The functional role of heteromers of G-protein-coupled receptors is a matter of debate. In the present study, we demonstrate that

Adenosine and dopamine signaling exert opposite effects in the basal ganglia, a brain region involved in sensory-motor integration. Thus, adenosine agonists induce motor depression and adenosine antagonists, such as caffeine, produce motor activation (1). These opposite effects result from specific antagonistic interactions between subtypes of adenosine and dopamine receptors in the striatum, the main input structure of the basal ganglia. In fact, striatal dopamine receptors and, to some extent, adenosine receptors are segregated in the two main populations of ␥-aminobutyric acid (GABA) efferent neurons. EXPERIMENTAL PROCEDURESCell Cultures-Maintenance of SH-SY5Y cells (parental and D 2 Rtransfected cells) as well as the pharmacological characterization and maintenance of D 2 R-and D 1 R-transfected mouse fibroblast Ltk Ϫ cells are described in detail elsewhere (7-9). For primary cultures, striata were removed from 16-day-old Sprague-Dawley rat embryos (B&K Universal) in Ca 2ϩ /Mg 2ϩ -free PBS supplemented with 20 units/ml penicillin and 20 g/ml streptomycin (Invitrogen). The tissue fragments were pooled and mechanically dissociated in SFM Neurobasal serum-free medium (Invitrogen), supplemented with B27 (Invitrogen), glutamine (2 mM; Invitrogen), penicillin/streptomycin (20 units/ml/20 g/ml; Invitrogen), and ␤-mercaptoethanol (25 M) (Invitrogen). Cells were collected by centrifugation at 100 ϫ g for 5 min and resuspended in fresh medium. The resulting single-cell suspension was seeded on 24-well plates coated with gelatin (Sigma) and poly-L-lysine (Sigma), and cells were grown at 37°C in saturation humidity with 5% CO 2 .Immunolabeling Experiments-Neuroblastoma cells were grown on glass coverslips coated with poly-L-lysine (Sigma) and exposed to vari-* This work was

Adenosine A 2A receptor (A 2A R)-dopamine D 2 receptor (D 2 R) heteromers are key modulators of striatal neuronal function. It has been suggested that the psychostimulant effects of caffeine depend on its ability to block an allosteric modulation within the A 2A R-D 2 R heteromer, by which adenosine decreases the affinity and intrinsic efficacy of dopamine at the D 2 R. We describe novel unsuspected allosteric mechanisms within the heteromer by which not only A 2A R agonists, but also A 2A R antagonists, decrease the affinity and intrinsic efficacy of D 2 R agonists and the affinity of D 2 R antagonists. Strikingly, these allosteric modulations disappear on agonist and antagonist coadministration. This can be explained by a model that considers A 2A R-D 2 R heteromers as heterotetramers, constituted by A 2A R and D 2 R homodimers, as demonstrated by experiments with bioluminescence resonance energy transfer and bimolecular fluorescence and bioluminescence com- Most evidence indicates that G protein-coupled receptors (GPCRs) form homodimers and heteromers. Homodimers seem to be a predominant species, and oligomeric entities can be viewed as multiples of dimers (1). It has been proposed that GPCR heteromers are constituted mainly by heteromers of homodimers (1, 2). Allosteric mechanisms determine a multiplicity of unique pharmacologic properties of GPCR homodimers and heteromers (1, 3). First, binding of a ligand to one of the receptors in the heteromer can modify the affinity of ligands for the other receptor (1, 3, 4). The most widely reproduced allosteric modulation of ligand-binding properties in a GPCR heteromer is the ability of adenosine A 2A receptor (A 2A R) agonists to decrease the affinity of dopamine D 2 receptor (D 2 R) agonists in the A 2A R-D 2 R heteromer (5). A 2A R-D 2 R heteromers have been revealed both in transfected cells (6, 7), striatal neurons in culture (6,8) and in situ, in mammalian striatum (9, 10), where they play an important role in the modulation of GABAergic striatopallidal neuronal function (9, 11).In addition to ligand-binding properties, unique properties for each GPCR oligomer emerge in relation to the varying intrinsic efficacy of ligands for different signaling pathways (1-3). Intrinsic efficacy refers to the power of the agonist to induce a functional response, independent of its affinity for the receptor. Thus, allosteric modulation of an agonist can potentially involve changes in affinity and/or intrinsic efficacy (1, 3). This principle can be observed in the A 2A R-D 2 R heteromer, where a decrease in D 2 R agonist affinity cannot alone explain the ability of an A 2A R agonist to abolish the decreased excitability of GABAergic striatopallidal neurons induced by high concentration of a D 2 R agonist (9), which should overcome the decrease in affinity. Furthermore, a differential effect of allosteric modulations of different agonist-mediated signaling responses (i.e., functional selectivity) can occur within GPCR heteromers (1, 2, 8 It has been hypothesized that the allos...

