Metabotropic glutamate type 5, dopamine D2 and adenosine A2a receptors form higher-order oligomers in living cells

G protein-coupled receptors are known to form homo- and heteromers at the plasma membrane, but the stoichiometry of these receptor oligomers are relatively unknown. Here, by using bimolecular fluorescence complementation, we visualized for the first time the occurrence of heterodimers of metabotropic glutamate mGlu₅ receptors (mGlu₅R) and dopamine D₂ receptors (D₂R) in living cells. Furthermore, the combination of bimolecular fluorescence complementation and bioluminescence resonance energy transfer techniques, as well as the sequential resonance energy transfer technique, allowed us to detect the occurrence receptor oligomers containing more than two protomers, mGlu₅R, D₂R and adenosine A_2A receptor (A_2AR). Interestingly, by using high-resolution immunoelectron microscopy we could confirm that the three receptors co-distribute within the extrasynaptic plasma membrane of the same dendritic spines of asymmetrical, putative glutamatergic, striatal synapses. Also, co-immunoprecipitation experiments in native tissue demonstrated the existence of an association of mGlu₅R, D₂R and A_2AR in rat striatum homogenates. Overall, these results provide new insights into the molecular composition of G protein-coupled receptor oligomers in general and the mGlu₅R/D₂R/A_2AR oligomer in particular, a receptor oligomer that might constitute an important target for the treatment of some neuropsychiatric disorders.     

Plasma membrane diffusion of G protein-coupled receptor oligomers

G protein-coupled receptors are known to form homo-and heteromers at the plasma membrane, but the molecular properties of these oligomers are relatively unknown. Here, we show a method that allows the diffusion of G protein-coupled receptors oligomers in the plasma membrane to be monitored in single cells by combining Bimolecular Fluorescence Complementation and Fluorescence Correlation Spectroscopy. With this approach we have measured, for the first time, the membrane diffusional characteristics of adenosine A(1) and A(2A) receptor homo-and heterodimers in Chinese Hamster Ovary cells. Interestingly, both homodimers display similar diffusion co-efficients (D) when expressed in living cells (D=5.0 and 4.8x10(-9) cm(2)/s, respectively) but the heterodimer formed by these receptors exhibit a significantly faster plasma membrane diffusion co-efficent (D=5.6x10(-9) cm(2)/s) when compared to the adenosine A(1) receptor tagged with the full-length yellow fluorescent protein (D=4.0x10(-9) cm(2)/s). Overall, these results demonstrate differences in plasma membrane diffusion between adenosine receptor homo-and heterodimers, providing new insights into the molecular plasticity of G protein-coupled receptor oligomerization.     

Adenosine A2A receptors assemble into higher-order oligomers at the plasma membrane

Oligomerization of G protein-coupled receptors (GPCRs) is known to play important roles in regulating receptor pharmacology and function. Whereas many bivalent GPCR interactions have been described, the stoichiometry and localization of GPCR oligomers are largely unknown. We have used bimolecular fluorescence complementation (BiFC) to study adenosine A_2A receptor (A_2AR) oligomerization. The data suggest specificity of the A_2AR/A_2AR interaction monitored by BiFC and proper sub-cellular localization of tagged receptors. Moreover, using a novel approach combining fluorescence resonance energy transfer and BiFC, we found that at least three A_2A receptors assemble into higher-order oligomers at the plasma membrane in Cath.A differentiated neuronal cells.

