Simon Keuerleber

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

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.

Recruitment of a cytoplasmic chaperone relay by the A2A-adenosine receptor.

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. The adenosine A2A receptor is a prototypical rhodopsin-like G protein-coupled receptor but has several unique structural features, in particular a long C terminus (of >120 residues) devoid of a palmitoylation site. It is known to interact with several accessory proteins other than those canonically involved in signaling. However, it is evident that many more proteins must interact with the A2A receptor, if the trafficking trajectory of the receptor is taken into account from its site of synthesis in the endoplasmic reticulum (ER) to its disposal by the lysosome. Affinity-tagged versions of the A2A receptor were expressed in HEK293 cells to identify interacting partners residing in the ER by a proteomics approach based on tandem affinity purification. The receptor-protein complexes were purified in quantities sufficient for analysis by mass spectrometry. We identified molecular chaperones (heat-shock proteins HSP90α and HSP70-1A) that interact with and retain partially folded A2A receptor prior to ER exit. Complex formation between the A2A receptor and HSP90α (but not HSP90β) and HSP70-1A was confirmed by co-affinity precipitation. HSP90 inhibitors also enhanced surface expression of the receptor in PC12 cells, which endogenously express the A2A receptor. Finally, proteins of the HSP relay machinery (e.g. HOP/HSC70-HSP90 organizing protein and P23/HSP90 co-chaperone) were recovered in complexes with the A2A receptor. These observations are consistent with the proposed chaperone/coat protein complex II exchange model. This posits that cytosolic HSP proteins are sequentially recruited to folding intermediates of the A2A receptor. Release of HSP90 is required prior to recruitment of coat protein complex II components. This prevents premature ER export of partially folded receptors.

From cradle to twilight: the carboxyl terminus directs the fate of the A(2A)-adenosine receptor.

The extended carboxyl terminus of the A(2A)-adenosine receptor is known to engage several proteins other than those canonically involved in signalling by GPCRs (i.e., G proteins, G protein-coupled receptor kinases/GRKs, arrestins). The list includes the deubiquinating enzyme USP4, α-actinin, the guanine nucleotide exchange factor for ARF6 ARNO, translin-X-associated protein, calmodulin, the neuronal calcium binding protein NECAB2 and the synapse associated protein SAP102. However, if the fate of the A(2A)-receptor is taken into account - from its birthplace in the endoplasmic reticulum to its presumed site of disposal in the lysosome, it is evident that many more proteins must interact with the A(2A)-adenosine receptor. There are several arguments that support the conjecture that these interactions will preferentially occur with the carboxyl terminus of the A(2A)-adeonsine receptor: (i) the extended carboxyl terminus (of 122 residues=) offers the required space to accommodate companions; (ii) analogies can be drawn with other receptors, which engage several of these binding partners with their C-termini. This approach allows for defining the nature of the unknown territory. As an example, we posit a chaperone/coat protein complex-II (COPII) exchange model that must occur on the carboxyl terminus of the receptor. This model accounts for the observation that a minimum size of the C-terminus is required for correct folding of the receptor. It also precludes premature recruitment of the COPII-coat to a partially folded receptor.

Expression of G Protein-Coupled Receptor 19 in Human Lung Cancer Cells Is Triggered by Entry into S-Phase and Supports G2–M Cell-Cycle Progression

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 cell-cycle. (i) Knockdown of Gpr19 mRNA by RNA interference reduced cell growth of human lung cancer cell lines. (ii) Cell-cycle progression through G2–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. (vi) E2f and Gpr19 expression correlated in lung cancer patient samples. To the best of knowledge, this is the first example of a GPCR showing cell-cycle-specific mRNA expression. Our data also validate GPR19 as a candidate target when overexpressed in lung cancer. Mol Cancer Res; 10(10); 1343–58. ©2012 AACR.

Reengineering the collision coupling and diffusion mode of the A2A-adenosine receptor: palmitoylation in helix 8 relieves confinement

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. The A2A-adenosine receptor undergoes restricted collision coupling with its cognate G protein Gs and lacks a palmitoylation site at the end of helix 8 in its intracellular C terminus. We explored the hypothesis that there was a causal link between the absence of a palmitoyl moiety and restricted collision coupling by introducing a palmitoylation site. The resulting mutant A2A-R309C receptor underwent palmitoylation as verified by both mass spectrometry and metabolic labeling. In contrast to the wild type A2A receptor, the concentration-response curve for agonist-induced cAMP accumulation was shifted to the left with increasing expression levels of A2A-R309C receptor, an observation consistent with collision coupling. Single particle tracking of quantum dot-labeled receptors confirmed that wild type and mutant A2A receptor differed in diffusivity and diffusion mode; agonist activation resulted in a decline in mean square displacement of both receptors, but the drop was substantially more pronounced for the wild type receptor. In addition, in the agonist-bound state, the wild type receptor was frequently subject to confinement events (estimated radius 110 nm). These were rarely seen with the palmitoylated A2A-R309C receptor, the preferred diffusion mode of which was a random walk in both the basal and the agonist-activated state. Taken together, the observations link restricted collision coupling to diffusion limits imposed by the absence of a palmitoyl moiety in the C terminus of the A2A receptor. The experiments allowed for visualizing local confinement of an agonist-activated G protein-coupled receptor in an area consistent with the dimensions of a lipid raft.

Restricted collision coupling of the adenosine A2A receptor is due to its agonist-induced confinement in the membrane

Background The A2A adenosine receptor is of interest because of several reasons. (i) It is a frequently blocked pharmacological target, because it is the site of action of caffeine. (ii) It has a long C-terminus that provides a docking site for several proteins, which direct the fate of the receptor from its synthesis to its lysosomal degradation. (iii) The A2A receptor can only promote activation of a limited number of available Gs molecules. This coupling mode was termed restricted collision coupling. (iv) Most G protein-coupled receptors carry one or several cysteine residues in their C-terminus which is subject to palmitoylation to anchor and stabilize the amphipathic helix 8; the A2A receptor lacks this palmitoylation site. We explored the hypothesis that there is a causal link between the absence of a palmitoyl moiety and restricted collision coupling.

Tracking the A2A adenosine receptor

Background The A2A adenosine receptor has become a drug target in the treatment of Parkinson’s disease, psychotic behavior and dementia. In addition, targeted deletion of this receptor in mice leads to hypertension, increased platelet aggregation, male aggressiveness and decreased susceptibility to ischemic brain damage. The potential clinical relevance of this receptor is obvious. The A2A adenosine receptor, a prototypical GPCR, is known to signal via restricted collision coupling with Gs. In addition, it is able to stimulate MAP kinase/ERK in a Gs-independent way but dependent on the lipid microenvironment of the membrane. Hence, we characterized the mobility and the targeting of the A2A receptor in nerve cells.

ARNO/cytohesin-2 regulates desensitization of the A2A adenosine receptor in rat pheochromocytoma cells

The A2A 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 A2A 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 A2A receptor signaling in rat pheochromocytoma (PC12) cells. These cells express the A2A 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 A2A 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 A2A 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 A2A receptor after agonist treatment. Our results indicate that the interaction with ARNO/cytohesin-2 stabilizes short-term desensitization of the A2A receptor to prevent excessive stimulation.