Adenosine deaminase (ADA), a protein whose deficit leads to severe combined immunodeficiency, binds to the cell surface by means of either CD26, A 1 adenosine receptors, or A2B adenosine receptors. The physiological role of these interactions is not well understood. Our results show that by a 3-fold reduction in the EC 50 for the antigen, ADA potentiated T cell proliferation in autologous cocultures with antigen-pulsed immature or mature dendritic cells. Costimulation was not due to the enzymatic activity but to the interaction of ADA-CD26 complexes in T cells with an ADAanchoring protein in dendritic cells. From colocalization studies, it is deduced that ADA colocalizing with adenosine receptors on dendritic cells interact with CD26 expressed on lymphocytes. This costimulatory signal in the immunological synapse leads to a marked increase (3-to 34-fold) in the production of the T helper 1 and proimmflamatory cytokines IFN-␥, TNF-␣, and IL-6. adenosine deaminase ͉ costimulation ͉ immunosynapse A denosine deaminase (ADA; EC 3.5.4.4) an enzyme involved in purine metabolism, catalyzes the hydrolytic deamination of adenosine or 2Ј-deoxyadenosine to inosine or 2Ј-deoxyinosine and ammonia. Congenital defect of ADA causes severe combined immunodeficiency, which is characterized by the absence of functional T and B lymphocytes in affected individuals (1). For many years, ADA was considered to be cytosolic, but it has been found on the cell surface of many cell types; therefore, it can be considered an ectoenzyme. In addition, ecto-ADA has been proposed to have a catalytic-independent function as a costimulatory molecule in lymphocytes (2).So far, two types of surface anchoring proteins for ecto-ADA have been described. The first type, with only one member, is CD26, a multifunctional protein of 110 KDa strongly expressed on epithelial cells (kidney proximal tubules, intestine, and bile duct) and on several types of endothelial cells and fibroblasts and on leukocyte subsets (3-5). The second type of ecto-ADA-binding proteins includes the adenosine receptors (AR) A 1 (A 1 R) (6) and A 2B (A 2B R) (7). The association between ADA and CD26 on the T cell surface has been proposed to have a costimulatory function during T cell antigen receptor-CD3 complex engagement (2). Because CD26 has a short cytoplasmatic tail, it needs partners to transduce the signal. Ishii et al. (8) have described that CD26-mediated signaling occurs through its association with CD45RO. At present, it is not known whether ADA generates a signal when it binds to AR. However, we have previously demonstrated that ADA binding to A 1 R or A 2B R is required for high efficiency affinity binding of the agonist and for efficient agonist-dependent signaling (6, 7).Dendritic cells (DC) are the most potent antigen-presenting cells (APC) specialized in the initiation of immune responses by directing the activation and differentiation of naïve T lymphocytes (9, 10). Immature DC (iDC) reside in most tissues to uptake antigen; they are engaged when exposed to danger ...

Identification of higher-order oligomers in the plasma membrane is essential to decode the properties of molecular networks controlling intercellular communication. We combined bioluminescence resonance energy transfer (BRET) and fluorescence resonance energy transfer (FRET) in a technique called sequential BRET-FRET (SRET) that permits identification of heteromers formed by three different proteins. In SRET, the oxidation of a Renilla luciferase (Rluc) substrate by an Rluc fusion protein triggers acceptor excitation of a second fusion protein by BRET and subsequent FRET to a third fusion protein. We describe two variations of SRET that use different Rluc substrates with appropriately paired acceptor fluorescent proteins. Using SRET, we identified complexes of cannabinoid CB(1), dopamine D(2) and adenosine A(2A) receptors in living cells. SRET is an invaluable technique to identify heteromeric complexes of more than two neurotransmitter receptors, which will allow us to better understand how signals are integrated at the molecular level.