Structured summary

MINT-6797156, MINT-6797142: A2AR (uniprotkb:P29274) physically interacts (MI:0218) with A2AR (uniprotkb:P29274) by bimolecular fluorescence complementation (MI:0809)

MINT-6797129: A2AR (uniprotkb:P29274) physically interacts (MI:0218) with A2AR (uniprotkb:P29274) by fluorescent resonance energy transfer (MI:0055)

     

Adenosine receptor containing oligomers: their role in the control of dopamine and glutamate neurotransmission in the brain

While the G protein-coupled receptor (GPCR) oligomerization has been questioned during the last fifteen years, the existence of a multi-receptor complex involving direct receptor-receptor interactions, called receptor oligomers, begins to be widely accepted. Eventually, it has been postulated that oligomers constitute a distinct functional form of the GPCRs with essential receptorial features. Also, it has been proven, under certain circumstances, that the GPCR oligomerization phenomenon is crucial for the receptor biosynthesis, maturation, trafficking, plasma membrane diffusion, and pharmacology and signalling. Adenosine receptors are GPCRs that mediate the physiological functions of adenosine and indeed these receptors do also oligomerize. Accordingly, adenosine receptor oligomers may improve the molecular mechanism by which extracellular adenosine signals are transferred to the G proteins in the process of receptor transduction. Importantly, these adenosine receptor-containing oligomers may allow not only the control of the adenosinergic function but also the fine-tuning modulation of other neurotransmitter systems (i.e. dopaminergic and glutamatergic transmission). Overall, we underscore here recent significant developments based on adenosine receptor oligomerization that are essential for acquiring a better understanding of neurotransmission in the central nervous system under normal and pathological conditions.     

Dimerization in the absence of higher-order oligomerization of the G protein-coupled secretin receptor

Oligomerization of G protein-coupled receptors has been proposed to affect receptor function and regulation; however, little is known about the molecular nature of such complexes. We previously utilized bioluminescence resonance energy transfer (BRET) to demonstrate that the prototypic Family B secretin receptor can form oligomers. We now explore the order of oligomerization present utilizing unique bimolecular fluorescence complementation and energy transfer techniques. The non-fluorescent carboxyl-terminal and amino-terminal halves of yellow fluorescent protein (YFP) were fused to the carboxyl terminus of the secretin receptor. These constructs bound secretin normally and signaled in response to secretin like wild type receptor. When co-expressed on COS cells, these constructs physically interacted to yield typical YFP fluorescence in biosynthetic compartments and at the plasma membrane, reflecting receptor homo-dimerization. However, the addition of another potential partner in form of Rlu- or CFP-tagged secretin receptor yielded no significant BRET or FRET signal, respectively, under conditions in which intact YFP-tagged secretin receptor yielded such a signal. Absence of higher-order receptor oligomers was further confirmed using saturation BRET techniques. Absence of significant resonance transfer to the secretin receptor homo-dimer was true for carboxyl-terminally-tagged secretin receptor, as well as for receptor incorporating the transfer partner into each of the three distinct intracellular loop domains. These results suggest that the secretin receptor can exist only as a structurally-specific homo-dimer, without being present as higher-order oligomers.     

Internalization and desensitization of adenosine receptors

Until now, more than 800 distinct G protein-coupled receptors (GPCRs) have been identified in the human genome. The four subtypes of the adenosine receptor (A(1), A(2A), A(2B) and A(3) receptor) belong to this large family of GPCRs that represent the most widely targeted pharmacological protein class. Since adenosine receptors are widespread throughout the body and involved in a variety of physiological processes and diseases, there is great interest in understanding how the different subtypes are regulated, as a basis for designing therapeutic drugs that either avoid or make use of this regulation. The major GPCR regulatory pathway involves phosphorylation of activated receptors by G protein-coupled receptor kinases (GRKs), a process that is followed by binding of arrestin proteins. This prevents receptors from activating downstream heterotrimeric G protein pathways, but at the same time allows activation of arrestin-dependent signalling pathways. Upon agonist treatment, adenosine receptor subtypes are differently regulated. For instance, the A(1)Rs are not (readily) phosphorylated and internalize slowly, showing a typical half-life of several hours, whereas the A(2A)R and A(2B)R undergo much faster downregulation, usually shorter than 1 h. The A(3)R is subject to even faster downregulation, often a matter of minutes. The fast desensitization of the A(3)R after agonist exposure may be therapeutically equivalent to antagonist occupancy of the receptor. This review describes the process of desensitization and internalization of the different adenosine subtypes in cell systems, tissues and in vivo studies. In addition, molecular mechanisms involved in adenosine receptor desensitization are discussed.     

