Dopamine D4 receptor, but not the ADHD-associated D4.7 variant, forms functional heteromers with the dopamine D2S receptor in the brain.

Polymorphic variants of the dopamine D(4) receptor have been consistently associated with attention-deficit hyperactivity disorder (ADHD). However, the functional significance of the risk polymorphism (variable number of tandem repeats in exon 3) is still unclear. Here, we show that whereas the most frequent 4-repeat (D(4.4)) and the 2-repeat (D(4.2)) variants form functional heteromers with the short isoform of the dopamine D(2) receptor (D(2S)), the 7-repeat risk allele (D(4.7)) does not. D(2) receptor activation in the D(2S)-D(4) receptor heteromer potentiates D(4) receptor-mediated MAPK signaling in transfected cells and in the striatum, which did not occur in cells expressing D(4.7) or in the striatum of knockin mutant mice carrying the 7 repeats of the human D(4.7) in the third intracellular loop of the D(4) receptor. In the striatum, D(4) receptors are localized in corticostriatal glutamatergic terminals, where they selectively modulate glutamatergic neurotransmission by interacting with D(2S) receptors. This interaction shows the same qualitative characteristics than the D(2S)-D(4) receptor heteromer-mediated mitogen-activated protein kinase (MAPK) signaling and D(2S) receptor activation potentiates D(4) receptor-mediated inhibition of striatal glutamate release. It is therefore postulated that dysfunctional D(2S)-D(4.7) heteromers may impair presynaptic dopaminergic control of corticostriatal glutamatergic neurotransmission and explain functional deficits associated with ADHD.

Real-time G-protein-coupled receptor imaging to understand and quantify receptor dynamics.

Understanding the trafficking of G-protein-coupled receptors (GPCRs) and their regulation by agonists and antagonists is fundamental to develop more effective drugs. Optical methods using fluorescent-tagged receptors and spinning disk confocal microscopy are useful tools to investigate membrane receptor dynamics in living cells. The aim of this study was to develop a method to characterize receptor dynamics using this system which offers the advantage of very fast image acquisition with minimal cell perturbation. However, in short-term assays photobleaching was still a problem. Thus, we developed a procedure to perform a photobleaching-corrected image analysis. A study of short-term dynamics of the long isoform of the dopamine type 2 receptor revealed an agonist-induced increase in the mobile fraction of receptors with a rate of movement of 0.08 µm/s For long-term assays, the ratio between the relative fluorescence intensity at the cell surface versus that in the intracellular compartment indicated that receptor internalization only occurred in cells co-expressing G protein-coupled receptor kinase 2. These results indicate that the lateral movement of receptors and receptor internalization are not directly coupled. Thus, we believe that live imaging of GPCRs using spinning disk confocal image analysis constitutes a powerful tool to study of receptor dynamics.

G-protein-coupled receptor heteromers: function and ligand pharmacology.

Almost all existing models for G-protein-coupled receptors (GPCRs) are based on the occurrence of monomers. Recent studies show that many GPCRs are dimers. Therefore for some receptors dimers and not monomers are the main species interacting with hormones/neurotransmitters/drugs. There are reasons for equivocal interpretations of the data fitting to receptor dimers assuming they are monomers. Fitting data using a dimer-based model gives not only the equilibrium dissociation constants for high and low affinity binding to receptor dimers but also a 'cooperativity index' that reflects the molecular communication between monomers within the dimer. The dimer cooperativity index (D(C)) is a valuable tool that enables to interpret and quantify, for instance, the effect of allosteric regulators. For different receptors heteromerization confers a specific functional property for the receptor heteromer that can be considered as a 'dimer fingerprint'. The occurrence of heteromers with different pharmacological and signalling properties opens a complete new field to search for novel drug targets useful to combat a variety of diseases and potentially with fewer side effects. Antagonists, which are quite common marketed drugs targeting GPCRs, display variable affinities when a given receptor is expressed with different heteromeric partners. This fact should be taken into account in the development of new drugs.

The heat shock cognate protein hsc73 assembles with A(1) adenosine receptors to form functional modules in the cell membrane.

