Background: The A2A receptor is known to accumulate in the endoplasmic reticulum.Results: Mass spectrometry identified molecular chaperones (HSP90 and HSP70) bound to the A2A receptor.Conclusion: Sequential recruitment of chaperones to the cytosolic face of the A2A receptor is consistent with a heat-shock protein relay assisting folding.Significance: The observations are consistent with a chaperone/COPII exchange model, where heat-shock proteins bound to the receptor preclude its premature ER export.

It has long been known that G protein-coupled receptors (GPCR) are subject to illegitimate expression in tumor cells. Presumably, hijacking the normal physiologic functions of GPCRs contributes to all biologic capabilities acquired during tumorigenesis. Here, we searched for GPCRs that were expressed in lung cancer: the mRNA encoding orphan G protein-coupled receptor 19 (GPR19) was found frequently overexpressed in tissue samples obtained from patients with small cell lung cancer. Several observations indicate that overexpression of Gpr19 confers a specific advantage to lung cancer cells by accelerating transition through the cellcycle. (i) Knockdown of Gpr19 mRNA by RNA interference reduced cell growth of human lung cancer cell lines. (ii) Cell-cycle progression through G 2 -M-phase was impaired in cells transfected with siRNAs directed against Gpr19 and this was associated with increased protein levels of cyclin B1 and phosphorylated histone H3. (iii) The expression levels of Gpr19 mRNA varied along the cell-cycle with a peak observed in S-phase. (iv) The putative control of Gpr19 expression by E2F transcription factors was verified by chromatin immunoprecipitation: antibodies directed against E2F-1 to -4 allowed for the recovery of the Gpr19 promoter. (v) Removal of E2F binding sites in the Gpr19 promoter diminished the expression of a luciferase reporter.

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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...

Background: The A2A receptor engages Gs by restricted collision coupling and lacks a palmitoyl moiety in its C terminus.Results: Engineering palmitoylated cysteine into the C terminus relieved restricted collision coupling and resulted in accelerated diffusion of the agonist-liganded A2A receptor.Conclusion: Restricted collision coupling arises from limits imposed on receptor diffusion.Significance: Agonist induced confinement of the A2A receptor in a structure consistent with a lipid raft.

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The A 2A adenosine receptor is a G protein-coupled receptor which desensitizes upon prolonged agonist stimulation. In order to understand the biological function of its unusually long C-terminus, we screened a human brain library for proteins capable of binding to the last 120 amino acids of the A 2A receptor. We identified a guanine nucleotide exchange factor for the small G protein ARF6 (ARNO/cytohesin-2) as a binding partner. In this study, we investigated the impact of ARNO on A 2A receptor signaling in rat pheochromocytoma (PC12) cells. These cells express the A 2A receptors endogenously. We created cell lines with inducible expression of ARNO or its catalytic inactive mutant E156K. Neither wild type ARNO nor the mutant had an effect on receptor expression, signaling via adenylyl cyclase after activation or long-term de-and resensitization kinetics. In order to investigate effects of ARNO on A 2A receptor short-term de-and resensitization we employed a FRET-based sensor to measure changes in cAMP in real time. Cells were transfected with plasmids encoding the regulatory and catalytic subunit of protein kinase A (PKA) fused to CFP and YFP, respectively. Accumulation of cAMP results in the dissociation of the PKA subunits, which can be measured in single cells as a loss of FRET. The presence of dominant negative ARNO accelerated the recovery of A 2A receptor after stimulation and led to a pronounced signaling response when cells were re-challenged with agonist. While membrane recruitment of ARNO was not affected by the mutation, we observed a difference in the recovery of the A 2A receptor after agonist treatment. Our results indicate that the interaction with ARNO/cytohesin-2 stabilizes short-term desensitization of the A 2A receptor to prevent excessive stimulation.