Netrin-1-independent adenosine A2b receptor activation regulates the response of axons to netrin-1 by controlling cell surface levels of UNC5A receptors

Growth cone response to the bifunctional guidance cue netrin-1 is regulated by the activity of intracellular signaling intermediates such as protein kinase C-alpha (PKCα) and adenylyl cyclase. Among the diverse cellular events these enzymes regulate is receptor trafficking. Netrin-1, itself, may govern the activity of these signaling intermediates, thereby regulating axonal responses to itself. Alternatively, other ligands, such as activators of G protein-coupled receptors, may regulate responses to netrin-1 by governing these signaling intermediates. Here, we investigate the mechanisms controlling activation of PKCα and the subsequent downstream regulation of cell surface UNC5A receptors. We report that activation of adenosine receptors by adenosine analogs, or activation of the putative netrin-1 receptor, the G protein-coupled receptor adenosine A2b receptor (A2bR) results in PKCα-dependent removal of UNC5A from the cell surface. This decrease in cell surface UNC5A reduces the number of growth cones that collapse in response to netrin-1 and converts repulsion to attraction. We show these A2bR-mediated alterations in axonal response are not because of netrin-1 because netrin-1 neither binds A2bR, as assayed by protein overlay, nor stimulates PKCα-dependent UNC5A surface loss. Our results demonstrate that netrin-1-independent A2bR signaling governs the responsiveness of a neuron to netrin-1 by regulating the levels of cell surface UNC5A receptor.     

Distinctive features of Trk neurotrophin receptor transactivation by G protein-coupled receptors.

Ligands for G protein-coupled receptors (GPCR) are capable of activating mitogenic receptor tyrosine kinases, in addition to the mitogen-activated protein (MAP) kinase signaling pathway and classic G protein-dependent signaling pathways involving adenylyl cyclase and phospholipase. For example, receptors for epidermal growth factor (EGF), insulin-like growth-1 and platelet-derived growth factor and can be transactivated through G protein-coupled receptors. Neurotrophins, such as NGF, BDNF and NT-3 also utilize receptor tyrosine kinases, namely TrkA, TrkB and TrkC. Recently, it has been shown that activation of Trk receptor tyrosine kinases can also occur via a G protein-coupled receptor mechanism, without involvement of neurotrophins. Adenosine and adenosine agonists can activate Trk receptor phosphorylation specifically through the seven transmembrane spanning adenosine 2A (A2A) receptor. Several features of Trk receptor transactivation are noteworthy and differ significantly from other transactivation events. Trk receptor transactivation is slower and results in a selective increase in activated Akt. Unlike the biological actions of other tyrosine kinase receptors, increased Trk receptor activity by adenosine resulted in increased cell survival. This article will discuss potential mechanisms by which adenosine can activate trophic responses through Trk tyrosine kinase receptors.     

Detection of antigen interactions ex vivo by proximity ligation assay: endogenous dopamine D2-adenosine A2A receptor complexes in the striatum

The existence of G protein-coupled receptor (GPCR) dimers and/or oligomers has been demonstrated in heterologous systems using a variety of biochemical and biophysical assays. While these interactions are the subject of intense research because of their potential role in modulating signaling and altering pharmacology, evidence for the existence of receptor interactions in vivo is still elusive because of a lack of appropriate methods to detect them. Here, we adapted and optimized a proximity ligation assay (PLA) for the detection in brain slices of molecular proximity of two antigens located on either the same or two different GPCRs. Using this approach, we were able to confirm the existence of dopamine D2 and adenosine A2A receptor complexes in the striatum of mice ex vivo.     