A(1) adenosine receptors (A(1)Rs) are G protein-coupled heptaspanning receptors that interact at the outer face of the plasma membrane with cell surface ecto-adenosine deaminase (ecto-ADA). By affinity chromatography the heat shock cognate protein hsc73 was identified as a cytosolic component able to interact with the third intracellular loop of the receptor. As demonstrated by surface plasmon resonance, purified A(1)Rs interact specifically with hsc73 with a dissociation constant in the nanomolar range (0.5 +/- 0.1 nM). The interaction between hsc73 and A(1)R led to a marked reduction in the binding of the ligands and prevented activation of G proteins, as deduced from (35)S-labeled guanosine-5'-O-(3-thio)triphosphate binding assays. Interestingly this effect was stronger than that exerted by guanine nucleotide analogs, which uncouple receptors from G proteins, and was completely prevented by ADA. As assessed by immunoprecipitation a high percentage of A(1)Rs in cell lysates are coupled to hsc73. A relatively high level of colocalization between A(1)R and hsc73 was detected in DDT(1)MF-2 cells by means of confocal microscopy, and no similar results were obtained for other G protein-coupled receptors. Colocalization between hsc73 and A(1)R was detected in specific regions of rat cerebellum and in the body of cortical neurons but not in dendrites or synapses. Remarkably, agonist-induced receptor internalization leads to the endocytosis of A(1)Rs by two qualitatively different vesicle types, one in which A(1)R and hsc73 colocalize and another in which hsc73 is absent. These results open the interesting possibility that signaling via G protein-coupled receptors may be regulated by heat shock proteins.

Cell surface adenosine deaminase: much more than an ectoenzyme.

During the last 10 years, adenosine deaminase (ADA), an enzyme considered to be cytosolic, has been found on the cell surface of many cells, therefore it can be considered an ectoenzyme. EctoADA, which seems to be identical to intracellular ADA and has a globular structure, does not interact with membranes but with membrane proteins. Two of these cell surface receptors for ectoADA have been identified: CD26 and A1 adenosine receptors (A1R). Apart from degradation of extracellular adenosine another functional role of ectoADA has been assigned. EctoADA is able to transmit signals when interacting with either CD26 or A1R. In this way, it acts as a co-stimulatory molecule which facilitates a variety of specific signalling events in different cell types. The heterogeneous distribution of the enzyme in the nervous system indicates that ectoADA may be a neuroregulatory molecule. On the other hand, ectoADA might act as a bridge between two different cells thus raising the possibility that it may be important for the development of the nervous system.

Kinetic mechanism of the hydroxypyruvate-lactate dehydrogenase-NADH system.

Chicken liver lactate dehydrogenase L-lactate : NAD+ oxidoreductase, EC1.1.1.27) reversibly catalyses the conversion of hydroxypyruvate to L-glycerate. The variation of the initial reaction rate with the substrate or coenzyme (NADH) concentration together with the inhibition caused by the reaction products and excess substrates, reveal that the kinetic mechanism of the reaction, with hydroxypyruvate as substrate, is of the rapid-equilibrium, ordered-ternary-complex type; NADH is the first substrate in the reaction sequence. Rate equations have been developed for the hydroxypyruvate.E.NADH system without inhibitors, with excess substrates, and with reaction products. Comparison of the rate equations obtained with those calculated theoretically from an ordered-ternary-complex mechanism reveals the existence of E.NAD.NADH,E.NAD-hydroxypyruvate and E.hydroxypyruvate complexes.

Kinetic formulations for the oxidation and the reduction of glyoxylate by lactate dehydrogenase.

Chicken liver lactate dehydrogenase (L-lactate : NAD+ oxidoreductase, EC 1.1.1.27) irreversibly catalyses the oxidation of glyoxylate (hydrated form) (I) to oxalate (pH = 9.6) and the reduction of (non-hydrated form) (II) to glycolate (pH = 7.4). (I) attaches to the enzyme in the pyruvate binding site and (II) attaches to the enzyme at the L-lactate binding site. The oxidation of (I) (pH = 9.6) is adapted to the following mechanism: (see book). The abortive complexes, E-NADH-I and E-NAD+-II, are responsible for the inhibition by excess substrate in the reduction and oxidation systems, respectively. When lactate dehydrogenase and NAD+ are preincubated, E-NAD+- NAD+ appears and causes inhibition by excess NAD+ in the glyoxylate-lactate dehydrogenase-NAD+ and L-lactate-lactate dehydrogenase-NAD+ systems; the second NAD+ molecule attaches to the enzyme at the L-lactate binding site.

