Ingrid Gsandtner

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.

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.

Subtype-Specific Differences in Corticotropin-Releasing Factor Receptor Complexes Detected by Fluorescence Spectroscopy

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 β-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 subtype-specific manner.

A Two-state Model for the Diffusion of the A2A Adenosine Receptor in Hippocampal Neurons: AGONIST-INDUCED SWITCH TO SLOW MOBILITY IS MODIFIED BY SYNAPSE-ASSOCIATED PROTEIN 102 (SAP102)*

Background: Agonist activation slows diffusion of the A2A receptor in the lipid bilayer. Results: In hippocampal neurons, the agonist-induced decrease in mobility was accounted for by both the hydrophobic receptor core and its extended C terminus, which recruited SAP102. Conclusion: The observations are consistent with two diffusion states of the A2A receptor in neurons. Significance: SAP102 regulates access of the A2A receptor to a compartment with restricted mobility. The A2A receptor is a class A/rhodopsin-like G protein-coupled receptor. Coupling to its cognate protein, Gs, occurs via restricted collision coupling and is contingent on the presence of cholesterol. Agonist activation slows diffusion of the A2A adenosine receptor in the lipid bilayer. We explored the contribution of the hydrophobic core and of the extended C terminus by examining diffusion of quantum dot-labeled receptor variants in dissociated hippocampal neurons. Single particle tracking of the A2A receptor(1–311), which lacks the last 101 residues, revealed that agonist-induced confinement was abolished and that the agonist-induced decrease in diffusivity was reduced substantially. A fragment comprising the SH3 domain and the guanylate kinase domain of synapse-associated protein 102 (SAP102) was identified as a candidate interactor that bound to the A2A receptor C terminus. Complex formation between the A2A receptor and SAP102 was verified by coimmunoprecipitation and by tracking its impact on receptor diffusion. An analysis of all trajectories by a hidden Markov model was consistent with two diffusion states where agonist activation reduced the transition between the two states and, thus, promoted the accumulation of the A2A receptor in the compartment with slow mobility. Overexpression of SAP102 precluded the access of the A2A receptor to a compartment with restricted mobility. In contrast, a mutated A2A receptor (with 383DVELL387 replaced by RVRAA) was insensitive to the action of SAP102. These observations show that the hydrophobic core per se does not fully account for the agonist-promoted change in mobility of the A2A receptor. The extended carboxyl terminus allows for regulatory input by scaffolding molecules such as SAP102.

A Tail of Two Signals: The C Terminus of the A2A-Adenosine Receptor Recruits Alternative Signaling Pathways

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 A2A-adenosine receptor, where the last 100 amino acids are of very modest relevance to Gs coupling. The C terminus was originally viewed mainly as the docking site for regulatory proteins of the β-arrestin family. These β-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 A2A receptor; translin-associated protein allows the A2A 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 A2A 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; A2A receptor agonists are candidate drugs for the treatment of schizophrenic symptoms. It is of obvious interest to explore a possible link.

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.

Heterologous expression of membrane proteins in cardiac myocytes

The cardiac isoform of the Na+ channel (NaV1.5) is known to accumulate in the endoplasmic reticulum (ER). This retention presumably reflects quality control in the ER. In order to understand the underlying mechanism, we heterologously expressed the human orthologue of the Na+ channel (NaV1.5) in neonatal primary rat and murine cardiomyocytes, in a cardiomyoblast cell line (H9c2) and in HEK293 cells (internal control). In HEK293 cells, NaV1.5 readily accumulated at the cell surface and gave rise to functional channels with the expected electrophysiological properties. In contrast, in cardiomyocytes and H9c2 cells, the NaV1.5 accumulated in the ER regardless of the transfection method employed (lipofection, nucleofection). As a positive control, we employed G protein-coupled β1-adrenergic, A1 and A2A adenosine receptors. In HEK293 cells, export of the A2A receptor is known to be enhanced by the deubiquinating enzyme USP4. Accordingly, we also co-expressed USP4 and the A2A receptor in the different cardiomyocyte preparations. However, in all instances, the membrane proteins were trapped within intracellular compartments. This was, in particular true for the NaV1.5, which, in many instances, accumulated in circular bodies, which are reminiscent of calnexin-rich organized smooth ER structures. Based on these findings, we conclude that (i) membrane proteins undergo stringent quality control in cardiac myocytes and (ii) ERexport of the NaV1.5 is limited by the availability of additional cardiomyocyte-specific components. Acknowledgements Supported by FWF M989-B09 (MR) and OAW DOC-fFORTE 22255 (IG). from 13th Scientific Symposium of the Austrian Pharmacological Society (APHAR). Joint Meeting with the Austrian Society of Toxicology (ASTOX) and the Hungarian Society for Experimental and Clinical Pharmacology (MFT) Vienna, Austria. 22–24 November 2007

Mapping the binding sites for accessory proteins on the A2A adenosine receptor

Among the four receptors for the purine nucleoside adenosine the A2A receptor subtype is the only one with an extended cytoplasmic domain and this is due to a remarkably long carboxyl terminal tail (C-tail). We have previously identified from an adult human brain cDNA library potential interaction partners which bind to the receptor C-tail and suggest that they impinge on receptor biology. (i) ARNO, the activator of the small GTP-binding protein ARF6, was shown to propagate sustained receptor signalling to ERK (extracellular signal regulated kinase); (ii) the ubiquitin-splitting protease USP4 controls receptor turnover; (iii) SAP (synapse-associated protein) 102 may form an anchor localizing the receptor in nerve cells. While ARNO binds to the membrane proximal portion, USP4 and SAP102 recognize more distal segments of the C-tail. In the case of SAP102 the recognition sequence could be narrowed down to a stretch of five amino acid residues (DVELL) which is conserved between species. A receptor where the DVELL sequence was changed to RVRAA has been characterized upon stable transfection of HEK293 cells (which express SAP102). Surface expression, radioligand binding and receptor-dependent stimulation of the effector adenylyl cyclase were similar to the wild type. In contrast, the agonist-dependent activation of ERK was attenuated in cells stably expressing the mutant receptor. Hence, SAP102 may be necessary to establish a signalling complex including ERK and the receptor as has been previously observed for the NMDA receptor in nerve cells. from 13th Scientific Symposium of the Austrian Pharmacological Society (APHAR). Joint Meeting with the Austrian Society of Toxicology (ASTOX) and the Hungarian Society for Experimental and Clinical Pharmacology (MFT) Vienna, Austria. 22–24 November 2007