It is well known that cocaine blocks the dopamine transporter. This mechanism should lead to a general increase in dopaminergic neurotransmission, and yet dopamine D 1 receptors (D 1 Rs) play a more significant role in the behavioral effects of cocaine than the other dopamine receptor subtypes. Cocaine also binds to σ-1 receptors, the physiological role of which is largely unknown. In the present study, D 1 R and σ 1 R were found to heteromerize in transfected cells, where cocaine robustly potentiated D 1 R-mediated adenylyl cyclase activation, induced MAPK activation per se and counteracted MAPK activation induced by D 1 R stimulation in a dopamine transporterindependent and σ 1 R-dependent manner. Some of these effects were also demonstrated in murine striatal slices and were absent in σ 1 R KO mice, providing evidence for the existence of σ 1 R-D 1 R heteromers in the brain. Therefore, these results provide a molecular explanation for which D 1 R plays a more significant role in the behavioral effects of cocaine, through σ 1 R-D 1 R heteromerization, and provide a unique perspective toward understanding the molecular basis of cocaine addiction.receptor heteromer | drug addiction A key molecular mechanism contributing to the development of addiction by drugs of abuse consist of the increase of the extracellular levels of dopamine in the striatum, particularly in its ventral portion, the nucleus accumbens (1, 2). Cocaine causes a rapid and strong increase in striatal extracellular dopamine by its ability to bind with high affinity to the dopamine transporter (DAT) and to inhibit its function (3-5). In the striatum, dopamine signaling is mediated mainly by dopamine D 1 and D 2 receptors (D 1 Rs and D 2 Rs, respectively), which are mostly segregated in two phenotypically different subtypes of GABAergic medium-sized spiny neurons (MSNs) (6). Activation of D 1 Rs is an absolute requirement for the induction of many of the cellular and behavioral responses to cocaine, as deduced from studies performed in D 1 R KO mice and from experiments with transgenic mice in which D 1 R-or D 2 R-expressing MSNs are visualized by the expression of fluorescent proteins (7-11).The σ-1 receptor, originally proposed as a subtype of opioid receptors, is now considered to be a nonopioid receptor with two transmembrane domains, one extracellular loop and cytosolic N and C termini (12). The σ 1 R is highly expressed in the brain, including the striatum, and its association with neurons is well established (12, 13). However, its biological function and even its main endogenous neurotransmitter remain enigmatic (12). Cocaine interacts with σ 1 Rs at pharmacologically relevant concentrations (12,14). In fact, reducing brain σ 1 R levels with antisense oligonucleotides attenuates the convulsive and locomotor stimulant actions of cocaine (15, 16), and σ 1 R antagonists mitigate the actions of cocaine in animal models (12,14). A recent study showed that σ 1 R agonists not only potentiate the reinforcing effects of cocaine, but they may be self...

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The results presented in this paper show that adenosine A 2A receptor (A 2A R) form homodimers and that homodimers but not monomers are the functional species at the cell surface. Fluorescence resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET) techniques have been used to demonstrate in transfected HEK293 cells homodimerization of A 2A R, which are heptaspanning membrane receptors with enriched expression in striatum. The existence of homodimers at the cell surface was demonstrated by time-resolved FRET. Although agonist activation of the receptor leads to the formation of receptor clusters, it did not affect the degree of A 2A R-A 2A R dimerization. Both monomers and dimers were detected by immunoblotting in cell extracts. However, cell surface biotinylation of proteins has made evident that more than 90% of the cell surface receptor is in its dimeric form. Thus, it seems that homodimers are the functional form of the receptor present on the plasma membrane.A deletion mutant version of the A 2A receptor, lacking its C-terminal domain, was also able to form both monomeric and dimeric species when cell extracts from transfected cells were analyzed by immunoblotting. This suggests that the C-terminal tail does not participate in the dimerization. This is relevant as the C-terminal tail of A 2A R is involved in heteromers formed by A 2A R and dopamine D2 receptors. BRET ratios corresponding to A 2A R-A 2A R homodimers were higher than those encountered for heterodimers formed by A 2A R and dopamine D2 receptors. As A 2A R and dopamine D2 receptors do indeed interact, these results indicate that A 2A R homodimers are the functional species at the cell surface and that they coexist with A 2A R/D2 receptor heterodimers. Keywords: 7TM receptors, dopamine receptors, G proteincoupled receptors, heteromerization, homomerization, receptor-receptor interactions. Heptaspanning membrane receptors (HSMRs) also known as G protein-coupled receptors (GPCRs) were previously considered to be monomeric proteins which only interacted with G proteins. However it has become clear that HSMRs are oligomeric structures formed by receptor homodimers, heterodimers and multimers and a variety of proteins interacting at the horizontal and the vertical level (Agnati et al. 2002Bouvier, 2001;Milligan and White 2001;Rios et al. 2001;Franco et al. 2003