Effects of the allosteric modulator SCH-202676 on adenosine and P2Y receptors

The G protein-coupled receptor allosteric modulator SCH-202676 (N-(2,3-diphenyl-1,2,4-thiadiazol-5-(2H)-ylidene)methanamine), which affects a wide range of structurally unrelated G protein-coupled receptors, has highly divergent effects on purine receptors. SCH-202676 inhibited radioligand binding to human adenosine A(1), A(2A), and A(3) receptors (IC(50) = 0.5-0.8 microM) and affected dissociation kinetics, but at the human P2Y(1) nucleotide receptor it had no effect. SCH-202676 (10 microM) selectively accelerated agonist dissociation at adenosine A(3) receptors and either slowed (adenosine A(1) receptors) or accelerated (adenosine A(2A) receptors) antagonist dissociation. Thus, SCH-202676 differentially modulated A(1), A(2A), and A(3) receptors as well as agonist- and antagonist-occupied receptors.     

Dimerization of G protein-coupled purinergic receptors: increasing the diversity of purinergic receptor signal responses and receptor functions

It is well accepted that G protein-coupled receptors (GPCRs) arrange into dimers or higher-order oligomers that may modify various functions of GPCRs. GPCR-type purinergic receptors (i.e. adenosine and P2Y receptors) tend to form heterodimers with GPCRs not only of the different families but also of the same purinergic receptor families, leading to alterations in functional properties. In the present review, we focus on current knowledge of the formation of heterodimers between metabotropic purinergic receptors that activate novel functions in response to extracellular nucleosides/nucleotides, revealing that the dimerization seems to be employed for ‘fine-tuning’ of purinergic signaling. Thus, the relationship between adenosine and adenosine triphosphate is likely to be more and more intimate than simply being a metabolite of the other.     

The extracellular N-terminal domain and transmembrane domains 1 and 2 mediate oligomerization of a yeast G protein-coupled receptor

G protein-coupled receptors (GPCRs) can form homodimers/oligomers and/or heterodimers/oligomers. The mechanisms used to form specific GPCR oligomers are poorly understood because the domains that mediate such interactions and the step(s) in the secretory pathway where oligomerization occurs have not been well characterized. Here we have used subcellular fractionation and fluorescence resonance energy transfer (FRET) experiments to show that oligomerization of a GPCR (alpha-factor receptor; STE2 gene product) of the yeast Saccharomyces cerevisiae occurs in the endoplasmic reticulum. To identify domains of this receptor that mediate oligomerization, we used FRET and endocytosis assays of oligomerization in vivo to analyze receptor deletion mutants. A mutant lacking the N-terminal extracellular domain and transmembrane (TM) domain 1 was expressed at the cell surface but did not self-associate. In contrast, a receptor fragment containing only the N-terminal extracellular domain and TM1 could self-associate and heterodimerize with wild type receptors. Analysis of other mutants suggested that oligomerization is facilitated by the N-terminal extracellular domain and TM2. Therefore, the N-terminal extracellular domain, TM1, and TM2 appear to stabilize alpha-factor receptor oligomers. These domains may form an interface in contact or domain-swapped oligomers. Similar domains may mediate dimerization of certain mammalian GPCRs.     

Activated cardiac adenosine A(1) receptors translocate out of caveolae

The cardiac affects of the purine nucleoside, adenosine, are well known. Adenosine increases coronary blood flow, exerts direct negative chronotropic and dromotropic effects, and exerts indirect anti-adrenergic effects. These effects of adenosine are mediated via the activation of specific G protein-coupled receptors. There is increasing evidence that caveolae play a role in the compartmentalization of receptors and second messengers in the vicinity of the plasma membrane. Several reports demonstrate that G protein-coupled receptors redistribute to caveolae in response to receptor occupation. In this study, we tested the hypothesis that adenosine A(1) receptors would translocate to caveolae in the presence of agonists. Surprisingly, in unstimulated rat cardiac ventricular myocytes, 67 +/- 5% of adenosine A(1) receptors were isolated with caveolae. However, incubation with the adenosine A(1) receptor agonist 2-chlorocyclopentyladenosine induced the rapid translocation of the A(1) receptors from caveolae into non-caveolae plasma membrane, an effect that was blocked by the adenosine A(1) receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine. An adenosine A(2a) receptor agonist did not alter the localization of A(1) receptors to caveolae. These data suggest that the translocation of A(1) receptors out of caveolae and away from compartmentalized signaling molecules may explain why activation of ventricular myocyte A(1) receptors are associated with few direct effects.     

