Adenosine A2A-dopamine D2 receptor-receptor heteromerization: qualitative and quantitative assessment by fluorescence and bioluminescence energy transfer

There is evidence for strong functional antagonistic interactions between adenosine A2A receptors (A2ARs) and dopamine D2 receptors (D2Rs). Although a close physical interaction between both receptors has recently been shown using co-immunoprecipitation and co-localization assays, the existence of a A2AR-D2R protein-protein interaction still had to be demonstrated in intact living cells. In the present work, fluorescence resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET) techniques were used to confirm the occurrence of A2AR-D2R interactions in co-transfected cells. The degree of A2AR-D2R heteromerization, measured by BRET, did not vary after receptor activation with selective agonists, alone or in combination. BRET competition experiments were performed using a chimeric D2R-D1R in which helices 5 and 6, the third intracellular loop (I3), and the third extracellular loop (E3) of the D2R were replaced by those of the dopamine D1 receptor (D1R). Although the wild type D2R was able to decrease the BRET signal, the chimera failed to achieve any effect. This suggests that the helix 5-I3-helix 6-E3 portion of D2R holds the site(s) for interaction with A2AR. Modeling of A2AR and D2R using a modified rhodopsin template followed by molecular dynamics and docking simulations gave essentially two different possible modes of interaction between D2R and A2AR. In the most probable one, helix 5 and/or helix 6 and the N-terminal portion of I3 from D2R approached helix 4 and the C-terminal portion of the C-tail from the A2AR, respectively.     

Variability of biochemical and clinical phenotype in X-linked liver glycogenosis with mutations in the phosphorylase kinase PHKA2 gene

X-linked liver glycogenosis (XLG) resulting from phosphorylase kinase (Phk) deficiency is one of the most common forms of glycogen storage disease. It is caused by mutations in the gene encoding the liver isoform of the Phk α subunit (PHKA2). In the present study, we address the issue of phenotypic and allelic heterogeneity in XLG. We have identified mutations in seven male patients. One of these patients represents the variant biochemical phenotype, XLG subtype 2 (XLG2), where Phk activity is low in liver but normal or even elevated in erythrocytes. He carries a K189E missense mutation, which adds to the emerging evidence that XLG2 is associated with missense mutations clustering at a few sites. Two patients display clinical phenotypes unusual for liver Phk deficiency, with dysfunction of the kidneys (proximal renal tubular acidosis) or of the nervous system (seizures, delayed cognitive and speech abilities, peripheral sensory neuropathy), respectively, in addition to liver glycogenosis. In the patient with kidney involvement, we have identified a missense mutation (P399S) and a trinucleotide deletion (2858del3) leading to the replacement of two amino acids by one new residue (N953/L954I), and a missense mutation has also been found in the patient with neurological symptoms (G1207W). These two cases demonstrate that PHKA2 mutations can also be associated with uncommon clinical phenotypes. Finally, in four typical XLG cases, we have identified three truncating mutations (70insT, R352X, 567del22) and an in-frame deletion of eight well-conserved amino acids (2452del24). Together, this study adds eight new mutations to the previously known complement of sixteen PHKA2 mutations. All known PHKA2 mutations but one are distinct, indicating pronounced allelic heterogeneity of X-linked liver glycogenosis with mutations in the PHKA2 gene. Received: 17 October 1997 / Accepted: 23 December 1997     

Role of Histidine Residues in Agonist and Antagonist Binding Sites of A1 Adenosine Receptor

Abstract: The influence of pH on the equilibrium dissociation constant and on kinetic association and dissociation constants was studied for adenosine receptor agonist L-N⁶-[adenine-2,8-³H, ethyl-2-³H]phenylisopropyladenosine ([³H]R-PIA) and antagonist 8-cyclopentyl-1,3-[³H]-dipropylxanthine ([³H]DPCPX). Two ionizable groups, of pK 7.0 and pK 7.4, are involved in the [³H]R-PIA associations with high- and low-affinity states of the receptor, and another group, of pK 6.0, is involved in the association with the low-affinity state. No ionizable group is involved in the dissociation process for the high-affinity state, whereas two ionizable groups, of pK 6.0 and 6.5, are involved in the low-affinity state. For [³H]DPCPX, three ionizable groups (pK 6.0, 7.4, and 8.0) are involved in the association process and only one group, (pK 6.0), is involved in the dissociation step. The apparent pK values obtained agree with histidine residues. We thus studied the effect of diethylpyrocarbonate (DEP), which reacts irreversibly with histidine residues, on agonist and antagonist binding to A₁ adenosine receptors from pig brain cortical membranes. DEP treatment of membrane reduced the affinity (K_D) and the total binding (R) of the agonist and the antagonist. Membrane preincubation with unlabeled ligand (R-PIA or DPCPX) prevented the effect of DEP modification observed when the same ligand, but with label, is added to the same membranes, but did not prevent the DEP modification on different, labeled ligand. The pattern of protective action of R-PIA, DPCPX, adenosine, and guanylylimidodiphosphate in DEP treatment and the displacement curves of radiolabeled agonist and antagonist by both unlabeled ligands indicated that the interaction site for agonist and antagonist binding is the same, although the complete mechanisms for recognition and binding differ.     

