as assessed by fluorescence resonance energy transfer microscopy in living cells). However, CRFR2, but not CRFR1, failed to undergo agonist-induced internalization. Likewise, agonist binding accelerated the diffusion rate of CRFR2 only (detected by fluorescence recovery after photobleaching and fluorescence correlation spectroscopy) but reduced the mobile fraction, which is indicative of local confinement. Fluorescence intensity distribution analysis demonstrated that the size of CRFR complexes was not changed. Disruption of the actin cytoskeleton abolished the agonist-dependent increase in CRFR2 mobility, shifted the agonist concentration curve for CRFR2 to the left, and promoted agonist-induced internalization of CRFR2. Our observations are incompatible with an agonist-induced change in monomer-oligomer equilibrium, but they suggest an agonist-induced redistribution of CRFR2 into a membrane microdomain that affords rapid diffusion but restricted mobility and that is stabilized by the actin cytoskeleton. Our data show that membrane anisotropy can determine the shape and duration of receptor-generated signals in a subtypespecific manner.Signal transduction via heterotrimeric G proteins is accomplished by a cycle of activation and deactivation of the G␣-subunit, which is achieved by receptor-catalyzed exchange of prebound GDP for GTP and GTP hydrolysis by the intrinsic GTPase of G␣, respectively. Superimposed on this GTPase cycle, there is a cycle of subunit dissociation and reassociation, in which the inactive heterotrimer G␣.GDP.␤␥ affords receptor docking, GTP binding drives subunit dissociation into G␣.GTP.Mg 2ϩ and G␤␥, and the GTPase-mediated hydrolysis promotes mutual inactivation of two G␣.GDP and G␤␥ by reassociation of the inactive heterotrimer G␣.GDP.␤␥. This model was established some 20 years ago, mainly by the study of reconstituted purified components (Freissmuth et al., 1989). However, since then, methods have become available that allow the tracking of the activity of individual components at the single-cell level. In several instances, these have led to observations that are incompat-

The A 2A adenosine receptor is a prototypical G s -coupled receptor, but it also signals, e.g. to mitogen-activated protein (MAP) kinase, via a pathway that is independent of heterotrimeric G proteins. Truncation of the carboxyl terminus affects the strength of the signal through these alternative pathways. In a yeast two-hybrid interaction hunt, we screened a human brain library for proteins that bound to the juxtamembrane portion of the carboxyl terminus of the A 2A receptor. This approach identified ARNO/cytohesin-2, a nucleotide exchange factor for the small (monomeric) G proteins of the Arf (ADP-ribosylation factor) family, as a potential interaction partner. We confirmed a direct interaction by mutual pull down (of fusion proteins expressed in bacteria) and by immunoprecipitation of the proteins expressed in mammalian cells. To circumvent the long term toxicity associated with overexpression of ARNO/cytohesin-2, we created stable cell lines that stably expressed the A 2A receptor and where ARNO/cytohesin-2 or the dominant negative version E156K-ARNO/ cytohesin-2 was inducible by mifepristone. Cyclic AMP accumulation induced by an A 2A -specific agonist was neither altered by ARNO/cytohesin-2 nor by the dominant negative version. This was also true for agonistinduced desensitization. In contrast, expression of dominant negative E156K-ARNO/cytohesin-2 and of dominant negative T27N-Arf6 abrogated the sustained phase of MAP kinase stimulation induced by the A 2A receptor. We therefore conclude that ARNO/cytohesin-2 is required to support the alternative, heterotrimeric G protein-independent, signaling pathway of A 2A receptor, which is stimulation of MAP kinase.Over the last decade, it has been increasingly accepted that G protein-coupled receptors also bind regulatory proteins other than G proteins, arrestins, and G protein-coupled kinases, which are involved in effector regulation and desensitization, respectively (1). These accessory proteins include components of signaling cascades and bind to the carboxyl termini of various G protein-coupled receptors (2). The A 2A adenosine receptor has an unusually long intracellular carboxyl-terminal tail, 122 amino acids in man, when compared, for instance, to 34 residues in the carboxyl terminus of the A 1 adenosine receptor. Circumstantial evidence suggests that accessory proteins bind to the carboxyl terminus of the A 2A receptor (3). A 2A receptors can activate mitogen-activated protein (MAP) 1 kinase by a G␣ s -independent signaling pathway; this can be seen both in endothelial cells where the receptor is endogenously expressed (4) and upon heterologous expression in HEK293 cells (5). Truncation of the carboxyl terminus does not impair the ability of the A 2A adenosine receptor to stimulate MAP kinase but blunts stimulation of cAMP accumulation. In addition, fulllength and truncated receptors differ in their constitutive (agonist-independent) activity; this difference is only seen in intact cells and is lost upon membrane preparation, suggesting the loss of one o...

G protein-coupled receptors are endowed with carboxyl termini that vary greatly in length and sequence. In most instances, the distal portion of the C terminus is dispensable for G protein coupling. This is also true for the A 2A -adenosine receptor, where the last 100 amino acids are of very modest relevance to G s coupling. The C terminus was originally viewed mainly as the docking site for regulatory proteins of the ␤-arrestin family. These ␤-arrestins bind to residues that have been phosphorylated by specialized kinases (G protein-coupled receptor kinases) and thereby initiate receptor desensitization and endocytosis. More recently, it has become clear that many additional "accessory" proteins bind to C termini of G protein-coupled receptors. The article by Sun et al. (p. 454) in the current issue of Molecular Pharmacology identifies translin-associated protein-X as yet another interaction partner of the A 2A receptor; translin-associated protein allows the A 2A receptor to impinge on the signaling mechanisms by which p53 regulates neuronal differentiation, but the underlying signaling pathways are uncharted territory. With a list of five known interaction partners, the C terminus of the A 2A receptor becomes a crowded place. Hence, there must be rules that regulate the interaction. This allows the C terminus to act as coincidence detector and as signal integrator. Despite our ignorance about the precise mechanisms, the article has exciting implications: the gene encoding for translin-associated protein-X maps to a locus implicated in some forms of schizophrenia; A 2A receptor agonists are candidate drugs for the treatment of schizophrenic symptoms. It is of obvious interest to explore a possible link.Adenosine is a retaliatory metabolite. This catch phrase succinctly summarizes the concept that adenosine is a cellular signal of metabolic distress: hypoxia leads to a decline in cellular ATP levels and to the release of adenosine. On the extracellular side, adenosine affords tissue protection by eliciting both short-term effects (e.g., cellular hyperpolarization, inhibition of Ca 2ϩ influx, vasodilation) and a delayed adaptive response (e.g., by triggering angiogenesis; see Linden, 2005). The widespread expression of adenosine receptors is also consistent with its role in mediating cellular protection: there are no tissues or organs that are not responsive to adenosine. The retaliatory action of adenosine results from the concerted stimulation of four adenosine receptors, termed A 1 -, A 2A -, A 2B -, and A 3 -adenosine receptors. These receptors differ in their affinity for adenosine, in the type of G proteins that they engage, and, hence, in the downstream signaling pathways that are activated in the receptive cells (Klinger et al., 2002a). Adenosine and NeuroprotectionAdenosine, however, is not only released as a signal of cellular distress; it also participates in the purinergic synaptic signaling network. ATP is a constituent of neurotransmitter-containing vesicles and is thus subject to Ca 2ϩ -...