Selective Reduction in A2 Adenosine Receptor Desensitization Following Antisense-Induced Suppression of G Protein-Coupled Receptor Kinase 2 Expression

Phosphorylation of G protein-coupled receptors (GPCRs) by GPCR kinases (GRKs) is considered to play a critical role in the desensitization of responses mediated by these receptors. To explore the role of GRK2 in A2 adenosine receptor desensitization, we attempted to reduce specifically GRK2 expression in NG108-15 cells by stable transfection with an antisense rat GRK2 cDNA sequence. This yielded up to a 69% loss of GRK2 when compared with plasmid-transfected control cells, which correlated with a reduction in kinase activity when measured by the ability of cell lysates to promote light-dependent phosphorylation of rhodopsin. Levels of GRK3 were the same in antisense and plasmid-transfected controls. On addition of the A2 adenosine receptor agonist 5'-(N-ethylcarboxamido)adenosine, cyclic AMP accumulation was greater in GRK2 antisense cells as compared with plasmid control cells. In contrast, cyclic AMP accumulation via agonist stimulation of either IP-prostanoid or secretin receptors or by addition of forskolin was not significantly different among all clones examined. The increase in A2 adenosine receptor response could not be explained by changes in A2A adenosine receptor expression, as assessed by ligand binding experiments with the radioligand 2-3H-labelled 4-[2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-++ +ylamino]ethyl]phenol ([3H]ZM241385). These data show for the first time a direct correlation between expression of GRK2 and desensitization of natively expressed A2 adenosine receptors in intact cells, suggesting that GRK2 plays a major role in the regulation of these receptors. Key Words: G protein-coupled receptor kinase-G protein-coupled receptor-Antisense-NG108-15 cells-A2 adenosine receptors-Desensitization.     

Signalling from adenosine receptors to mitogen-activated protein kinases

The purine nucleoside adenosine acts via four distinct adenosine receptor subtypes: the adenosine A(1), A(2A), A(2B), and A(3) receptor. They are all G protein-coupled receptors (GPCR) coupling to classical second messenger pathways such as modulation of cAMP production or the phospholipase C (PLC) pathway. In addition, they couple to mitogen-activated protein kinases (MAPK), which could give them a role in cell growth, survival, death and differentiation. Although each of the adenosine receptors can activate one or more of the MAPKs, the mechanisms appear to differ substantially, both between receptor subtypes in the same cell type and between the same receptor in different cell types.     

Molecular cloning and expression of an adenosine A2b receptor from human brain.

A novel receptor cDNA was isolated from a human hippocampal cDNA library. The encoded polypeptide contains structural features consistent with its classification as a G protein-coupled receptor and shares 45% homology with the human A1 and A2a adenosine receptors. Chinese hamster ovary K1 cells expressing this receptor showed marked stimulation of adenylate cyclase when treated with 1mM adenosine. There was no response to ligands selective for A1 and A2a receptors but the general adenosine agonist N-ethylcarboxyamidoadenosine (NECA) caused a 10 fold increase in cyclic AMP accumulation with an EC50 of approximately 0.9 microM. This effect was inhibited by the adenosine receptor antagonist theophylline. Specific binding of A1 and A2a selective agonists and NECA was not detected. It is proposed that the novel receptor is a human brain adenosine A2b receptor subtype.     