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.     

Mycobacterial Dihydrofolate Reductase Inhibitors Identified Using Chemogenomic Methods and In Vitro Validation

The lack of success in target-based screening approaches to the discovery of antibacterial agents has led to reemergence of phenotypic screening as a successful approach of identifying bioactive, antibacterial compounds. A challenge though with this route is then to identify the molecular target(s) and mechanism of action of the hits. This target identification, or deorphanization step, is often essential in further optimization and validation studies. Direct experimental identification of the molecular target of a screening hit is often complex, precisely because the properties and specificity of the hit are not yet optimized against that target, and so many false positives are often obtained. An alternative is to use computational, predictive, approaches to hypothesize a mechanism of action, which can then be validated in a more directed and efficient manner. Specifically here we present experimental validation of an in silico prediction from a large-scale screen performed against Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis. The two potent anti-tubercular compounds studied in this case, belonging to the tetrahydro-1,3,5-triazin-2-amine (THT) family, were predicted and confirmed to be an inhibitor of dihydrofolate reductase (DHFR), a known essential Mtb gene, and already clinically validated as a drug target. Given the large number of similar screening data sets shared amongst the community, this in vitro validation of these target predictions gives weight to computational approaches to establish the mechanism of action (MoA) of novel screening hit.     

Successful prediction of the hydrogen bond network of the 3-oxo-12alpha-hydroxy-5beta-cholan-24-oic acid crystal from resolution of the crystal structure of deoxycholic acid and its three 3,12-dihydroxy epimers

Crystal structures of p-xylene-crystallized deoxycholic acid (3alpha,12alpha-dihydroxy-5beta-cholan-24-oic acid) and its three epimers (3beta,12alpha-; 3alpha,12beta-; and 3beta,12beta-) have been solved. Deoxycholic acid forms a crystalline (P21) complex with the solvent with a 2:1 stoichiometry whereas crystals of the three epimers do not form inclusion compounds. Crystals of the 3beta,12beta-epimer are hexagonal, whereas the 3alpha,12beta-and 3beta,12alpha-epimers crystallize in the P2(1)2(1)2(1) orthorhombic space group. The three hydrogen bond sites (two hydroxy groups, i. e. O3-H, and O12-H, and the carboxylic acid group of the side chain, O24bO24a-H) simultaneously act as hydrogen bond donors and acceptors. The hydrogen bond network in the crystals was analyzed and the following sequences have been observed: two chains (abcabc... or acbacb... ) and two rings (abc or acb), which constitute a complete set of all the possible sequences which can be drawn for an intermolecular hydrogen bond network formed by three hydrogen bond donor/acceptor sites forming crossing hydrogen bonds. The orientation of O3-H (alpha or beta) determines the sequence of the acceptor and the donor groups involved in the pattern: O24a --> O12 --> O3 --> O24b when it is alpha and O24a --> O3 --> O12--> O24B when it is beta. These observations were used to predict the hydrogen bond network of p-xylene-crystallized 3-oxo,12alpha-hydroxy-5beta-cholan-24-oic acid. This compound has two hydrogen bond donor and three potential hydrogen bond acceptor sites. According to the previous sequence set, this compound should crystallize in the monoclinic P21 system, should form a complex with the solvent, O24b should not participate in the hydrogen bond network, and the chain sequence O24a --> O12 --> O3 would be followed. All predictions were confirmed experimentally.     

Complexes of Cu with mixed-donor phenanthroline-containing macrocycles: analysis of their structural, redox and spectral properties in the context of Type-1 blue copper proteins biomimetic models

The macrocycles L¹-L³ having N₂S₂O-, N₂S₂-, and N₂S₃-donor sets, respectively, and incorporating the 1,10-phenanthroline unit interact in EtOH and MeCN solutions with Cu^II to give 1:1 [M(L)]²⁺ complex species. The compounds [Cu(L¹)(ClO₄)]ClO₄ (1), [Cu(L²)(ClO₄)]ClO₄ ·  (2) and [Cu(L³)](ClO₄)₂ (3) were isolated at the solid state and the first two also characterised by X-ray diffraction studies. The conformation adopted by L¹ and L² in the cation complexes reveals the aliphatic portion of the rings folded over the plane containing the heteroaromatic moiety with the ligands encapsulating the metal centre within their cavity by imposing, respectively, a square-based pyramidal and a square planar geometry. In both complexes, the metal ion completes its coordination sphere by interacting with a ClO₄⁻ ligand. The compound [Cu(L³)₂](PF₆)₂ (4) containing a 1:2 cation complex was also isolated at the solid state: EPR spectroscopy measurements suggest the presence of a CuN₄ chromophore in this complex. The EPR and electronic spectral features of 1-4 have been studied and their redox properties examined in comparison with those observed for Type-1 blue copper proteins.The reactivity of L¹-L³ has also been tested toward stoichiometric amounts of the Cu^I salt [CuCl(PPh₃)₃].     