Relationship between hydroxypyruvate and the production of oxalate in vitro.

Chicken liver lactate dehydrogenase (L-lactate:NAD+ oxidoreductase, EC1.1.1.27) catalyses the reversible reduction reaction of hydroxypyruvate to L-glycerate. It also catalyses the oxidation reaction of the hydrated form of glyoxylate to oxalate and the reduction of the non-hydrated form of glyoxylate to oxalate and the reduction of the non-hydrated form to glycolate. At pH 8, these latter two reactions are coupled. The coupled system equilibrium is attained when the NAD+/NADH ratio is greater than unity. Hydroxypyruvate binds to the enzyme at the same site as the pyruvate. When there are substances with greater affinity to this site in the reaction medium and their concentration is very high, hydroxypyruvate binds to the enzyme at the L-lactate site. In vitro and with purified preparation of lactate dehydrogenase, hydroxypyruvate stimulates the production of oxalate from glyoxylate-hydrated form and from NAD; the effect is due to the fact that hydroxypyruvate prevents the binding of non-hydrated form of glyoxylate to the lactate dehydrogenase in the pyruvate binding site. At pH 8, THE L-glycerate stimulates the production of glycolate from glyoxylate-non-hydrated form and NADH since hydroxypyruvate prevents the binding of glyoxylate-hydrated form to the enzyme

Effect of salts on D-glycerate dehydrogenase kinetic behavior.

Bovine liver D-glycerate dehydrogenase (D-glycerate:NAD (NADP) oxidoreductase, EC 1.1.1.29) adapts its kinetic behaviour to a sequential mechanism. The presence of NaCl causes an appreciable variation in the Km and V values. relative to the both substrates in the hydroxypyruvate/D-glycerate dehydrogenase/NADH system, which does not happen in the D-glycerate/D-glycerate dehydrogenase/NAD system. The former system is inhibited by high concentrations of NaCl and activated by low salt concentrations. The hydroxypyruvate concentration causing substrate inhibition increases as the concentration of NaCl increases; excess NADH inhibition is independent of the salt concentration. The variation of the initial rates of both systems, in the presence of chlorides having monovalent and divalent cations, or sodium halides, Na2SO4 and NaNO3 (at constant ionic strength) suggests that the anions have a specific action on the enzyme. An increase in the NaCl concentration causes a displacement of the optimum D-glycerate dehydrogenase pH (with hydroxypyruvate and NADH as substrates) towards the acid area. The enzyme stability, at varying pH, varies with the salt concentration.

Solubilization and molecular characterization of the nitrobenzylthioinosine binding sites from pig kidney brush-border membranes.

The nitrobenzylthioinosine binding sites from luminal membranes of proximal tubule of pig kidney were solubilized by treatment of the brush-border membrane vesicles with the zwitterionic detergent CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate) in 2% solution. The high yield solubilization of a stable form of the transporter took place in the presence of adenosine in the medium of incubation with the detergent and the additional presence of glycerol as stabilizer. The solubilization of the NBTI-sensitive nucleoside transporter from pig kidney brush-border membranes did not change the nitrobenzylthioinosine (NBTI) binding characteristics; the only major change was a 3-fold decrease in the affinity. The carrier molecule was cross-linked to [3H]NBTI and by electrophoretic characterization under reducing conditions it displayed a molecular mass of 65 kDa. Treatment of the samples at low temperature prior to electrophoresis gave rise to the appearance of further bands corresponding to dimeric and tetrameric forms which interacted non-covalently. The removal of the N-linked oligosaccharides by treatment with endoglycosidase F shifted the molecular mass to 57 kDa. The chromatographic behaviour of the solubilized transporter was similar to that of human erythrocytes and differed from that found in pig erythrocytes. Since the molecular mass of the monomer before and after treatment with endoglycosidase F is the same for pig erythrocytes and pig kidney luminal membranes, the different chromatographic behaviour might result from tissue differences due to transcriptional variations or to posttranscriptional modifications of the transporter molecule.