Adenosine inhibits calcium channel currents via A1 receptors on salamander retinal ganglion cells in a mini-slice preparation

The effects of adenosine on high-voltage-activated calcium channel currents in tiger salamander retinal ganglion cells were investigated in a mini-slice preparation. Adenosine produced a concentration-dependent decrease in the amplitude of calcium channel current with a maximum inhibition of 26%. The effects of adenosine on calcium channel current were both time- and voltage-dependent. In cells dialyzed with GTP-gamma-s, adenosine caused a sustained and irreversible inhibition of calcium channel current, suggesting involvement of a GTP-binding protein. The inhibitory effect of adenosine on calcium channel current was blocked by the A1 antagonist 8-cyclopentyltheophylline (DPCPX, 1-10 microm), but not by the A2 antagonist 3-7-dimethyl-1-propargylxanthine (DMPX, 10 microm), and was mimicked by the A1 agonist N6-cyclohexyladenosine (CHA, 1 microm) but not by the A2 agonist 5'-(N-cyclopropyl) carbox-amidoadenosine (CPCA, 1 microm). Adenosine's inhibition of calcium channel current was not affected by the L-type calcium channel blocker nifedipine (5 microm). However, adenosine's inhibition of calcium channel current was reduced to approximately 10% after application of omega-conotoxin GVIA (1 microm), suggesting that adenosine inhibits N-type calcium channels. These results show that adenosine acts on an A1 adenosine receptor subtype via a G protein-coupled pathway to inhibit the component of calcium channel current carried in N-type calcium channels.     

Signaling through a G Protein-coupled receptor and its corresponding G protein follows a stoichiometrically limited model

The bradykinin receptor is a G protein-coupled receptor (GPCR) that is coupled to the Galpha(q) family of heterotrimeric G proteins. In general, a GPCR can exert intracellular signals either by transiently associating with multiple diffusing G protein subunits or by activating a G protein that is stably bound to the receptor, thus generating a signal that is limited by the stoichiometry of the complex. Here we have distinguished between these models by monitoring the association of type 2 bradykinin receptor (B(2)R) and the Galpha(q)/Gbetagamma heterotrimer in living human embryonic kidney 293 cells expressing fluorescent-tagged proteins. Stable B(2)R-Galpha(q) x Gbetagamma complexes are observed in resting cells by fluorescence resonance energy transfer from either Galpha(q)-eCFP or eCFP-Gbetagamma to B(2)R-eYFP. Stimulating the cells with bradykinin causes detachment of B(2)R from the G protein subunits as the receptor internalizes into early endosomes, with a corresponding elimination of B(2)R-G protein fluorescence resonance energy transfer because Galpha(q) and its associated Gbetagamma remain on the plasma membrane. Single point and scanning fluorescence correlation spectroscopy measurements show that a portion of B(2)R molecules diffuses with a mobility corresponding to dimers or small oligomers, whereas a second fraction diffuses in higher order molecular assemblies. Our studies support a model in which receptors are pre-coupled with their corresponding G proteins in the basal state of cells thereby limiting the response to an external signal to a defined stoichiometry that allows for a rapid and directed cellular response.     

G protein-coupled receptor oligomerization for what?

Although the G protein-coupled receptor (GPCR) oligomerization has been questioned during the last decade, under some premises the existence of a supramolecular organization of these receptors begins now to be widely accepted by the scientific community. Indeed, GPCR oligomers may enhance the diversity and performance by which extracellular signals are transferred to the G proteins in the process of receptor transduction, although the mechanism that underlie this phenomenon remains still unexplained. Recently, a trans-conformational switching model has been proposed as a mechanism allowing direct inhibition of receptor activation. Thus, heterotropic receptor–receptor allosteric regulations are behind the GPCR oligomeric function. Accordingly, we revise here how GPCR oligomerization impinge in several important receptor functions like biosynthesis, plasma membrane diffusion or velocity, pharmacology and signaling. Overall, the rationale of receptor oligomerization might lie in the cellular need of sensing complex extracellular signals and to translate into a simple computational mode.