Adenosine A(1) receptor in cultured neurons from rat cerebral cortex: colocalization with adenosine deaminase

Adenosine A(1) receptors (A(1)Rs) have been characterized in primary cultures of neurons from cerebral cortex. The specific adenosine A(1) antagonist 8-cyclopentyl-1,3-[(3)H]dipropylxanthine bound to both membranes and intact cells. When saturation experiments were performed in membranes, a K(D) value of 0.76 nM and a B(max) of 57 fmol/mg of protein were obtained. Competition assays revealed a pharmacological profile characteristic of A(1)Rs. The presence of this receptor was further confirmed by RT-PCR analysis. The expression of the receptor showed no significant changes during the period of culture studied, up to 12 days in vitro. A(1)R agonist inhibited forskolin-stimulated adenylyl cyclase, showing the functional coupling of these receptors with the effector. alphaG(i1, 2) protein level, detected by immunoblot, presented an increase during the period of culture. This increase correlated with an increase in the mRNA level of alphaG(i1) but not alphaG(i2). By immunochemical assays, it is shown that these receptors are expressed in both the neuronal cell body and the proximal dendrites. Colocalization of A(1)Rs with microtubule-associated protein 2 and cell surface adenosine deaminase was shown by confocal microscopy. The high degree of colocalization observed between A(1)Rs and ectoadenosine deaminase in neurons could suggest an important role of the enzyme in adenosine-mediated neuromodulation.     

Adenosine A2A receptor stimulation potentiates nitric oxide release by activated microglia

The absence of adenosine A2A receptors, or its pharmacological inhibition, has neuroprotective effects. Experimental data suggest that glial A2A receptors participate in neurodegeneration induced by A2A receptor stimulation. In this study we have investigated the effects of A2A receptor stimulation on control and activated glial cells. Mouse cortical mixed glial cultures (75% astrocytes, 25% microglia) were treated with the A2A receptor agonist CGS21680 alone or in combination with lipopolysaccharide (LPS). CGS21680 potentiated lipopolysaccharide-induced NO release and NO synthase-II expression in a time- and concentration-dependent manner. CGS21680 potentiation of lipopolysaccharide-induced NO release was suppressed by the A2A receptor antagonist ZM-241385 and did not occur on mixed glial cultures from A2A receptor-deficient mice. In mixed glial cultures treated with LPS + CGS21680, the NO synthase-II inhibitor 1400W abolished NO production, and NO synthase-II immunoreactivity was observed only in microglia. Binding experiments demonstrated the presence of A2A receptors on microglial but not on astroglial cultures. However, the presence of astrocytes was necessary for CGS21680 potentiating effect. In light of the reported neurotoxicity of microglial NO synthase-II and the neuroprotection of A2A receptor inhibition, these data suggest that attenuation of microglial NO production could contribute to the neuroprotection afforded by A2A receptor antagonists.     

Characterization of a C3a receptor in rainbow trout and Xenopus: the first identification of C3a receptors in nonmammalian species

Virtually nothing is known about the structure, function, and evolutionary origins of the C3aR in nonmammalian species. Because C3aR and C5aR are thought to have arisen from the same common ancestor, the recent characterization of a C5aR in teleost fish implied the presence of a C3aR in this animal group. In this study we report the cloning of a trout cDNA encoding a 364-aa molecule (TC3aR) that shows a high degree of sequence homology and a strong phylogenetic relationship with mammalian C3aRs. Northern blotting demonstrated that TC3aR was expressed primarily in blood leukocytes. Flow cytometric analysis and immunofluorescence microscopy showed that Abs raised against TC3aR stained to a high degree all blood B lymphocytes and, to a lesser extent, all granulocytes. More importantly, these Abs inhibited trout C3a-mediated intracellular calcium mobilization in trout leukocytes. A fascinating structural feature of TC3aR is the lack of a significant portion of the second extracellular loop (ECL2). In all C3aR molecules characterized to date, the ECL2 is exceptionally large when compared with the same region of C5aR. However, the exact function of the extra portion of ECL2 is unknown. The lack of this segment in TC3aR suggests that the extra piece of ECL2 was not necessary for the interaction of the ancestral C3aR with its ligand. Our findings represent the first C3aR characterized in nonmammalian species and support the hypothesis that if C3aR and C5aR diverged from a common ancestor, this event occurred before the emergence of teleost fish.