Dipropylcyclopentylxanthine triggers apoptosis in Jurkat T cells by a receptor-independent mechanism.

1,3-Dipropyl-8-cyclopentylxanthine (DPCPX), a xanthine analog used as selective antagonist of adenosine receptors, caused apoptosis in a human leukemia T cell line. Jurkat cells treated with DPCPX underwent apoptosis as demonstrated by flow cytometry, by DNA fragmentation and by accumulation of histones, H2A, H2B, H3 and H4, in the nucleoplasm of cells. Cell cycle and cell sorting analyses indicated an arrest of cells in G(2)/M followed by the appearance of apoptotic cells in G(1) and G(2)/M phases. The mechanism of programmed cell death does not seem to be mediated by signal transduction events at the plasma membrane since it did not involve activation of cell membrane receptors and modification of the intracellular levels of Ca(2+) or cAMP. Apoptosis by incorporation into DNA of a derivative of DPCPX is suggested in basis of the presence of radioactivity label in the DNA obtained from cells preincubated with [(3)H]DPCPX.

Cytokines regulate membrane adenosine deaminase on human activated lymphocytes.

CD26 is a lymphocyte marker that can anchor adenosine deaminase (ADA) on the T cell surface. We found that ADA is regulated by cytokines on the cell surface during T cell activation. By means of flow cytometry, immunofluorescence, and immunoblotting techniques, we found that interleukin (IL)-2 and IL-12 up-regulate ecto-ADA and CD26 expression. In clear contrast, IL-4 led to down-regulation of lymphocyte surface ADA without modifying the level of CD26. Moreover, neither circulating ADA transcription nor mRNA translation was regulated by cytokines. These results, along with absence of total-ADA modulation, the variable amount of ADA found in purified plasma membranes, and the different effect of Brefeldin A on the surface presence of ADA and CD26 indicated that cytokines regulate the translocation of ADA towards the cell surface through a mechanism not involving CD26. Ecto-ADA protected activated lymphocytes from the toxic effects of extracellular adenosine. Therefore, this cell surface ADA control might constitute part of the fine immunoregulatory mechanism of adenosine-mediated signaling through purinergic receptors in leukocytes.

The binding of [3H]R-PIA to A1 adenosine receptors produces a conversion of the high- to the low-affinity state.

Kinetic evidence for negative cooperativity on the binding of [3H]R-PIA to A1 adenosine receptors was obtained from dissociation experiments at different ligand concentrations and from the equilibrium isotherm. The dissociation curves indicate that there is an apparent ligand-induced transformation of high- to low-affinity states of the receptor. At concentrations of 18.2 nM R-PIA or higher there was only found the low-affinity state of the receptor. In view of these results equilibrium binding data were analyzed by the usual two-state model (assuming that there is an interconversion between them) and by the negative cooperativity model employing the Hill equation.

The HIV-1 gp120 inhibits the binding of adenosine deaminase to CD26 by a mechanism modulated by CD4 and CXCR4 expression.

HIV-1 external envelope glycoprotein gp120 inhibits adenosine deaminase (ADA) binding to its cell surface receptor in lymphocytes, CD26, by a mechanism that does not require the gp120-CD4 interaction. To further characterize this mechanism, we studied ADA binding to murine clones stably expressing human CD26 and/or human CD4, and transiently expressing human CXCR4. In this heterologous model, we show that both recombinant gp120 and viral particles from the X4 HIV-1 isolate IIIB inhibited the binding of ADA to wild-type or catalytically inactive forms of CD26. In cells lacking human CXCR4 expression, this gp120-mediated inhibition of ADA binding to human CD26 was completely dependent on the expression of human CD4. In contrast, when cells were transfected with human CXCR4 the inhibitory effect of gp120 was significantly enhanced and was not blocked by anti-CD4 antibodies. These data suggest that the interaction of gp120 with CD4 or CXCR4 is required for efficient inhibition of ADA binding to CD26, although in the presence of CXCR4 the interaction of gp120 with CD4 may be dispensable.

Adenosine deaminase affects ligand-induced signalling by interacting with cell surface adenosine receptors.

Adenosine deaminase (ADA) is not only a cytosolic enzyme but can be found as an ecto-enzyme. At the plasma membrane, an adenosine deaminase binding protein (CD26, also known as dipeptidylpeptidase IV) has been identified but the functional role of this ADA/CD26 complex is unclear. Here by confocal microscopy, affinity chromatography and coprecipitation experiments we show that A1 adenosine receptor (A1R) is a second ecto-ADA binding protein. Binding of ADA to A1R increased its affinity for the ligand thus suggesting that ADA was needed for an effective coupling between A1R and heterotrimeric G proteins. This was confirmed by the fact that ASA, independently of its catalytic behaviour, enhanced the ligand-induced second messenger production via A1R. These findings demonstrate that, apart from the cleavage of adenosine, a further role of ecto-adenosine deaminase on the cell surface is to facilitate the signal transduction via A1R.

Evidence for adenosine/dopamine receptor interactions: indications for heteromerization.

Evidence has been obtained for adenosine/dopamine interactions in the central nervous system. There exists an anatomical basis for the existence of functional interactions between adenosine A(1)R and dopamine D(1)R and between adenosine A(2A) and dopamine D(2) receptors in the same neurons. Selective A(1)R agonists affect negatively the high affinity binding of D(1) receptors. Activation of A(2A) receptors leads to a decrease in receptor affinity for dopamine agonists acting on D(2) receptors, specially of the high-affinity state. These interactions have been reproduced in cell lines and found to be of functional significance. Adenosine/dopamine interactions at the behavioral level probably reflect those found at the level of dopamine receptor binding and transduction. All these findings suggest receptor subtype-specific interactions between adenosine and dopamine receptors that may be achieved by molecular interactions (e.g., receptor heterodimerization). At the molecular level adenosine receptors can serve as a model for homomeric and heteromeric protein-protein interactions. A1R forms homodimers in membranes and also form high-order molecular structures containing also heterotrimeric G-proteins and adenosine deaminase. The occurrence of clustering also clearly suggests that G-protein- coupled receptors form high-order molecular structures, in which multimers of the receptors and probably other interacting proteins form functional complexes. In view of the occurrence of homodimers of adenosine and of dopamine receptors it is speculated that heterodimers between these receptors belonging to two different families of G-protein-coupled receptors can be formed. Evidence that A1/D1 can form heterodimers in cotransfected cells and in primary cultures of neurons has in fact been obtained. In the central nervous system direct and indirect receptor-receptor interactions via adaptor proteins participate in neurotransmission and neuromodulation and, for example, in the establishment of high neural functions such as learning and memory.

Interactions among adenosine deaminase, adenosine A(1) receptors and dopamine D(1) receptors in stably cotransfected fibroblast cells and neurons.

The role of adenosine deaminase in the interactions between adenosine A(1) and dopamine D(1) receptors was studied in a mouse fibroblast cell line stably cotransfected with human D(1) receptor and A(1) receptor cDNAs (A(1)D(1) cells). Confocal laser microscopy analysis showed a high degree of adenosine deaminase immunoreactivity on the membrane of the A(1)D(1) cells but not of the D(1) cells (only cotransfected with human D(1) receptor cDNAs). In double immunolabelling experiments in A(1)D(1) cells and cortical neurons a marked overlap in the distribution of the A(1) receptor and adenosine deaminase immunoreactivities and of the D(1) receptor and adenosine deaminase immunoreactivities was found. Quantitative analysis of A(1)D(1) cells showed that adenosine deaminase immunoreactivity to a large extent colocalizes with A(1) and D(1) receptor immunoreactivity, respectively. The A(1) receptor agonist caused in A(1)D(1) cells and in cortical neurons coaggregation of A(1) receptors and adenosine deaminase, and of D(1) receptors and adenosine deaminase. The A(1) receptor agonist-induced aggregation was blocked by R-deoxycoformycin, an irreversible adenosine deaminase inhibitor. The competitive binding experiments with the D(1) receptor antagonist [(3)H]SCH-23390 showed that the D(1) receptors had a better fit for two binding sites for dopamine, and treatment with the A(1) receptor agonist produced a disappearance of the high-affinity site for dopamine at the D(1) receptor. R-Deoxycoformycin treatment, which has previously been shown to block the interaction between adenosine deaminase and A(1) receptors, and which is crucial for the high-affinity state of the A(1) receptor, also blocked the A(1) receptor agonist-induced loss of high-affinity D(1) receptor binding. The conclusion of the present studies is that the high-affinity state of the A(1) receptor is essential for the A(1) receptor-mediated antagonistic modulation of D(1) receptors and for the A(1) receptor-induced coaggregates of A(1) and adenosine deaminase, and of D(1) and adenosine deaminase. Thus, the confocal experiments indicate that both A(1) and D(1) receptors form agonist-regulated clusters with adenosine deaminase, where the presence of a structurally intact adenosine deaminase bound to A(1) receptors is important for the A(1)-D(1) receptor-receptor interaction at the level of the D(1) receptor recognition.

Immunolocalization of A1 adenosine receptors in mammalian spermatozoa.

The presence of A1 adenosine receptors (A1AR) in mammalian spermatozoa was previously demonstrated by radiochemical and immunochemical detection. This study was performed to investigate the cellular location of the A1AR to determine whether these receptors were somehow connected with ecto-adenosine deaminase and to evaluate their function in calcium uptake. By immunofluorescence staining we showed that in mammalian spermatozoa A1AR were constantly localized in the acrosomal region. This finding was confirmed by immunogold detection. Confocal analyses with anti-A1 and anti-ADA antibodies showed a high degree of co-localization. Calcium loading assay showed that this association was functional and affected calcium accumulation in mammalian spermatozoa. Therefore, we concluded that the acrosomal localization of A1AR was a constant feature in mammalian sperm. Moreover, these A1 receptors were functionally coupled to ecto-ADA and were able to modulate calcium uptake into an IP3-gated store.(J Histochem Cytochem 48:1163-1171, 2000)

Calcium mobilization in Jurkat cells via A2b adenosine receptors.

1. A functional study of cell surface A2b adenosine receptors was performed on the T cell leukaemia line, Jurkat. 2. A2b receptors were coupled both to the adenylate cyclase system and to intracellular calcium channels. In fact, the agonist of A2b receptors, 5'-N-ethylcarboxamidoadenosine (NECA), led to a transient accumulation of intracellular calcium by an inositol phosphate-independent mechanism. 3. The NECA-induced accumulation of cGMP was not responsible for the calcium mobilization via A2b receptors. 4. The calcium response elicited by activation of A2b receptors was independent of that evoked by activation of the T cell receptor. 5. These findings not only delineate a novel transduction mechanism for adenosine but also support a specific role for adenosine in modulating signals elicited via the T cell receptor.

Characterization of adenosine receptors in brush-border membranes from pig kidney.

1. The adenosine receptors from pig kidney proximal tubules have been studied in membrane vesicle preparations derived from either luminal (brush-border membranes-BBM-) or basolateral (BL) sides. There was a substantial amount of A2-like NECA binding in both preparations, but the A1 subtype of adenosine receptors was not found in either BBM or BL membranes. The use of [3H]-CGS21680 which is a more specific ligand for A2a receptors revealed true adenosine receptors in the BBM. 2. The kinetic parameters for [3H]-CGS21680 binding to pig renal BBM were: Bmax = 1.48 pmol mg-1 protein and Kd = 150 nM. In the presence of Gpp(NH)p the affinity decreased (Kd = 220 nM), whereas the addition of Mg2+ induced a marked increase in affinity (Kd = 83 nM). These equilibrium constants are higher than those found for the A2a adenosine receptors present in pig brain striatal membranes (Kd = 12 nM), and are close to those found in rat renal BBM (Kd = 90 nM). 3. The order of potency of agonist and antagonists was not consistent with the presence of either A1 or A2 receptors, but it was very similar to the agonist order of potency for the A3 receptor subtype. Furthermore, the blockade of the [3H]-CGS21680 binding by both cholera and pertussis toxin further supports the view that the subtypes present in BBM are neither A1 nor A2. 4. Overall the results suggest the presence in BBM of an A3 receptor, or of a new subtype of adenosine receptor, which is linked to G proteins sensitive to both cholera and pertussis toxins.